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Article

Application of Immobilized Microorganism Gel Beads in Black-Odor Water with High Nitrogen and Phosphorus Removal Performance

by
Fengbin Zhao
1,†,
Shumin Liu
2,†,
Xin Fang
3,* and
Ning Yang
2
1
College of Transportation Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
2
State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
3
School of Business, Macau University of Science and Technology, Taipa, Macao 999078, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Water 2024, 16(17), 2534; https://doi.org/10.3390/w16172534
Submission received: 1 August 2024 / Revised: 29 August 2024 / Accepted: 3 September 2024 / Published: 7 September 2024
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

:
Black-odor water, which is caused by the excessive accumulation of nitrogen and phosphorus in water, is a significant problem. Immobilized microorganisms are considered to be an effective technical solution, but there are still many key parameters to be determined, such as organic matter dissolution, insufficient stability, and insufficient phosphorus removal capacity, among other problems. In this study, the optimum raw material ratios of immobilized microorganism gel beads were determined by means of a response surface experiment. The optimal ratio of raw materials was 5% polyvinyl alcohol (PVA), 1% sodium alginate (SA), and 6% bacterial powder. In addition, the nitrogen and phosphorus removal performance of the materials was improved by loading inorganic compounds, such as 0.5 wt.% zeolite, 0.5 wt.% iron powder, and 0.2 wt.% activated carbon. Tolerance analysis determined that these gel beads could maintain a good performance in a series of harsh environments, such as during intense agitation, at high temperatures, and at low pH values, etc. The total nitrogen (TN), ammonia nitrogen (NH3-N), and phosphorus (TP) removal efficiencies were 88.9%, 90%, and 95%.

Graphical Abstract

1. Introduction

Black-odor water is an extremely serious water pollution phenomenon, which results in water being a deep black color and emitting a fetid odor; it has long affected residents living in areas experiencing this phenomenon [1,2]. Many studies have proven that physical methods, chemical methods, and biological methods can produce a control effect on black-odor water. However, physical methods usually consume a great deal of energy and require large amount of space. Chemical methods are easy to operate and generate quick reactions, but they can cause secondary pollution [3]. Biological methods can be divided into two categories: phytoremediation and microbial remediation [4]. Phytoremediation aims to improve water quality by restoring submerged plants [5]; however, projects aiming to restore submerged plants have high failure rates due to insufficient transparency [6]. Microorganisms have better environmental adaptability and rapid proliferation, making them useful for black-odor water treatment [7]. The traditional microbial technology is faced with two limitations. First, this type of technology is affected by the flow and erosion of rivers, which make it difficult to maintain enough biomass [8]. Second, the high levels of heavy metals and ammonia nitrogen and the insufficient levels of dissolved oxygen in rivers often lead to the failure of microbial treatment methods [9]. Microbial immobilization technology provides a better solution to the bottleneck.
The application of microbial immobilization technology involves the use of carrier materials and immobilization methods [10]. At present, there are five commonly used immobilization carriers: inorganic carriers, organic carriers, composite carriers, modified carriers, and new carriers. Inorganic carriers are inexpensive and have a long life span, but their small specific surface area often leads to the shedding of microorganisms [11]. Organic materials have high biological density, and their porous structure plays a significant role in maintaining a high biochemical reaction rate [12]. Albert Magrí [13] obtained PVA carriers to generate anaerobic ammoxidation with a high packing density (~20%), resulting in a nitrogen removal rate in treated livestock wastewater of up to 80%, but this specific application was still affected by problems related to the poor durability. Modified materials and new carriers can solve specific application problems, but difficult manufacturing processes and high application costs limit their wide application [14]. The emergence of organic and inorganic composite materials solves the shortcomings of single microbial immobilization materials. Among them, immobilized materials comprising a PVA + SA composite are becoming more widely used in wastewater treatment. SA can change the surface properties of the composites, reduce the aggregation tendency between the carrier particles [15], and even eliminate the toxicity of a boric acid-saturated solution [16]. In addition, PVA-SA that is cross-linked with boric acid has better technical advantages in terms of the cross-linking rate, strength, and durability [17,18]. Therefore, PVA + SA has been applied in sewage treatment applications as an immobilized microbial carrier. However, the excessive consumption of organic matter, and insufficient phosphorus removal capacity in wastewater treatment processes, have plagued the application of microbial immobilization technology in surface water treatment and need to be further explored [19].
The excellent performance of immobilized microorganism-based materials depends on diverse immobilization methods [20,21]. The adsorption method has mild reaction characteristics and does not require chemicals [22]. However, microorganisms demonstrate a weak ability to bind to the carrier [23]. The entrapment method involves the embedding of microorganisms in carrier materials, but the temperature of the embedding process affects the activity of the microorganisms [24]. The covalent method uses the reaction group between microbial cells and carriers to form a chemical covalent bond [25]. The binding ability is strong, but this method is complex, and the reaction conditions often cause significant damage to the structure of the microbial cells [26]. The cross-linking method has the advantages of high binding strength, good stability, and a strong anti-interference ability [27], but it has a significant impact on microbial cell activity. The combined immobilization method [28] makes up for the shortcomings of other single immobilization methods, such as the entrapment cross-linking method, adsorption entrapment method, adsorption entrapment cross-linking method, etc. [29]. Entrapment cross-linking methods have been proven to obtain stable and efficient ammonia nitrogen removal efficiency when PVA + SA is used as a carrier [30].
At the same time, immobilized microorganisms have significantly improved the biological activity and stability of microbial wastewater treatment. Bustos Terrones et al. [31] used SA to embed activated sludge and put it into a fixed bed reactor for continuous aeration. The effective lifespan of microorganisms was 12 h, and the removal rates of COD and total phosphorus were 68.54% and 93.33%, respectively. Quan et al. [32] put PVA-SA whole cell anaerobic ammonium oxidation sludge embedded particles into a stirred tank reactor (SRT); after 22 days of reactor start-up, it reached a stable operating state, with higher denitrification efficiency than other immobilized anaerobic ammonia oxidation reactors. Kiran et al. [33] conducted intermittent and continuous studies on the removal of heavy metals using SA immobilized sulfate reducing bacteria (SRB). The experimental results showed that the removal efficiency of heavy metals was highest at 48 h, with a removal rate of up to 95%. Tang Meizhen et al. [34] used the embedding method to fix a low-temperature bacterium Pseudomonas flava WD-3 isolated and purified on SA-PVA composite carrier. Within three months, it maintained good microbial activity and stability. Lin et al. [35] immobilized the TNT degrading strain on a carrier containing kaolin, PVA, SA, and cell suspension, resulting in higher bacterial activity. Hong Mei et al. [36] maintained good stability when using PVA-SA composite gel to immobilize microorganisms to treat groundwater polluted by chlorobenzene, and the degradation rate of chlorobenzene could reach 78.16% after 15 days. Even though there are many experiments on the stability and biological activity of gel beads, the treatment of surface water is faced with many adverse factors, such as high difficulty in nitrogen and phosphorus removal, insufficient dissolved oxygen in water, and so on. The application of biological activity and stability of microbial immobilization technology is still worth exploring.
Although there are some relatively mature research results on the materials and methods required for microbial immobilization, the results on the effects of the application process for black-odor water treatment are still insufficient, particularly those related to the dissolution of microbially immobilized materials in organic matter, which leads to a significant increase in COD, insufficient phosphorus removal capacity, insufficient environmental adaptability, and insufficient tolerance and stability of the immobilized materials, limiting the application of the technology. In addition, most of the applications of microbially immobilized materials involve their direct addition into rivers, and there is no effective application process to ensure good treatment efficiency.
Based on the above, this study explores the optimal proportions of PVA, SA, and bacterial dose in a new immobilization microorganism material system through the response surface methodology. The dissolution of organic matter from these carriers was also investigated by adjusting a series of factors, such as the cross-linking reaction time, the CaCl2 concentration, and the PVA viscosity. In order to improve nitrogen and phosphorus removal, the dosing methods for inorganic materials, such as zeolite, iron powder, and activated carbon, were discussed. Meanwhile, based on the hostile environment of black-odor water, the tolerance analysis of the immobilized microorganism material was carried out in the extreme conditions of intense agitation, pH, and temperature. Finally, we propose a fluidized process, the nitrogen and phosphorus removal capacity of black-odor water is analyzed, and the changes that took place in the bacterial community are explored. It is expected that the results published here can provide better applications of microbial immobilization technology for the treatment of surface water pollution.

2. Materials and Methods

2.1. Compound Strains and Amplification Preparation

The microbial agent NCMa (a new compound microbial agent) used in this experiment was a mixed strain of many microbial agents in a certain proportion purchased from Defeng Biological Co., Ltd. (Guangzhou, China). The main strains of NCMa were Saccharopolyspora, Thermoactiomyces, Aeribacillus, Flavobacterium, Nitrosomonas, Hydrogenophaga, Acinetobacter, Aeromonas, and Pseudomonas, etc. The culture medium used for the strain amplification experiments is given in Table S1, and the experimental process is also introduced in the Supporting Information. High-efficiency nitrifying composite strain powder was obtained by freeze centrifugation and freeze-drying technology.

2.2. Response Surface Methodology Optimization Experiments

The PVA, SA, and bacterial strain dosages have a significant influence on the strength of the gel beads, the activity of the microbial agents, and the ability to form spherical gel beads. In this study, these three variables were determined at three different levels using the Design Expert 8.05 software combined with the Box method [37]. The three independent variables were PVA dose (X1), SA dose (X2), and bacterial dose (X3). The independent variable coded values were −1 (lowest level), 0 (middle level), and 1 (highest level). The actual values chosen from previous studies [38] and the corresponding coded values of those independent variables are given in Table 1. For the response observations, the ammonia nitrogen (NH3-N) removal rate (24 h) was the evaluation index during throughout the 24 h operation period. The complete design consisted of 16 experimental groups, which are listed in Table S2.

2.3. Preparation of Microbial Immobilized Gel Beads

The 5% PVA (2699, viscosity 75~80 mPa·s) was dissolved in 80 mL of hot water at 85 °C. Then, 1% SA powder (viscosity approximately 250 mPa·s) was added to the mixer under stirring conditions for 4 h. After that, the PVA-SA mixture was cooled to room temperature. The 6% NCMa dry powder was added to the PVA-SA mixture, and stirring continued until the mixture was uniform. The mixed liquid was dripped to the cross-linking solution (saturated boric acid with 4% calcium chloride) by a peristaltic pump. After the cross-linking reaction (15 h), the wet immobilized gel beads were formed. Then, rinse gel beads with deionized water for three times, and the wet gel beads were dried in an oven at 25 °C to obtain dried gel beads. The non-immobilized gel beads were prepared using the same method without the addition of bacterial strain powder.

2.4. Performance Optimization of Immobilized Gel Beads

The organic dissolution of immobilized microgel beads is the main reason for the increased COD levels in the water [39]. In order to improve the stability, the dissolution of organic matter was investigated from three aspects: cross-linking reaction time (3 h, 15 h, 30 h, 60 h), calcium chloride concentration (0%, 2%, 4%), and PVA viscosity (PVA 1799, PVA2499, PVA 2699, PVA-aladdin). The total organic carbon (TOC) in the solution represented organic matter dissolution during the shaker test [40]. To improve nitrogen and phosphorus removal performance, a certain proportion of zeolite, iron powder, and activated carbon were loaded in the immobilized gel beads. The effects of nitrogen and phosphorus removal by the immobilized microorganisms before and after inorganic particles were loaded analyzed.

2.5. Characterization of Immobilized Microorganism Materials

Scanning electron microscopy (SEM, PHILIPS-XL30 SEM, Eindhoven, The Netherlands) was used to observe the surface and internal morphology of the immobilized microorganism materials. A confocal laser scanning microscope (CLSM, Eclipse 80i, Philadelphia, PA, USA) was used to characterize the biological activity of the immobilized microorganism materials. Before the observations, the dried immobilized carrier was ground in an agate mortar, and the positive film was extruded and tested using an infrared spectrometer.

2.6. Tolerance Analysis

To create a fierce operation environment, 100 regular-shaped gel beads were put into a 250 mL conical flask with 200 mL deionized water. Then, the conical flask was subjected to intense rocking in a shaker with a shaking speed of 180 r/min. The numbers of intact and broken gel beads were recorded every other day to describe the intensity. In addition, a good repeatability experiment that used the NH3-N removal rate as the evaluation index could help to reduce operation costs when used in practical applications. An amount of 1.00 g immobilized gel beads was added into a 250 mL conical flask with 100 mL 20 mg/L ammonia nitrogen wastewater. Then, the conical flask was rocked in a shaker at a shaking rate (180 r/min) of 25 °C. These gel beads were retrieved and washed three times every 72 h. The NH3-N concentration was tested simultaneously.

2.7. Water Purification Effect of Immobilization Materials

Urban black-odor water was taken from Xupu Port in Shanghai, China, as shown in Figure S1. The cylindrical glass reactor (2.5 L) was designed for the black-odor water purification experiment as shown in Figure 1. The water quality of the black-odor water is shown in Table S3. Specifically, 20.00 g of the dry immobilized gel beads was added into the glass reactor with an aeration rate of 10.0 L/min. Then, water was pumped into the reactor through a peristaltic pump. The hydraulic retention time (HRT) was sustained at 24 h for the first 48 h. After that, the HRT was adjusted to 12 h. The water quality is analyzed every 24 h. After the effluent quality was stable, the structure changes of microbial community in the influent and effluent were analyzed (I11umina, San Diego, CA, USA).
In addition, the NH3-N and phosphate (phosphate radical, PO43−-P) were measured by Auto Discrete Analyzers (SEAL Discrete Chemistry Analyzer AQ2, Bradford, UK). TOC and Total Nitrogen (TN) were examined using a TOC (TOC-VCPN, Kyoto, Japan). Chemical oxygen demand (COD) and total phosphorus (TP) were determined by the US Hach method using the model DRB 200 digestion instrument (Hach, Loveland, CO, USA) and DR 1900 spectrophotometer (Hach, Loveland, CO, USA).

3. Results and Discussion

3.1. Optimization of Immobilized Microorganism Gel Beads by Response Surface

The response surface optimization experiments of the 16 experimental groups were carried out using the PVA dosage, SA dosage, and bacterial dosage as independent variables χ1, χ2, χ3, and the NH3-N removal rate was the response value Y. Through the analysis of the simulation algorithm software, the relationship between each factor and response value was expressed by the following quadratic multiple regression equation:
Y = 17.91791 + 0.90580 χ1 + 8.42911 χ2 + 9.93148 χ3 − 0.59722 χ1χ2 − 0.24474 χ1χ3 − 0.71999 χ2χ3 − 0.34183 χ32
The response surface analysis of each coefficient variance is listed in Table 2. The R2 of the model was 0.9435, which indicated that the model had a good fit and had a high accuracy. The effects of the PVA content and bacterial agent content on the ammonia nitrogen degradation of the immobilized microbial materials were significant because the p values of χ1, χ3, χ1χ3, χ2χ3 and χ2χ3 were all less than 0.05. The SA content had an insignificant effect on the NH3-N degradation of the immobilized microbial materials due to the high p value (p > 0.1) of χ2.
As shown in Figure 2a, when the bacterial dose was 5% and when the PVA content was 0~6%, the more SA that was added, the higher the NH3-N removal rate was. However, the NH3-N removal rate first increased and then decreased as the SA content increased when PVA content was high (6~10%), indicating that the high PVA content would lead to tighter gel beads and weaken NH3-N degradation. On the contrary, the linear polymer SA was able to reduce the steric hindrance, improving the permeability of the immobilized gel beads and the NH3-N removal rate [41]. However, the bacterial dose had a positive correlation with NH3-N removal (Figure 2b). Interestingly, when the PVA content was fixed at 5%, it was positively correlated with the bacterial dose on the NH3-N removal rate when the SA content was low (less than 1%). Additionally, the NH3-N removal rate first increased and then decreased when the bacterial dose increased when the SA content ranged from 1% to 4%. This is because non-denitrifying bacteria occupy too much biological space in the gel beads when the microbial dosage is increased. This is related to the complex porous characteristics of SA, which can form a variety of microbial growth environments in the anaerobic zone, facultative anaerobic zone and aerobic zone inside the gel beads, and has a complete micro-denitrification reaction process inside a single particle [42].
The optimization function in the Design Expert 8.0.5 software was used to optimize the preparation conditions of the immobilized microorganism materials. The optimization conditions are defined in Table S4. The top 10 optimized results are listed in Table 3. The best results aligned with the highest NH3-N removal rate (63.51%), where the PVA dose was 5%, the SA dose was 1%, and the bacterial dose was 6%.

3.2. Characterization of Immobilized Microorganism Gel Beads

The surfaces of the immobilized gel beads without the bacterial strain were relatively flat (Figure 3a). However, the surfaces of the immobilized microorganism gel beads were quite wrinkly (Figure 3b,c). The bacterial particles were found to have a uniform configuration in the internal structure of the gel beads (Figure 3d), contributing mass transfer and metabolism.
Through fluorescent staining, the living cells/dead cells in the dried cell beads could be detected through CLSM images. The living microorganisms that were present on the material were clearly seen under a fluorescence microscope. The green fluorescence (Figure 3e) represents the active microorganisms, and the red fluorescence (Figure 3f) represents the dead microorganisms. Figure 3g shows the overall number of microorganisms in both red and green color. In general, the activity of the immobilized microorganisms was very well preserved, as indicated by the bright green color.

3.3. The Optimization Design of Immobilized Microorganism Gel Beads

3.3.1. The Dissolution of Organic Matter from the Gel Beads

The optimization of the SA, PVA, and bacterial dose was calculated by the quadratic multiple regression equation. However, the COD of the wastewater sample with these gel beads would increase due to the dissolution of the TOC in these beads [43]. In order to deal with this situation, the cross-linking reaction time, the CaCl2 concentration, and the PVA viscosity were explored. The cross-linking time had a significant effect on the strength and biological activity of the gel beads [44], significantly increasing the surface strength and internal tightness of the gel beads [45]; however, this also led to a decrease in the biological activity. Therefore, choosing suitable a cross-linking time to reduce the TOC dissolution and to maintain enough microbial activity is very important [46]. During the cross-linking process, there were two main reaction processes: One was the reaction between PVA and boric acid, and the other was the reaction between SA and the CaCl2 solution. As shown in Figure 4a, the TOC elution amount decreased as the reaction time extended, indicating that the stability of the gel beads was enhanced. In addition, the TOC elution amount at 15 h was similar to that at 30 h, when the operation time was 720 h. If the cross-linking time was too short, the mechanical strength of the immobilized material would deteriorate, but the long cross-linking time would reduce the mass transferability [47,48]. Moreover, the immobilized microorganism activity would be inhibited in a saturated boric acid solution (pH value < 4.0). To sum up, to avoid the excessive loss of microbial activity, the best cross-linking reaction time should be controlled at 15 h.
The mechanical strength, mass transfer performance, and biological activity of the immobilized microorganisms would be affected by the CaCl2 concentration [49]. With the increase in the CaCl2 solution, a dense cross-linked structure was formed on the surface of the gel ball [50]. When the CaCl2 was 0%, the TOC elution reached its maximum at 36 h. When the CaCl2 in the cross-linking solution was 2% and 4%, the TOC elution reached its maximum at 168 h. When the operation was conducted over the course of 720 h, the TOC elution without CaCl2 was 33% higher than that of gel beads with 2% CaCl2 and 47% higher than that of gel beads with 4% CaCl. Studies have shown that a high Ca2+ concentration could shorten the contact distance between the hydroxyl group and the carboxyl group in SA molecules, promoting intermolecular hydrogen bond generation [51], resulting in a tight layer of the carriers. The carrier prepared by the 4% CaCl2 cross-linking solution has the least amount of organic matter dissolution, making the immobilized carrier more stable. Therefore, 4% CaCl2 is the best choice when trying to reduce TOC dissolution.
Meanwhile, PVA-1799 (20~26 mPa·s), PVA-2499 (48~60 mPa·s), PVA-2699 (75~80 mPa·s), and PVA-aladdin (80~110 mPa·s) were used as the supporting materials in the immobilized materials system under different viscosities. It was found that the TOC elution concentration of PVA-aladdin was much higher than that of the other three PVA materials (Figure 4c), indicating that the PVA viscosity was a major factor leading to the dissolution of organic matter. Compared to PVA at other viscosities, there were acetyl groups with infrared activity on PVA-aladdin, and the existence of acetyl groups affects the stability of the molecular structure after PVA reacts with boric acid [52]. In addition, through the observation during the experiment, it was found that pva-1799 and pva-2499 showed weak adhesion to the mixed microbial agents, resulting in the precipitation of the microbial agents. As such, the PVA-2699, which was the cheapest, was chosen as the optimum supporting material.

3.3.2. Loading Inorganic Particles to Improve Properties

The trailing phenomenon occurred throughout the preparation of these gel beads, affecting the performance and the aesthetics of the gel beads. The stickiness of the carrier could be improved with the addition of zeolite, which would guarantee the pelletizing effect. The nitrogen removal efficiency was enhanced by the immobilized microorganism material that had been loaded with zeolite (Figure 5a–c). The NH3-N removal rate was significantly accelerated because the zeolite demonstrated a special NH3-N adsorption performance, promoting the internal nitrification and denitrification reaction inside the gel beads. The water with initial ammonia nitrogen concentration of 50 mg/L was completely degraded after 216 h, which was 96 h shorter than that of the control group. The addition of zeolite did not have a significant effect on the gel beads’ ability to remove phosphate. At 360 h, the removal rate of phosphate was 67% and that of control group was 59%.
However, the TP removal rate was strengthened to a large extent by loading the microorganism materials with iron powder (Figure 5d–f). This resulted in hydrogen atoms being produced in the water by means of nitrification, and these hydrogen atoms reacted with the iron powder to form ferrous ions and iron ions, enriching the phosphate in the water and allowing it to form precipitates. The coexistence of the zero-valent iron and the phosphorus-accumulating bacteria could produce a synergistic phosphorus removal effect with the adsorption–precipitation–degradation process. The NH3-N and TN removal rates were obviously accelerated because microorganism growth and reproduction require the consumption of nitrogen sources.
Although activated carbon has a huge specific surface area, it did not obviously improve the phosphate removal effect with the addition of the activated carbon-composited immobilized materials (Figure 5g–i). It was difficult for its action effect to be induced to form a synergistic effect with the microorganisms for nitrogen and phosphorus removal. However, the addition of the activated carbon improved the mass transfer performance of the immobilized materials and increased the internal pore size, which was beneficial for the exchange of the dissolved oxygen and nutrients between the immobilized bacteria and the outside, improving the denitrification effect of the immobilized microorganism materials.

3.4. Tolerance Analysis of the Gel Beads

For the practical application of immobilized microorganism gel beads for black-odor water treatment, the technological parameters should be controlled to ensure optimum microorganism activity. As such, tolerance analysis was conducted to explore the suitable operation conditions when used in practical applications, such as the intensity, repeatability, pH value, and temperature, etc.
As shown in Figure 6a, the operation cycle of these gel beads under intense conditions was up to 60 days. The gel beads remained completely intact for the first two weeks. The proportion of intact gel beads was more than 80% before the 40th day, indicating that these gel beads had good intensity. As shown in Figure 6b, the NH3-N removal rate first increased and then decreased, but the NH3-N removal rate of 12 consecutive experiments was more than 80%, indicating that the immobilized microorganisms adapted to the new environment quickly and that they maintained high levels of activity for about 600 h. However, from the sixth consecutive experiment, the NH3-N removal rate began to decrease, suggesting that a proportion of the gel beads had been damaged after the 15th day of operation (Figure 6b), leading to the loss of the active immobilized microorganisms. Although the 15 days stabilization time has a greater advantage than that of the CaCl2 cross-linking gel ball in the SBBR process [42], SA is a natural polymer polysaccharide material with poor biodegradation resistance. With the increase of the concentration of microorganisms in the reactor, the carrier has degraded, which is the disadvantage of SA as the immobilized carrier. In future research, it is necessary to find its modification methods and alternative products. Polyvinyl alcohol sodium alginate immobilized copper green microcystis for phosphorus adsorption [53].
To evaluate the adaptive capacity of the immobilized microorganisms to extreme environments, the temperature and the pH value of the black-odor water were explored. The NH3-N removal rates of the immobilized materials were higher than those of non-immobilized materials at low (10 °C) and high (40 °C) temperatures (Figure 7a). As the HRT increased, the NH3-N removal rate of the immobilized materials was similar to that of non-immobilized materials at 15~35 °C. The phosphorus removal rate of the immobilized microbial materials was more than 97%, and the phosphorus concentration was less than 0.5 mg/L in each temperature range. For the non-immobilized materials, the phosphate removal rate increased as the hydraulic retention time increased; when t = 30 °C, the phosphate removal rate increased to up to 27%, and the removal effect was not obvious. In conclusion, the immobilized material improves the ability of microbial nitrogen and phosphorus removal, and expands the effective temperature range of microorganisms. The temperature adaptation range of the non-immobilized bacteria was 15~35 °C, and the temperature adaptation range of the immobilized microbial materials was 10~40 °C. Although it has become a consensus in the scientific community that low temperatures can inhibit the activity of microorganisms [54] the further improvement of microbial water purification performance at low temperatures through microbial immobilization technology has a broader application space in winter river and lake water quality control.
There was a good linear relationship between the bacterial concentration and the absorbance at 600 nm, which was reflected by the OD600 value. When the HRT was 12 h, the OD600 values of the non-immobilized materials were all higher than those of the immobilized materials at a series of pH values. However, as the HRT value increased, the OD600 of the immobilized materials also increased, and even at pH = 2 and pH = 12, the adaptive capacity of the immobilized materials to the wastewater at different pH values was enhanced due to the protection of the carrier (Figure 7b). However, when the pH < 4 and pH > 10, the OD 600 value of non-immobilized materials decreased as the hydraulic retention time increased. This shows that the adaptation range of the microorganisms treated by immobilization technology to pH is improved. In an alkaline environment with pH < 10, ammonium ions can be converted into ammonia nitrogen and escape from the water, which is in accordance with the chemical hydrolysis equilibrium principle. Therefore, in high-pH environments, the ammonia nitrogen removal rates of both are high (Figure S2). The phosphate removal rates of the immobilized microbial materials and unfixed microbial agents were significantly different. When the pH of the immobilized microbial material was 2~10, the removal rate was more than 95%. For unfixed microbial agents, when pH = 8, the phosphate removal rate reached 31% (Figure S2). In conclusion, immobilized microorganisms increase their adaptability to extreme environments such as temperature and pH. Most studies show that most ammonia oxidizing bacteria (nitrite bacteria) are suitable to grow in an environment with a pH value of 7.5~8.0 [55], and phosphorus removal bacteria and denitrifying bacteria are also suitable to grow in a slightly alkaline environment. However, the gel beads in this study significantly improved the pH tolerance of nitrogen removal bacteria, and still have good nitrogen and phosphorus removal effects in strong acid and alkali environments, with significant technical advantages.

3.5. The Performance of Nitrogen and Phosphorus Removal in Black-Odor Water

The influent NH3-N concentration of the black-odor water was above 30.9 mg/L, and the TN concentration was above 44.7 mg/L. In the stable effluent period, the total effluent nitrogen was about 5 mg/L, and the removal rate reached about 88.9% (Figure 8a). The NH3-N removal rate was up to 90%, and the effluent NH3-N concentration was below 2 mg/L (Figure 8b). Within 48 h of starting the reactor, the NH3-N and TN removal rates were higher in the first 12 h, because the SA in the gel beads has a porous structure, which can adsorb organics and ammonia nitrogen in the body [56], the adsorption of the immobilized gel beads as the water inflow increased, and the adsorption was saturated. After 24 h, the concentration of nitrogen pollutants began to gradually decline, reaching the lowest level at 48 h, indicating that the immobilized materials had an obvious effect on the removal of NH3-N and TN in black-odor water. The phosphate removal rate was maintained above 95% in the stabilized effluent operation, with a small fluctuation of the phosphate concentration (below 0.2 mg/L), indicating that there were immobilized microorganism gel beads in the black-odor water, which demonstrated high nitrogen and phosphorus removal performance.

3.6. Microbial Colony Analysis

The treatment of the black-odor water with these gel beads resulted in changes in the microbial community. The five most common genera in the raw water were Arcobacter, Cloacibacterium, Hydrogenophaga, Acidovorax, and Aeromonas (Figure 9). Moreover, the optimum microbial colonies with the highest activity in the effluent water were Flavobacterium, Zoogloea, Aeromonas, comamons, Limnohabitans, and Hydrogenophaga. Among them, Arcobacter [57,58] and Acidovorax [59], which belong to Ascomycota and Cloacibacterium [60,61] and are part of Bacteriodetes, were frequently detected in the sewage, and they were hardly detected in the effluent, showing that the quality of the black and odorous water changed significantly after being treated with the microbial immobilized materials. Limnohabitans, a biological water quality indicator, was very sensitive to the acidity and salinity of the wastewater. As such, compared to raw water, the relative abundance of Limnohabitans in the effluent water increased to a large extent, suggesting that the pollution load of the black-odor wastewater had been reduced due to the improved water quality.

4. Conclusions

This research proposes a novel model that is able to the NH3-N removal rate according to the addition of different raw materials during the preparation of immobilized gel beads. The optimum raw material ratio was able to be determined by means of a response surface experiment. The optimal raw material ratio was 5% PVA, 1% SA, and 6% bacterial preparation, which resulted in an NH3-N removal rate of up to 63.44%. Additionally, in order to reduce the TOC elution experiments, 4% CaCl2 and PVC-2699 were added to enhance the mechanical strength of the gel beads. In addition, the ability of the material to remove nitrogen and phosphorus was improved by loading the material with inorganic particles such as 0.5 wt.% zeolite, 0.5 wt.% iron powder, and 0.2 wt.% activated carbon. The tolerance analysis determined that these gel beads could maintain a good performance in a series of harsh environments, such as during intense agitation, at high temperatures, and at low pH value, etc. The immobilized material showed high microbial activity and good performance in terms of nitrogen and phosphorus removal. The TN, NH3-N, and TP were able to be removed from black-odor water at efficiency rates of 88.9%, 90%, and 95%, respectively.
The production process of immobilized microbial gel beads proposed in this study showed good performance and great application potential in stability and biological activity. However, it is difficult for immobilized microorganisms to have long-term stability like traditional activated sludge processes. In order to realize the widespread use of this technology, more research work needs to be done in the modification of SA (to reduce the biodegradation of gel beads), the research and development of substitutes for porous materials such as SA, and the optimization of nitrogen and phosphorus removal strains with self reproducing ability.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w16172534/s1, Table S1: Composition of culture medium; Table S2: Experimental design of immobilized microorganism; Table S3: Water quality of the black odor water; Table S4: Optimization of scheme conditions; Table S5: Water quality used in response surface orthogonal test; Figure S1: Picture of black odor water sampling site; Figure S2: Removal of nitrogen and phosphorus pollutants by immobilized microorganism and unfixed agent under different pH conditions; Figure S3: The concentration of three metal elements of influent and effluent.

Author Contributions

Methodology, S.L.; Investigation, N.Y.; Writing—original draft, F.Z.; Writing—review & editing, X.F. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the scientific research by the Science and Technology Committee of Shanghai (18DZ1206505).

Data Availability Statement

Data is contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Schematic diagram of the simulated device for immobilized microorganisms to treat contaminated water.
Figure 1. Schematic diagram of the simulated device for immobilized microorganisms to treat contaminated water.
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Figure 2. The response surface 3D maps of NH3-N removal rates with the variation sources (5% bacterial dose (a), 1% SA content (b), and 5% PVA content (c)).
Figure 2. The response surface 3D maps of NH3-N removal rates with the variation sources (5% bacterial dose (a), 1% SA content (b), and 5% PVA content (c)).
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Figure 3. The covalent immobilization of microbial cells on microchannel surfaces. SEM of common carrier (a) and immobilized microorganism (b) surface; SEM diagram of immobilized gel beads (surface structure (c) and internal structure (d)); the CLSM images of the granules of immobilized beads (living cells (green) (e), dead cells (red) (f), and combined image of living and dead cells (g)).
Figure 3. The covalent immobilization of microbial cells on microchannel surfaces. SEM of common carrier (a) and immobilized microorganism (b) surface; SEM diagram of immobilized gel beads (surface structure (c) and internal structure (d)); the CLSM images of the granules of immobilized beads (living cells (green) (e), dead cells (red) (f), and combined image of living and dead cells (g)).
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Figure 4. The concentration of TOC change curves of the gel beads with time under different cross-linking time conditions (a), concentrations of calcium chloride (b), and PVA viscosities (c).
Figure 4. The concentration of TOC change curves of the gel beads with time under different cross-linking time conditions (a), concentrations of calcium chloride (b), and PVA viscosities (c).
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Figure 5. Total amounts of nitrogen (a), ammonia nitrogen (b), and phosphorus (c) removed by immobilized microorganism loaded with zeolite; total nitrogen (d), ammonia nitrogen (e), and phosphorus (f) removal by immobilized microorganisms loaded by iron powder; total nitrogen (g), ammonia nitrogen (h), and phosphorus (i) removal by immobilized microorganism loaded with activated carbon.
Figure 5. Total amounts of nitrogen (a), ammonia nitrogen (b), and phosphorus (c) removed by immobilized microorganism loaded with zeolite; total nitrogen (d), ammonia nitrogen (e), and phosphorus (f) removal by immobilized microorganisms loaded by iron powder; total nitrogen (g), ammonia nitrogen (h), and phosphorus (i) removal by immobilized microorganism loaded with activated carbon.
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Figure 6. The proportion of complete particles and damaged particles in the immobilized microorganism particle oscillation experiment (a) should be changed, and the relationship between the repeated use of immobilized microorganisms and the removal rate of ammonia nitrogen (b).
Figure 6. The proportion of complete particles and damaged particles in the immobilized microorganism particle oscillation experiment (a) should be changed, and the relationship between the repeated use of immobilized microorganisms and the removal rate of ammonia nitrogen (b).
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Figure 7. Removal of nitrogen and phosphorus pollutants by immobilized microorganisms and non-immobilized agent under different temperature conditions (a) and OD600 changes by immobilized microorganisms and non-immobilized agent under different pH conditions (b).
Figure 7. Removal of nitrogen and phosphorus pollutants by immobilized microorganisms and non-immobilized agent under different temperature conditions (a) and OD600 changes by immobilized microorganisms and non-immobilized agent under different pH conditions (b).
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Figure 8. The total nitrogen (a), ammonia nitrogen (b), and phosphate (c) concentration changes in black and odorous water.
Figure 8. The total nitrogen (a), ammonia nitrogen (b), and phosphate (c) concentration changes in black and odorous water.
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Figure 9. Changes in microbial community structure of the black-odor water before and after treatment (genus level).
Figure 9. Changes in microbial community structure of the black-odor water before and after treatment (genus level).
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Table 1. Factor level table for immobilized microorganism optimization experiments.
Table 1. Factor level table for immobilized microorganism optimization experiments.
Independent VariablesSymbolsLevels
Coding ValuesTrue Values−101
PVA doseΧ1χ105%(w/v)10%(w/v)
SA doseΧ2χ202%(w/v)4%(w/v)
Bacterial doseΧ3χ307.5%(w/v)15%(w/v)
Table 2. Regression analysis of regression model with ammonia nitrogen removal rate as response value.
Table 2. Regression analysis of regression model with ammonia nitrogen removal rate as response value.
Variation SourcesSquare SumDegree of FreedomMean SquareF ValueProb > F
Model5475.927782.2721.46<0.0001
χ1902.441902.4424.760.0008
χ20.05910.0594.420.9687
χ32061.4212061.4256.55<0.0001
χ1χ2142.671142.673.910.0793
χ1χ3336.931336.939.240.0140
χ2χ3466.551466.5512.800.0060
χ321565.8611565.8642.950.0001
Residual error328.09936.45
Lack of fit310.95562.1914.510.0113
Table 3. Top 10 optimization solutions.
Table 3. Top 10 optimization solutions.
No.PVA (%)SA (%)Bacterial Dose (%)Removal Rate of NH3-N (%)
15.001.006.0063.5106
25.000.946.0063.4415
35.121.006.0063.3614
45.251.006.0063.2201
55.000.726.0063.1992
65.000.416.0062.8455
75.001.005.8362.8362
85.000.306.0062.7271
95.000.196.0062.6017
105.000.076.0062.4639
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Zhao, F.; Liu, S.; Fang, X.; Yang, N. Application of Immobilized Microorganism Gel Beads in Black-Odor Water with High Nitrogen and Phosphorus Removal Performance. Water 2024, 16, 2534. https://doi.org/10.3390/w16172534

AMA Style

Zhao F, Liu S, Fang X, Yang N. Application of Immobilized Microorganism Gel Beads in Black-Odor Water with High Nitrogen and Phosphorus Removal Performance. Water. 2024; 16(17):2534. https://doi.org/10.3390/w16172534

Chicago/Turabian Style

Zhao, Fengbin, Shumin Liu, Xin Fang, and Ning Yang. 2024. "Application of Immobilized Microorganism Gel Beads in Black-Odor Water with High Nitrogen and Phosphorus Removal Performance" Water 16, no. 17: 2534. https://doi.org/10.3390/w16172534

APA Style

Zhao, F., Liu, S., Fang, X., & Yang, N. (2024). Application of Immobilized Microorganism Gel Beads in Black-Odor Water with High Nitrogen and Phosphorus Removal Performance. Water, 16(17), 2534. https://doi.org/10.3390/w16172534

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