Treatment of High-Ammonia-Nitrogen Wastewater with Immobilized Ammonia-Oxidizing Bacteria Alcaligenes sp. TD-94 and Paracoccus sp. TD-10

Abstract

In this study, modified granular activated carbon (GAC) and immobilized cells were used to improve the biological efficiency of high-ammonia-nitrogen wastewater treatment using microorganisms. The results showed that using sodium-hydroxide-modified activated carbon (NaOH-GAC) greatly increased the immobilized numbers of the ammonia-oxidizing bacteria Alcaligenes sp. TD-94 and Paracoccus sp. TD-10. Using NaOH-GAC increased the number of immobilized cells by 63.27% over GAC. Compared with free cells, those immobilized on modified activated carbon were more effective in the removal of high ammonia nitrogen levels from wastewater. In wastewater with an ammonia nitrogen concentration of 100 mg·L⁻¹, the ammonia nitrogen removal efficiencies of NaOH-GAC-immobilized cells and free cells within 24 h were 100% and 4.17%, respectively. After 45 cycles, NaOH-GAC-immobilized cells maintained an ammonia nitrogen removal efficiency of 79.24%. After 70 days of storage at 4 °C, the ammonia nitrogen removal efficiency was still as high as 100%. The removal efficiencies of ammonia nitrogen (NH₄⁺-N), total nitrogen (TN), and chemical oxygen demand (COD) in high-ammonia-nitrogen wastewater from petrochemical enterprises reached 99.27%, 88.39%, and 69.85%, with removal rates of 75.21, 69.43, and 1117.40 mg·L⁻¹·d⁻¹, respectively. The findings demonstrated that NaOH-GAC improved the capacity of the biological treatment to remove ammonia nitrogen from wastewater and provide a practical option for the remediation of environmental pollution.

1. Introduction

Since the beginning of the 21st century, water pollution has been increasing, resulting in a compound water shortage situation in terms of both quality and quantity, and the issue of water security is limiting global development [1,2]. The national sewage discharge in China reached 57.136 billion t in 2020, up 22.44% from the end of the 12th Five-Year Plan, according to the National Bureau of Statistics. This discharge includes 25.648 million tons of chemical oxygen demand (COD) emissions and 984,000 t of ammonia nitrogen (NH₄⁺-N) emissions [3]. One of the main issues with traditional wastewater treatment, characterized by huge discharge, complicated composition, challenging treatment, and high toxicity, is the difficulty associated with the treatment of wastewater containing nitrogen. Water bodies suffer environmental harm as a result of the release of high-ammonia-nitrogen wastewater, which is hazardous to aquatic life and causes red tides and water blooms [4]. Therefore, researchers have focused much effort on developing strategies for the quick removal of ammonia contaminants from wastewater. The main methods used for ammonia nitrogen removal from water bodies include physical (adsorption, purging, vapor extraction [5], ion exchange [6], etc.), chemical (chemical precipitation [7], fold point chlorination [8], electrochemical oxidation [9], etc.), and biological methods (nitrification–denitrification [10], short-course nitrification–denitrification, anaerobic ammonia oxidation [11], etc.). Biological nitrogen removal is generally considered the most cost-effective method among the various techniques for removing nitrogen from wastewater with high ammonia levels. Unlike physical and chemical nitrogen removal methods, this method is simple, efficient, complete, and does not generate secondary pollution, among other advantages [12].

Biological nitrogen removal usually includes two major processes: nitrification and denitrification, wherein the nitrification process includes ammonia oxidation (NH₄⁺-N→NO₂⁻-N) and nitrite oxidation (NO₂⁻-N→NO₃⁻-N). Nitrification is accomplished by different microbial species using different enzymes. First, ammonia-oxidizing bacteria (AOB) catalyze the conversion of ammonia (NH₃) to hydroxylamine (NH₂OH) by ammonia monooxygenase (AMO), which is then oxidized to nitrite (NO₂⁻-N) by hydroxylamine oxidase (HAO). Then, nitrite oxidoreductase (NXR) in nitrite-oxidizing bacteria (NOB) catalyzes the second step by oxidizing NO₂⁻-N to nitrate (NO₃⁻-N) [13]. Ammonia oxidation, as the rate-limiting step of the nitrification process, is the key process in the biological removal of nitrogen from wastewater [14]. Usually, AOB are mostly autotrophic bacteria. Further research has discovered microorganisms capable of heterotrophic ammonia oxidation, including Alcaligenes, Pseudomonas, Paracoccus, Rhodococcus, Acinetobacter, Bacillus, Acidovorax, Hydrogenophaga, etc. [15,16,17]. Compared with conventional ammonia-oxidizing bacteria, heterotrophic ammonia-oxidizing bacteria are less affected by carbon sources and have better nitrogen removal capacity [18].

Through the study of ammonia-oxidizing bacteria, it was found that mixing two or more strains in a certain ratio may provide a better ammonia nitrogen removal effect than a single strain [19]. However, free microorganisms can also suffer from instability, are easily lost, and their removal capacity is influenced by the environment [20]. The immobilization of microbial cells can overcome these drawbacks, representing a promising new biotechnology. Because of its substantial specific surface area, well-developed pore structure, abundance of surface functional groups, simplicity of supply, and affordability [21,22], granular activated carbon (GAC) is an excellent immobilization material. Generally speaking, minimal ammonia nitrogen is removed by the adsorption capacity of GAC, and most of the removal is by microorganisms attached to GAC [23]. Therefore, there is a need to modify GAC and develop new materials with high adsorption capacities. Among them, acid treatment [24], metal or metal oxide impregnation [25], and modification of activated carbon followed by treatment with NH₄⁺-N have been widely studied, while the sodium hydroxide alkaline treatment of GAC followed by adsorption of NH₄⁺-N has been less studied.

Alcaligenes sp. TD-94 and Paracoccus sp. TD-10 are two strains of heterotrophic ammonia-oxidizing bacteria isolated by the Laboratory of Applied Microbiology and Biotechnology (LAMB), Changzhou University. In order to improve the biotransformation efficiency and the recycling of cells, immobilized cells were prepared using NaOH-GAC as a carrier in this study. We characterized the carriers and immobilized cells using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR), compared the ammonia nitrogen removal capacity of immobilized cells and free cells, evaluated the reuse performance and storage stability performance of immobilized cells, and verified the treatment effect of immobilized cells in actual high-ammonia-nitrogen wastewater. This research offers fundamental information to support the use of immobilized microbial technologies for the treatment of aquatic environments.

2. Materials and Methods

2.1. Microbial Strains and Reagents

The strains used in the experiments were Alcaligenes sp. TD-94 and Paracoccus sp. TD-10, which were stored in the LAMB, Changzhou University [19]. The GAC was purchased from Shanghai Sinopharm Chemical Reagent Co. Analytical-grade ammonium chloride, anhydrous sodium acetate, magnesium sulfate heptahydrate, ferrous sulfate heptahydrate, sodium chloride, sodium hydroxide, and other chemical reagents were all acquired from Shanghai Sinopharm Chemical Reagent Co.

2.2. Preparation of Carriers and Immobilization of Cells

The GAC was washed three times with distilled water before being dried for 12 h at 105 °C. The purpose of this procedure was to remove contaminants from the surface of the activated carbon. The pretreated GAC was modified with 1 mol·L⁻¹ NaOH after drying and passing through a 40–80 mesh sieve. The ratio of GAC to modifier was 1:10 (w/w), and the modification time was 24 h. The modified GAC was washed at least five times with an equal volume of distilled water and finally dried at 105 °C to a constant weight. The GAC obtained via NaOH modification is described herein as NaOH-GAC.

The pure cultures of Alcaligenes sp. TD-94 and Paracoccus sp. TD-10 were cultured for 24 h at 160 rpm in a constant-temperature oscillating incubator at 28 °C. The medium in which the strains were grown was as follows: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, and 1000 mL distilled water. After incubation, the microbial cells in the supernatant were extracted by centrifuging at 8000 rpm for 10 min. The next step was to obtain cell suspensions by washing them three times with 0.9% sterile saline. The two strains were adjusted to the same cell concentration and mixed according to the optimal ratio of TD-94:TD-10 = 4:1 after measuring the optical density (OD₆₀₀).

Sterilized NaOH-GAC was added to the cell suspension, and the mixture was incubated in a constant-temperature oscillating incubator at 160 rpm at 28 °C. The immobilized cells were collected after 4 h and gently washed with 0.9% sterile saline at least three times to eliminate the organisms that were not firmly adsorbed. The supernatant was used for biomass determination, and the prepared immobilized cells were stored at 4 °C for backup.

2.3. High-Ammonia-Nitrogen Wastewater Removal Experiments—Simulated Wastewater

2.3.1. Ammonia Nitrogen Removal Capacity of Immobilized Cells

One gram of NaOH-GAC with immobilized cells was added to a 250 mL conical flask containing 50 mL of simulated wastewater and placed in a constant-temperature oscillating incubator at 28 °C and 160 rpm. The formulation of simulated wastewater was as follows: 0.382 g ammonium chloride, 2 g anhydrous sodium acetate, 0.2 g dipotassium hydrogen phosphate, 0.12 g sodium chloride, 0.01 g ferrous sulfate heptahydrate, 0.05 g magnesium sulfate heptahydrate, 0.01 manganese sulfate monohydrate, 1000 mL distilled water, pH 7.0 to 7.4. Meanwhile, the same masses of GAC-immobilized cells, free cells, NaOH-GAC, and GAC, and a control group without immobilized cells, carrier, or free cells were compared with 1 g NaOH-GAC-immobilized cells under the same conditions. Samples were taken at regular intervals to test the concentration of NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N. The calculation formula was as follows:

$$\mathrm{A}\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}=\frac{\mathrm{A}-\mathrm{B}}{\mathrm{T}}$$

(1)

$$\mathrm{A}\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{r}\mathrm{e}\mathrm{m}\mathrm{o}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}=\frac{\mathrm{A}-\mathrm{B}}{\mathrm{A}}\times 100\%$$

(2)

$$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{b}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{i}\mathrm{o}\mathrm{d}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\mathrm{C}-\mathrm{D}$$

(3)

where A represents the initial ammonia concentration (mg·L⁻¹), B represents the residual ammonia concentration (mg·L⁻¹), C represents the ammonia nitrogen removal efficiency for immobilized cells (%), D represents the ammonia nitrogen removal efficiency for adsorption (%), and T represents the reaction time (d). These experiments were replicated three times.

2.3.2. Reusability and Stability of Immobilized Cells

The reusability of immobilized cells for ammonia nitrogen removal was evaluated. Immobilized cells were placed in 50 mL of simulated wastewater medium with 100 mg·L⁻¹ ammonia nitrogen and biodegraded for 24 h. The previous medium was poured out and the immobilized cells were washed with 0.9% sterile saline before adding 50 mL of fresh medium. In addition, the storage stability of the immobilized cells was also assessed. The prepared immobilized cells were stored in a refrigerator at 4 °C for 35 and 70 d, then removed and injected into wastewater with an ammonia nitrogen concentration of 100 mg·L⁻¹, and the ammonia nitrogen removal capacity of the immobilized cells was tested.

2.4. High-Ammonia-Nitrogen Wastewater Removal Experiments—Actual Wastewater

To verify the effect of the modified activated carbon with immobilized cells in actual high-ammonia-nitrogen wastewater, wastewater was collected from the wastewater treatment center of the Maoming Branch of the Sinopec Corporation; the physicochemical characteristics of this wastewater are shown in

Table 1. Physicochemical characteristics of the high-ammonia-nitrogen wastewater.

Projects	COD/mg·L−1	NH4+-N/mg·L−1	Ca2+/mg·L−1	Mg2+/mg·L−1	Hardness/mg·L−1	Alkalinity/mg·L−1	pH
Value	500–800	150–300	80–200	30–50	120–250	900–1400	7.5–8.5

. Four treatment groups were set up for the experiment (

Table 2. Experimental grouping table of actual high-ammonia-nitrogen wastewater treatment experiment.

Group	Treatment
1	High-ammonia-nitrogen wastewater and modified activated carbon with immobilized cells
2	High-ammonia-nitrogen wastewater and free cells
3	High-ammonia-nitrogen wastewater and modified activated carbon
4	High-ammonia-nitrogen wastewater

), including the immobilized cell treatment group (1), the free cell treatment group (2), the modified activated carbon group (3), and the blank control group (4). Before the initiation of the reaction, appropriate amounts of nutrient factors, such as sodium acetate [26], metal ions, etc., were added to each treatment group. The experiment was conducted continuously for 10 d in 250 mL conical flasks, incubated at 28 °C in a constant-temperature shaking incubator, and the wastewater was changed every 2 d. NH₄⁺-N, NO₂⁻-N, NO₃⁻-N, total nitrogen (TN), COD, and acidity (pH) were monitored throughout the experiment.

2.5. Characterization and Analytical Methods

Scanning electron microscopy (SEM) was used to analyze the morphology of the carrier material before and after the modification and immobilization of cells.

Structural changes before and after GAC modification and before and after immobilization of cells were analyzed using Fourier-transform infrared spectroscopy (FTIR). GAC before and after immobilization was mixed with potassium bromide at 1:100, ground into powder under infrared light, and then formed into transparent flakes using the press method. The spectral scan range was from 4000 to 500 cm⁻¹ with a resolution of 2 cm⁻¹.

The biomass of immobilized cells was determined via colony plate counting. NH₄⁺-N was determined according to Nessler’s reagent spectrophotometric method [27], NO₂⁻-N was determined using the N-(1-naphthyl)ethylenediamine spectrophotometric method [28], NO₃⁻-N was determined using the ultraviolet spectrophotometric method [29], and NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N parameters were measured using a UV–visible spectrophotometer (Shanghai Onra Instruments Ltd., EU-2200, Shanghai, China). An analysis of TN and COD was performed using a multiparameter water quality meter (Beijing Lianhua Technology Ltd., 5B-3B, Beijing, China). The pH values were determined using a Sartorius acidity meter (Sartorius Scientific Instruments (Beijing) Ltd., PB-30, Goettingen, Germany). Three replicate experiments were performed for each sample, and all water samples were filtered with a syringe filter (aqueous system, 0.22 µm) prior to analysis.

3. Results and Discussion

3.1. Scanning Electron Microscopy of Carriers and Immobilized Cells

The morphological characteristics of the carrier material before and after alteration, as well as before and after cell adsorption, were revealed by SEM. The surface morphology and pore structure of the activated carbon changed after modification, as can be observed in Figure 1a,b, which shows the SEM images of the material before and after modification. The surface of the activated carbon became rougher and the pore structure increased after modification, which was more conducive to the attachment and immobilization of bacteria and to the provision of storage space for the microbial metabolism of substrates [30]. The SEM images of immobilized microbial cells are shown in Figure 1c,d. It was clear that the cells were successfully immobilized on the carrier material and the number of NaOH-GAC-immobilized microorganisms was much larger than that of GAC-immobilized microorganisms. The SEM results demonstrated that the ability of the modified granular activated carbon to immobilize microbial cells was increased by about 63.27% (

Table 3. Immobilized biomass.

Procedures	Biomass/pc·g−1
1	1.8 × 108
2	4.9 × 108

(1: GAC-immobilized cells; 2: NaOH-GAC-immobilized cells).

) and also showed that the surface roughness and pore structure of the carrier material determined the immobilized biomass.

3.2. Fourier-Transform Infrared Analysis

The infrared spectra of pristine activated carbon and sodium-hydroxide-modified activated carbon and of modified activated carbon after adsorption of bacteria are shown in Figure 2. It is clear from the figure that the presence of a peak at 3407 cm⁻¹ for all samples was associated with the stretching vibration of the hydroxyl [31]. The peak appearing at 1558 cm⁻¹ was attributed to the stretching vibration of the C=C bond of the aromatic ring [21]. The peak at 1140 cm⁻¹ narrowed after the adsorption of microorganisms onto the modified activated carbon, which was presumed to be caused by the C-O stretching vibration of the alcohol hydroxyl group [32]. The absorption peak at 873 cm⁻¹ represented the C-H bond plane bending vibration [33]. The primary characteristic absorption peaks of the virgin and modified activated carbons were nearly identical, indicating that the modification did not change the skeletal structure of the activated carbon, which was consistent with previous reports [34]. Comparing the GAC with the NaOH-GAC and the adsorption of bacteria based on NaOH-GAC, the most significant change in peak shape was observed at 1140 cm⁻¹. A possible reason is that the modification affected the alcohol hydroxyl group, and the C-O bond on the alcohol hydroxyl group was involved in the adsorption of bacteria. In addition, the change in the C=C bond of the aromatic ring suggested that this bond may also be involved in the adsorption of bacteria.

3.3. Treatment of High-Ammonia-Nitrogen Wastewater Using Immobilized Cells

The rates of removal of ammonia nitrogen by free cells and immobilized cells are shown in Figure 3. It was apparent that NaOH-GAC-immobilized cells exhibited a higher ammonia removal capacity compared to unmodified-GAC-immobilized cells and free cells. When the initial concentration of ammonia nitrogen was 100 mg·L⁻¹, the ammonia nitrogen removal rate of the NaOH-GAC-immobilized cells reached 100 mg·L⁻¹·d⁻¹ within 24 h, and the removal efficiency was 100%. This indicated that the microbial cells immobilized onto the modified GAC were more effective at removing ammonia nitrogen than the experimental group without the modification treatment. The reason for this result is that, on the one hand, the change in the surface properties of the modified GAC, which increased the number of adsorbed microorganisms (Figure 3d) and enhanced their metabolic activity [35]; on the other hand, the ammonia oxidation process consumes a certain amount of alkalinity, and too low a pH value cannot guarantee sufficient alkalinity, which affects the growth of ammonia-oxidizing bacteria [36]. The use of alkali-modified activated carbon can increase the pH value of the carrier and provide an alkaline growing environment for ammonia-oxidizing bacteria, thus, improving the efficiency of ammonia nitrogen removal.

The ammonia removal efficiency of free cells was low throughout the process; the ammonia removal rate of this treatment was 4.17 mg·L⁻¹·d⁻¹, and the ammonia removal efficiency of the reaction after 24 h was only 4.17%. This perfectly exemplifies the enormous potential of immobilized microbial technology for the treatment of wastewater containing ammonia nitrogen. The reason immobilized cells were more advantageous than free cells in treating wastewater is twofold: first, immobilization of microbial cells can increase the cell density per unit volume and maintain higher biomass, thus, improving biological nitrogen removal capacity [37]; second, immobilization enhances the ability of microbial cells to resist the effects external toxic substances or an unfavorable environment [38]. In addition, GAC without microbial cells and NaOH-GAC were used as control experiments. The GAC alone had a weak adsorption capacity for ammonia nitrogen compared to the NaOH-GAC, which had an improved adsorption capacity for ammonia nitrogen. The ammonia nitrogen removal efficiency of the NaOH-GAC was 9.73% in 24 h. The results demonstrated that adsorption and biodegradation synergistically enhanced the ammonia nitrogen removal process. The contributions of adsorption and biodegradation to ammonia nitrogen removal were 9.73% and 90.27%, respectively.

3.4. Stability of the Immobilized Cells

3.4.1. Reusability

Immobilized microbial cells can be reused at the end of a reaction, so the process is relatively inexpensive. To investigate the reusability and stability of immobilized cells, batch experiments with simulated high-ammonia-nitrogen wastewater were repeated, and the reuse results are shown in Figure 4. The immobilized cells showed high metabolic activity for 45 cycles and were used satisfactorily. The NaOH-GAC-immobilized cells achieved nearly 100% ammonia removal efficiency even after 40 repetitions in simulated wastewater with an initial ammonia nitrogen concentration of 100 mg·L⁻¹ and retained 79.24% of the initial activity after 45 cycles. In contrast, the efficiency of nitrogen removal by GAC-immobilized cells was slightly reduced after 45 cycles, and the ammonia nitrogen removal efficiency was 72.99%. This result indicates that the NaOH-GAC-immobilized cells have excellent reusability. In contrast, most previous investigations have developed immobilized cells that can only be reused 5 to 10 times [39,40,41]. Immobilized yeast cells studied by Cervantes et al. were successfully reused for at least 20 cycles to remove D-glucose and D-fructose from FOS syrup [42]. The cells immobilized with NaOH-GAC in this experiment could be reused for at least 40 cycles without loss of biological activity, and the reuse performance was higher than that reported in most previous studies.

3.4.2. Storage Stability

Storage stability should be taken into account when immobilized cells are mass-produced, which requires that the produced immobilized microorganisms maintain a certain number of viable organisms and that losses are minimized during storage and transportation. Therefore, this study evaluated the nitrogen removal performance of NaOH-GAC-immobilized cells stored at 4 °C for different periods, and the results are shown in Figure 5. NaOH-GAC-immobilized cells were used to treat simulated wastewater after 35 d of storage at 4 °C, and the ammonia nitrogen removal efficiency reached nearly 100% with little effect on the nitrogen removal performance. After being stored for 70 d, the immobilized cells also achieved nearly 100% ammonia removal efficiency in the simulated wastewater, but there was a lag period in the microbial cells’ activity, resulting in a delay in nitrogen removal time [43]. In conclusion, the immobilized cells had good storage stability and were able to return to their initial biological activity after 70 d of storage. This result was better than the storage stability reported by Chen et al. for immobilized recombinant E. coli after storage at 4 °C [44].

3.5. Treatment of High-Ammonia-Nitrogen Wastewater from a Petrochemical Enterprise Using Immobilized Cells

The NaOH-GAC-immobilized cells were added to the high-ammonia-nitrogen petrochemical enterprise wastewater from the wastewater treatment center of the Maoming Branch of the Sinopec Corporation for continuous experiments to verify the treatment effect of the immobilized cells in an actual wastewater environment. In the first stage of the reaction, the treatment of wastewater with the immobilized cells reached the maximum removal rates of NH₄⁺-N, TN, and COD, which were present in concentrations ranging from 151.52, 157.08, and 3199.40 mg·L⁻¹ to 1.10, 18.23, and 964.60 mg·L⁻¹ (Figure 6a,d,e). The removal rates reached 75.21, 69.43, and 1117.40 mg·L⁻¹·d⁻¹, with removal efficiencies of 99.27%, 88.39%, and 69.85%, respectively. This was followed by the combination of wastewater and free cells with removal efficiencies of 49.18%, 44.65%, and 52.15% for NH₄⁺-N, TN, and COD at 37.26, 35.07, and 834.30 mg·L⁻¹·d⁻¹, respectively. The NH₄⁺-N, TN, and COD removal rates for the wastewater and modified activated carbon combination were 31.34, 31.42, and 674.05 mg·L⁻¹·d⁻¹, with removal efficiencies of 41.37%, 40.00%, and 42.14%, respectively. Under the same experimental conditions, the NH₄⁺-N and TN removal effects of immobilized cells were better than those of free cells, the nitrogen removal efficiency was almost doubled, and the COD removal ability was more prominent. The immobilization technology facilitated a significant increase in the number of bacteria per unit volume, which consumed a large amount of the carbon source, thus reducing COD. The nitrite nitrogen was continuously converted to nitrate nitrogen with the addition of immobilized cells, and after five consecutive experiments, there was hardly any further accumulation of nitrite and nitrate nitrogen in the reaction system (Figure 6c). The pH of the treatment groups with immobilized and free cells was maintained at around 9.0 throughout the experimental period, which was beneficial for the growth, reproduction, and metabolic activities of ammonia-oxidizing bacteria [45].

In conclusion, the treatment group with immobilized cells was the most effective and stable throughout the 10 d continuous experiment, which was attributed to the combined effect of the physical adsorption and biodegradation of the modified activated carbon. In the first stage, some of the ammonia nitrogen pollutants were adsorbed onto the modified activated carbon; when the ammonia-oxidizing bacteria started to grow in the system, the removal of ammonia nitrogen mainly depended on biodegradation, and the modified activated carbon enhanced the biodegradation [35]. A previous study used glass pumice as a carrier to immobilize Rhodopseudomonas palustris P1 for the removal of NH₄⁺-N from the loose water of aquaculture ponds, achieving a removal rate of 0.44 mg·L⁻¹·d⁻¹ [46], and the ammonia nitrogen removal efficiency of the modified activated carbon with immobilized cells used in this study was significantly better.

4. Conclusions

In this study, NaOH-GAC-immobilized cells were used to treat high-ammonia-nitrogen wastewater. The immobilized biomass increased by about 63.27% after modification of the activated carbon. NaOH-GAC-immobilized cells achieved nearly 100% ammonia nitrogen removal efficiency within 24 h, which was much higher than the removal efficiency of the free cells (4.17%). The contributions of biodegradation and adsorption to ammonia nitrogen removal were 90.27% and 9.73%, respectively. In addition, the immobilized cells had good reusability and storage stability and could be reused at least 45 times, with a strong nitrogen removal performance after 70 d of storage at 4 °C. The immobilized cells were applied to actual high-ammonia-nitrogen wastewater samples from a petrochemical enterprise, and the removal rates of NH₄⁺-N, TN, and COD reached 75.21, 69.43, and 1117.40 mg·L⁻¹·d⁻¹ after 48 h of reaction, with removal efficiencies of 99.27%, 88.39%, and 69.85%, respectively. The results were stable after five consecutive experiments. The outcomes of this trial were successful, and they offer a theoretical direction for scaled-up trials in the plant, which will have great potential for application and promotion. The present findings indicate that cell immobilization on modified activated carbon is a promising alternative for the treatment of wastewater containing ammonia nitrogen. Future work on the synergistic mechanism of ammonia-oxidizing bacteria in the reaction process will be required to improve the biological treatment process through exogenous pathways [47].