A novel wastewater-quality-improver, Sodium Alginate Embedded Microbe-treated Zeolite (SAEMZ), was proposed. The strains used are screened from black-odorous water and have high-efficiency NH4+-N degradation performance. The Gram-positive bacteria, belonging to Achromobacter sp., was determined through the screening and identification for this strain, whose removal rate of NH4+-N can reach 88.06%, to decrease the NH4+-N concentration from 61.83 mg/L to 7.80 mg/L, and its optimal growth conditions are pH 7–8, rotation speed 150–210 r/min, temperature 25–35 °C. The SAEMZ's removal effect on NH4+-N was considered in this research from aspects of reusability, storage stability, and the effects of dosage, coexisting ions, and wastewater's concentration. The increase of the SAEMZ's dosage effectively improved the NH4+-N removal rate; Ca2+ in the solution promoted the NH4+-N removal rate, while Mg2+ and Mn2+ inhibited it. Also, the NH4+-N removal rate improved slightly with Fe2+ concentration's increase and then decreased significantly; with the increase of the wastewater dilution factor, the NH4+-N removal rate showed an upward trend and with the increase of the SAEMZ's reuse times, it decreased. Therefore, recycle times should be controlled to less than 3 times in practical application; the SAEMZ still maintains its physiological stability, high mechanical strength, and good storage stability after being stored at 4 °C for 120 days.
The S1 is a highly efficient NH4+-N-degrading strain, belonging to Achromobacter sp.
Proposal of a novel agent, Sodium Alginate Embedded Microbe-treated Zeolite (SAEMZ).
The SAEMZ can remove ammonia nitrogen efficiently.
SAEMZ has excellent physical stability, mechanical strength, and storage stability.
With the ceaseless improvement of social economics and industrialization, water pollution is becoming increasingly serious. Therefore, water pollution control and water environment restoration are vital and challenging problems that urgently need to be studied and resolved (Cao et al. 2020). According to environmental statistics in 2013, the North Canal Basin is dominated by organic and nutrient pollution with 5,932 tons direct discharge environmental quality of NH4+-N, accounting for 61% of all domestic sources of emissions, resulting in the serious exceedance of NH4+-N to become the most prominent pollutant in the basin (Pan 2017). Xie Wenli (Xie et al. 2018) found that the Taige Canal has severe NH4+-N pollution, mainly caused by the tributary pollution, agricultural pollution and domestic pollution, and the worst water quality during the dry season, leading to more significant impact on the eutrophication status of Taihu Lake Zhushan Bay according to water quality monitoring data of the Taige Canal from 2013 to 2017. The direct discharge of high-concentration NH4+-N wastewater into the water body without treatment will exacerbate the eutrophication of the water body and pose a threat to aquatic organisms and even human health (Wang et al. 2016). Therefore, it is of great significance to control the content of ammonia nitrogen in water to restore the self-purification ability of the ecosystem and river landscape (Jiang et al. 2013).
Using microorganisms to decompose and transform pollutants in water is an economical, healthy, and effective way to control water quality. Microbial immobilization technology, including the adsorption method, embedding method, adsorption-embedding method, embedding-crosslinking method, adsorption-embedding-crosslinking method, and other composite immobilization methods (Lu et al. 2012), is a kind of bio-enhanced treatment technology by using physical or chemical means to immobilize free microorganisms with specific functions on carrier materials, so as to increase the effective microbial concentration and the ability to degrade pollutants (Cao et al. 2002). Embedding and immobilization technology traps and fixes microbial cells in a grid with macromolecular gel polymer voids or natural macromolecular polysaccharides, and uses the embedded carrier's structure to make microbial cells highly dense and maintain biological activity (He et al. 2004). Embedding and immobilization can not only prevent cell loss but also allow reaction products and substrates to enter and exit the carrier freely (Lin et al. 2013a, 2013b). Also, the embedding method's immobilized pellets have the advantages of high mechanical strength, substantial impact load, non-easy falling out of immobilized microorganisms, and the convenient solid-liquid separation with unique superiority in water treatment (Chang et al. 2005; Kim et al. 2012).
The traditional immobilization techniques are carrier binding and gel embedding, both of which immobilize microorganisms on a certain type of carrier material for further utilization. However, it is difficult for the immobilized microorganism to resist the impact of adverse environment when the combination between the carrier and the microorganism is not close or the stability of the embedded gel bead is poor (Chen et al. 2010). Clay minerals such as zeolite achieve the merits of rigidity, chemical inertia, and low cost, while SA (sodium alginate) is easy to gel under mild conditions (Chen et al. 2010). Combining the characteristics of both, the microorganisms are first loaded onto an inorganic carrier material and then immobilized by encapsulation. The immobilized particles obtained by this method have a unique spatial structure, which can effectively reduce the loss of microorganisms and maintain their biological activity, making them good for application (Lu et al. 2019). In addition, free bacteria are prone to lose their degrading activity in a high-concentration pollutant environment. After immobilization, the bacteria can not only maintain the vigor at the original pollutant concentration, and keep good removal effects, but also continuously perform multiple degradation cycles (Ahmad et al. 2012). Notably, zeolite, as a carrier material for immobilized microbial agents, provides sufficient attachment points for microorganisms, NH4+-N and other nutrients are also easily adsorbed on the surface of the carrier, increasing the opportunity for microorganisms to fully contact with pollutants and nutrients, which facilitates the growth and metabolic process of microorganisms.
In general, the effect of immobilized microorganisms on the removal of pollutants is relatively stable. However, in the application, due to the complex natural water environment and many interference factors, there are still many factors that limit its ability to remove ammonia. For example, immobilized microorganisms’ dosage, ammonia nitrogen's concentration in the water body and coexisting ions. When the immobilized particles react with cations used as chelating agents and anti-gelling cations (such as dissolved phosphorus, sodium bicarbonate, and EDTA citrate), calcium alginate gel will be unstable. While in a complex water environment, metal ions are also essential factors that affect the growth of microorganisms and the synthesis of metabolites (Yang & Tang 2008). A proper amount of metal ions can promote enzyme activity to a certain extent, but simultaneously, excessive, or too little metal ions concentration in the environment will easily influence the enzyme activity, thereby affecting the growth and metabolism of microorganisms.
Therefore, to better control the NH4+-N's content in the water body, the SAEMZ for efficiently removing NH4+-N was prepared in the paper, and the research work was mainly completed as follows:
Microorganisms with high NH4+-N removal ability were screened from black-odorous water bodies and identified by morphology and molecular chemistry;
Appropriate carriers and embedding materials were selected to immobilize the strains, and a new embedding immobilized microbial agent was prepared for the efficient removal of NH4+-N from water bodies;
The optimal application conditions of the SAEMZ were determined by exploring the influence of factors such as dosage, dilution ratio of wastewater and co-existing ions (Ca2+, Mg2+, Mn2+ and Fe2+) on the NH4+-N removal effect of the SAEMZ, and the reusability and storage stability of the SAEMZ were investigated.
MATERIALS AND METHODS
C6H12O6·H2O, CaCl2, Na2HPO4, KH2PO4, MgSO4, MnSO4·H2O, FeSO4·7H2O, Agar powder, Beef extract, Peptone, NaCl, NH4Cl, KNO3, NaOH, KI, HgI, KNaC4H6O6·4H2O, 95% Ethanol, Ammonium Oxalate Crystal Violet Dye, Iodine solution, Safranin Dye, Glutaraldehyde, Absolute ethanol, SA (sodium alginate), Zeolite. Among them, NH4Cl and KNO3 are of excellent grade purity, and other drugs are of analytical grade.
Experimental materials’ preparation
Black-odorous water mixed bacteria liquid: polyethylene bottles were used to collect black-odorous water samples below 20 cm above the water surface of the Xunsi River section, leaving no headspace. Through being filtered with medium-speed by filter paper, the filtrate was stood for 2 h. The supernatant is the mixed bacterial liquid with black-odorous water, it was stored in a sterile Erlenmeyer flask and put in a refrigerator at 4 °C as spare.
Simulated ammonia nitrogen wastewater: Glucose and NH4Cl were used to adjust the C/N of the simulated ammonia nitrogen wastewater. The remaining components are: 0.1 g/L CaCl2, 1 g/L Na2HPO4, 1 g/L KH2PO4, 0.05 g/L MgSO4.
Bacteria suspension: Two-ring of bacteria stored on the inclined plane in a 50 mL bacterial culture medium were picked, and placed in a constant temperature shaker with a rotating speed of 150 r/min and 30 °C for 24 h activation. The activated bacterial solution in a centrifuge tube was taken out for centrifugal operation at 4,000 r/min for 10 min. Then, the supernatant was discarded, and washed with ultrapure water to obtain a bacterial suspension.
Nutrient solution: 1 g/L glucose, 0.1 g/L CaCl2, 0.3 g/L Na2HPO4, 0.5 g/L KH2PO4, 0.05 g/L MgSO4, 0.0025 g/L MnSO4 ·H2O, 0.0025 g/L FeSO4·7H2O.
Medium selected: 0.02 g/L NH4Cl, and the remaining components were the same as the nutrient solution.
Bacterial medium: 5 g/L beef extract, 10 g/L peptone, 5 g/L NaCl.
The above medium was adjusted to pH 7.2–7.4 with 1 mol/L NaOH, and sterilized at 121 °C for 20 min. To prepare a solid medium, add 20 g/L agar and make a solid flat plate or test tube slope after sterilization.
Bacterial strains and their growth conditions
Cultivation of bacterial strains
The black-odorous water mixed bacteria liquid was added into the selection medium. 10 mL fresh nutrient solution shall be replenished every 2 days, while increasing NH4Cl solution gradually. Initially, 2 mL of 1,000 mg/L NH4Cl solution was added to the culture flask. Henceforth, the added NH4Cl solution was 5 mL more than the previous one every 2 days. During the cultivation for 20 days in a constant temperature shaker at 150 r/min and 30 °C, the morphology of microbial colonies under a biological microscope shall be observed constantly.
The bacterial liquid after 20 days cultivation was spread on the solid selective medium and cultured upside down in a constant temperature incubator at 30 °C. Two days later, round light-yellow colonies grew on the surface of the medium, and a single colony was picked and inoculated again until the subsequent colony morphology remained unchanged. The screened and cultivated bacteria were stored on a solid medium slant at 4 °C.
Screening of bacterial strains
The two-ring cultured ammonia nitrogen removal bacteria was taken and placed in simulated ammonia nitrogen wastewater, to conduct reaction at 150 r/min and 30 °C. NH4+-N's concentration in the supernatant was measured at intervals of 24 hours.
Identification of bacteria
Gram staining was performed on the selected strains, and the staining and cell morphology characteristics were observed under a microscope. The Ezup column type bacterial genomic DNA extraction kit was used to extract the strain's genomic DNA and perform DNA electrophoresis detection. PCR amplification was performed with bacterial universal primers and electrophoresis detection (1.5% agarose gel, 1 × TAE, 150 V, 100 mA, 20 min electrophoresis observation).
The primer sequences used are as follows:
Forward primer: 27F 5′-AGAGTTTGATCMTGGCTCAG-3′
Reverse primer: 1492R 5′-GGTTACCTTGTTACGACTT-3′
The PCR reaction system is shown in Table 1.
|Reaction component .||Volume (μL) .|
|10 × PCR buffer||2|
|Primer F (10 μM)||0.5|
|Primer R (10 μM)||0.5|
|dNTP (each 10 mM)||0.5|
|Taq Plus DNA Polymerase (5 U/μL)||0.5|
|Reaction component .||Volume (μL) .|
|10 × PCR buffer||2|
|Primer F (10 μM)||0.5|
|Primer R (10 μM)||0.5|
|dNTP (each 10 mM)||0.5|
|Taq Plus DNA Polymerase (5 U/μL)||0.5|
The PCR reaction conditions: pre-denaturation at 95 °C for 5 min, denaturation at 94 °C for 30 s, renaturation at 57 °C for 30 s, extension at 72 °C for 90 s, 30 cycles, and extension at 72 °C for 8 min.
The 16S rDNA sequence of strain S1 was input into National Center for Biotechnology Information (NCBI) for BLAST alignment. Sequences with higher similarity were selected for homology comparison, and the phylogenetic tree was constructed by using the neighbor-joining method of MEGA 5.05.
The bacterial suspension (2%) was added to the bacterial culture medium with pH 7, and placed in a constant temperature oscillator for 24 h (150 r/min, 30 °C). The cultured bacterial suspension in a 50 mL centrifuge tube was taken out for centrifugal operation at 4,000 r/min for 15 min. The supernatant was discarded, washed with ultrapure water, and centrifuged again, and the washing was repeated twice to obtain wet bacteria. Then it was placed in a 60 °C blast drying oven to dry to constant weight to weigh and calculate its biomass (g/L). The controlled variable method was adopted to study the influence factors of bacterial growth: ① pH: 5.5, 6, 6.5, 7, 7.5, 8, 8.5; ② Speed (r/min): 90, 120, 150, 180, 210; ③ Temperature (°C): 20, 25, 30, 35, 40;
The effect of C/N on the NH4+-N's removal rate
Glucose and NH4Cl were used to adjust the C/N of the simulated ammonia nitrogen wastewater to 3, 5, 10, 15, 20, 25, 30. 2% bacterial suspension was added to 20 mg/L simulated ammonia nitrogen wastewater (initial C/N is 20, pH is 7), and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C), the supernatant was taken to determine the removal rate of NH4+-N.
Immobilized method and SEM
The immobilized microorganism adopts the sodium alginate embedding method and uses zeolite as the carrier, was called sodium alginate embedded microbe-treated zeolite, and abbreviated as SAEMZ.
The 0.2 g (1%) zeolite, sterilized and dried to a constant weight, and 3 mL (15%) of the microbial suspension are fully mixed, and then shaken and adsorbed at 150 r/min and 30 °C for 2 h. After standing for 24 hours, it was added in 20 mL (2.5%) SA solution. After mixing thoroughly, an injection was used to drop the mixture slowly and evenly into a 2% CaCl2 solution by squeezing and dropping, which shall be placed in the refrigerator, cross-linked at 4 °C for 24 h, removed and washed with ultrapure water three times to obtain the SAEMZ. The prepared SAEMZ should be stored in a refrigerator at 4 °C as spare. The preparation process is shown in Figure 1.
The prepared SAEMZ was observed by SEM, and the SEM photos of the SAEMZ's surface (magnification 75 times) and inside (magnification m 10,000 times) were taken.
Research on SAEMZ's influencing factors
The effect of dosage
0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 g of SAEMZ were respectively added to the 50 mL, 20 mg/L simulated ammonia nitrogen wastewater and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C). The supernatant was taken to determine the removal rate of NH4+-N.
The effect of coexisting ions
2.5 g of SAEMZ was added into 50 mL, 20 mg/L simulated ammonia nitrogen wastewater, and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C), then the supernatant was taken to determine the removal rate of NH4+-N. Among them, ① Ca2+ gradient is 0, 10, 30, 50 mg/L; ② Mg2+ gradient is 0, 10, 30, 50 mg/L; ③ Fe2+ gradient is 0, 2, 5, 10 mg/L; ④ Mn2+ gradient is 0, 2, 5, 10 mg/L.
The effect of wastewater dilution ratio
The simulated ammonia nitrogen wastewater with an initial NH4+-N concentration of 80 mg/L was diluted 0, 2, 4, 5, and 10 times. Then, 2.5 g of SAEMZ was added to a 50 mL diluted water sample, and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C). The supernatant was taken to determine the removal rate of ammonia nitrogen.
Reusability experiment of SAEMZ
2.5 g of SAEMZ was added into 50 mL, 20 mg/L of simulated ammonia nitrogen wastewater, and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C). The supernatant was taken to determine the NH4+-N removal rate. After removing SAEMZ and being washed with ultrapure water 3 times, they were put into use again. The above steps shall be repeated.
Storage stability experiment of SAEMZ
SAEMZ (2.5 g) stored at 4 °C for the different time were added to the 50 mL, 20 mg/L simulated ammonia nitrogen wastewater, respectively, and reacted in a thermostatic oscillator for 48 h (150 r/min, 30 °C). The supernatant was taken to measure the removal rate of NH4+-N.
Determination of NH4+-N
The content of NH4+-N in water was determined by "Water quality—Determination of ammonia nitrogen—Nessler's reagent spectrophotometry" (HJ 535–2009).
Three parallel samples were set in each group of experiments.
RESULTS AND DISCUSSION
Screening of bacterial strains
The strain obtained after cultivation and screening was tested for the NH4+-N removal effect, and the strain was named S1. The experimental results are shown in Figure 2.
Figure 2 shows that strain S1 can removal NH4+-N efficiently. With significant increase of NH4+-N's removal rate by S1 within 0–72 h, the NH4+-N residual concentration decreased from 61.83 mg/L to 7.80 mg/L. When the reaction time exceeds 72 h, NH4+-N's removal trend by S1 is relatively gentle, to achieve the 7.38 mg/L remaining NH4+-N concentration during equilibrium state with 88.06% final removal rate.
Identification of the strain S1
The colony morphology and staining characteristics have been displayed in Figure 3, Figure 3(a) reflects the colony on the plate as moist, small, and protruding, opaque, light yellow, with neat edges, and the colony shape is round. The strain S1 after Gram staining was observed under a microscope, and the results are depicted in Figure 3(b); the bacteria are short rod-shaped, and regarded as Gram-positive bacteria (G+).
The DNA of strain S1 was used as a template for PCR amplification. 16S rDNA PCR amplification product electrophoresis detection results are shown in Figure 4(a). The PCR product channel of strain S1 has no heterozygous bands and bright bands. Compared with the left image of Figure 4(a), the length of the target gene fragment in the right image was about 1.5 kb.
The purified PCR product was sequenced by Sangong Bioengineering (Shanghai) Co., Ltd. The 16S rDNA gene sequence length of strain S1 was 1,459 bp.
The 16S rDNA sequence of strain S1 was input into NCBI for BLAST comparison (Sun et al. 2016), and the sequence with high homology selected to build a phylogenetic tree using MEGA 5.05. The phylogenetic tree of the 16S rDNA sequence of strain S1 is represented in Figure 4(b). Figure 4(b) reveals that strain S1 has more than 99% homology with the Achromobacter pulmonis PI3-03, Achromobacter sp. EP17, Achromobacter sp. W-SL-1, and Achromobacter sp. W-1. According to the phylogenetic tree's information and the morphological observation results of the bacteria, strain S1 is predetermined to be Achromobacter sp.
SEM characterization of SAEMZ
Figure 5(a) shows that the surface structure of the SAEMZ is relatively compact, which can effectively reduce bacteria's leakage and the entry of some macromolecules harmful to microorganisms. Figure 5(b) indicates that the inner surface of the SAEMZ is uneven, the inside is wrinkled without complete agglomeration. After zeolite adsorbs microorganisms, there are still many small holes and narrow channels on the surface, conducive to the transmission of water, oxygen, and substrates, and providing space for the life activities of microorganisms inside the particles, so that the microorganisms maintain normal physiological metabolism inside the particles (Hu 2010; Qin 2011). In addition, large specific surface area provides it with ample adsorption sites, to improve SAEMZ's removal on NH4+-N.
Factors affecting growth characteristics
The effect of pH on bacterial growth
The pH will directly affect the growth of microorganisms and their enzyme activities, pH too high or too low is either instrumental to the growth and reproduction of bacteria. The main influences of pH on bacteria are as follows: First, it affects the cell surface charge and membrane permeability, which affects the uptake of nutrients by bacteria; Secondly, it affects organic substances' ionization degree. Most non-ionic compounds are more likely to penetrate cells, and changes in their ionization may impede the entry of nutrients or expose bacteria to toxic substances; thirdly, unsuitable pH reduces enzyme activity and affects the rate of bacterial proliferation and metabolism, which in turn affects the biochemical processes within the bacterial cell (Zhou 2008). pH on bacterial growth is provided in Figure 6(a).
Figure 6(a) presents that the biomass is very low at pH 5.5 and 8.5 and that the strain biomass increases gradually with increasing pH in the pH range of 6–7.5. Available studies have found that the pH environment suitable for HN-AD bacteria is neutral or weakly alkaline, while acidic or strongly alkaline environments can lead to sluggish growth and affect nitrification and denitrification efficiency (Huang et al. 2015). The results of this study are consistent with existing reports that a weak alkaline environment is favorable for bacterial growth, with strain S1 reaching a maximum biomass of 3.2 g/L at a pH of 7.5.
The effect of rotating speed on bacterial growth
The stain S1 to remove NH4+-N is an aerobic process, and dissolved oxygen's concentration directly affects the growth activity of aerobic strains. A moderate increase in dissolved oxygen concentration can effectively promote the denitrification ability of aerobic denitrifying strains. However, when the dissolved oxygen concentration is too high it is easy to oxidize the microbial cells, causing a reduction in the strain's denitrification ability (Yang & Cui 2019). Dissolved oxygen's concentration in the reaction solution was controlled by changing the constant temperature oscillator's rotation speed. Rotation speed's effect on bacterial growth is shown in Figure 6(b).
Figure 6(b) indicates that the biomass of strain S1 was lower at speeds below 120 r/min. Biomass was higher in the 150–210 r/min range, all exceeding 3 g/L, and reaching a maximum biomass of 3.2 g/L at 150 r/min. When the rotation speed exceeds 150 r/min, the strain's biomass will decrease slightly. When the rotation speed is too high, a large shear force produced by the eccentric oscillation affects the biological activity, due to the difficulty for the microbial cells to resist the shear force's destruction.
The effect of temperature on bacterial growth
Temperature affects the rate of bacteria proliferation and metabolism. Increasing the temperature within a suitable temperature range is conducive to the increase of the enzymatic reaction rate, thus thereby correspondingly increasing the growth rate and the bacteria's metabolic rate. Overheating or undercooling is not conducive to microorganisms' growth, and too high ambient temperature has a lethal effect on bacteria.
As shown in Figure 6(c), changes in temperature had a greater impact on bacterial growth. The biomass at 20 °C was low and increased from 1.1 g/L to 3.3 g/L with increasing temperature. When the temperature exceeded 30 °C, the biomass decreased significantly as the temperature increased, with 2.1 g/L and 1.3 g/L at 35 and 40 °C respectively. The experimental results show that the optimum temperature for strain growth should be 25–35 °C.
The effect of C/N on the strain's removal efficiency of NH4+-N
Figure 6(d) displayed that the NH4+-N's removal rate of strain S1 was very low at C/N 3 and 5, probably because the carbon source was not sufficient currently and the microorganism did not require enough energy to limit the growth process, thus affecting its NH4+-N removal rate. As the C/N increased, the NH4+-N removal rate increased significantly, from 5% to 63.78%. Further increase in the initial concentration of glucose as an easily degradable carbon source would possibly improve the biomass concentration of strain S1, thus facilitating the removal of NH4+-N. NH4+-N removal was better in the range of 20–30 for C/N, which all exceeded 60% removal. In addition to carbon source consumption during nitrification, a low carbon source provided during denitrification also prevents the bacteria from synthesizing sufficient denitrifying enzymes, thus affecting the degree of denitrification and leading to the accumulation of the intermediate product nitrite (Xiu et al. 2011), and the carbon source, as a limiting factor, causes the microorganisms to carry out endogenous denitrification by consuming their own protoplasm, leading to a reduction in cytoplasm and the simultaneous production of NH3 (Xin et al. 2007).
Considering that in practical application, if the carbon source is dosed at high C/N it will cause an increase in carbon source cost and COD of the water body. In addition, too much carbon can be embedded in the enzyme structure, affecting the enzyme activity, thereby affecting the nitrification performance (Song et al. 2013). Therefore, a C/N of 20 was selected as a suitable reaction condition for strain S1, and the removal rate of NH4+-N was 63.67% under this C/N condition.
Research on influencing factors
The effect of dosage on NH4+-N removal efficiency
The different dosage of SAEMZ on the NH4+-N removal efficiency was explored, and the experimental results are shown in Figure 7(a).
Figure 7(a) shows that when the dosage is small, the SAEMZ has less NH4+-N adsorption. And owing to bounded capacity of the bacteria on assimilation and nitrification for NH4+-N, the NH4+-N removal rate is low. In pace with the increase in dosage, the removal rate of NH4+-N increased significantly, reaching the maximum removal rate of 85.20% at the dosage of 2.5 g. When the dosage exceeded 2.5 g, the removal rate of NH4+-N decreased again, possibly because the bacteria in the SAEMZ have a high concentration of bacteria after reproduction, and the inadequate nutrients cannot meet the needs of bacterial growth, thereby affecting the bacterial activity.
Xuelian Zhao (Zhao et al. 2012) used immobilized nitrifying bacteria as the main material to treat the water samples with artificial ammonia nitrogen configuration, and the best NH4+-N removal effect was achieved when the dosage was 8.5%. While Jilun Shao (Shao et al. 2015) used heterotrophic nitrifying bacterium Burkholderia sp. YX02 as the target and used polyvinyl alcohol (PVA) and sodium alginate (SA) as carriers to immobilize the strain, the removal rate of NH4+-N was best when the dosage was 5%-8%. Both are analogous to our study, and in our paper, the NH4+-N's effect is best when the dosage is 5%, which is relatively more economical and environmentally friendly, in line with green energy development.
The effect of coexisting ions on the removal efficiency of NH4+-N
Calcium alginate is the outer carrier of immobilized microorganism material. Its abundant groups can adsorb toxic pollutants through electrostatic or hydrogen bonding, and the zeolite added in the inner layer can capture the fine suspended matter entering through the pores. Adsorption of the carrier material is one of the critical reasons for the microenvironmental enhancement mechanism of immobilized particles, and whether the adsorption is reversible or not will affect the bacterial agent's adsorption capacity (Li et al. 2007). After many negatively charged carboxyl groups in sodium alginate are combined with metal ions, immobilized particles' adsorption is reduced and their ability to protect microorganisms from toxic substances is significantly weakened. The adsorption reaction on active group is difficult to restore their original ability by desorption (Li et al. 2006). Therefore, the effect of metal ions on immobilized microorganism is almost irreversible. The effects of Ca2+, Mg2+, Fe2+, and Mn2+ on the removal rate of SAEMZ were investigated by coexisting ion experiments, and the results are shown in Figure 8.
(1) The effect of Ca2+ on the removal efficiency of NH4+-N
Ca2+ affects bacterial cell membrane permeability, and its increased concentration advances the mitochondrial membrane potential to promote the phosphorylation process, which provides energy for cell proliferation, and maintains stable growth of strains (Zhou et al. 2005).
Figure 8(a) illustrates that Ca2+ has a certain promoting effect on the removal of NH4+-N by SAEMZ. Within the experimental concentration range, as the concentration of Ca2+ increases, the removal rate first increases and then decreases, possibly because the addition of Ca2+ strengthens the calcium alginate structure in the outer layer of the SAEMZ and enhances the SAEMZ's mechanical properties. Therefore, the NH4+-N removal efficiency is better when the Ca2+ concentration is 30 mg/L. However, higher Ca2+ concentration can be toxic to microorganisms, leading to apoptosis and cell necrosis, and the dialysis of high-concentration CaCl2 solution will also dehydrate microbial cells (Simpson et al. 2004), which will affect the physiological activities of microorganisms.
(2) The effect of Mg2+ on the removal efficiency of NH4+-N
Mg2+ plays an important role in glycolysis, respiration, and oxidative phosphorylation, and is an activator of various kinases (Yang & Tang 2008). It also has a certain effect on the stability of cell structures such as ribosomes and cell membranes, influencing cell membrane stiffness and cell wall synthase activity (Li et al. 2002), and regulating cell proliferation and differentiation.
From Figure 8(b), Mg2+'s addition leads to a decrease in NH4+-N's removal efficiency, but to a lesser extent. Studies have shown that Mg2+ can significantly promote heterotrophic nitrifying bacteria's growth and increase the removal rate of NH4+-N (Kim et al. 2005). However, in this experiment, Mg2+ inhibits the NH4+-N removal process of SAEMZ, probably because higher concentrations of Mg2+ in solution currently have a much weaker effect on microbial growth promotion than on the destruction of calcium alginate structure.
(3) The effect of Fe2+ on the removal efficiency of NH4+-N
Iron and manganese are trace elements for biological growth. Fe2+ has a significant contribution to heterotrophic nitrification process and activates hydroxylamine oxidase (HAO) (Song & Xu 2008), which causes the intermediate product hydroxylamine to release electrons and phosphorylation energy (Wang et al. 2003). Figure 8(c) shows that low concentrations of Fe2+ (2 mg/L, 5 mg/L) can slightly boost the NH4+-N removal rate of SAEMZ, and the NH4+-N removal rate is significantly reduced at Fe2+ concentration of 10 mg/L. This phenomenon was similar to the nitrogen removal by heterotrophic nitrification-aerobic denitrification strain YL (Providencia rettgeri), where Fe2+ at 10 mg/L slightly promotes the growth and denitrification process of strain YL (Zhao et al. 2010). However, the suitable Fe2+ concentration range for SAEMZ is smaller, probably because strain S1 is more sensitive to the increase of Fe2+ concentration.
(4) The effect of Mn2+ on the removal efficiency of NH4+-N
Mn2+ participates in the activity of bacterial superoxide dismutase and serves as a cofactor for certain enzymes. The existence of Mn2+ can enhance the permeability of cell membranes, beneficial to the absorption of nutrients by microorganisms, thereby promoting the biochemical process (Wang et al. 2003).
Figure 8(d) presents that the NH4+-N's removal efficiency tends to decrease as the Mn2+ concentration increases. Studies have shown that a certain concentration of Mn2+ stimulates the activity of ammonia monooxygenase (AMO), thus promoting the removal of NH4+-N during heterotrophic nitrification, and Mn2+ also promotes ammonia assimilation by enhancing the activity of glutamate synthase (GS) (Zhao et al. 2017). However, within the concentration range of this experiment, Mn2+ harmed the removal of NH4+-N by SAEMZ. Analysis of the reason may be that microorganisms are more sensitive to the effects of heavy metals. At this time, the Mn2+ concentration range has exceeded the limit of microorganisms. High concentrations of Mn2+ have apparent toxic effects on microorganisms, so the NH4+-N removal effect is poor.
The effect of wastewater dilution multiple on the removal efficiency of NH4+-N
The simulated ammonia nitrogen wastewater was diluted at different multiples and put into the SAEMZ for experiments. The effect of wastewater dilution multiples on the removal efficiency of NH4+-N is shown in Figure 7(b).
As shown in Figure 7(b), the removal of ammonia nitrogen by SAEMZ in simulated ammonia nitrogen wastewater tended to increase as the dilution of the wastewater increased. When the initial NH4+-N concentration is high (the dilution factors are 0 and 2 times), the removal efficiency of NH4+-N from the wastewater by SAEMZ is low. When the initial NH4+-N concentrations are 20 mg/L, 16 mg/L, and 8 mg/L (dilution multiples of 4, 5, and 10), the SAEMZ's removal efficiency of NH4+-N in the simulated ammonia nitrogen wastewater exceeded 80%. It can be concluded that the appropriate concentration of NH4+-N removal by SAEMZ under the experimental conditions is 8–20 mg/L, which is in line with existing research (Li et al. 2010).
Reuse is an important advantage of SAEMZ, which can decrease cell waste, reduce time, and culture costs. The reusability experiments were carried out on the SAEMZ, and the results are shown in Figure 7(c).
In the first three reuses, the removal efficiency of NH4+-N dropped from 83.98% to 69.13%. With four reuses, the removal rate dropped significantly to 10%, while at five reuses, the removal effect of SAEMZ on NH4+-N was almost lost. A small number of white flocculent substances can be observed in the reacted NH4+-N wastewater, probably due to the partial dissolution of the bacteria in the SAEMZ and the outer carrier material, which form microbial flocs. The main reason for this is that the immobilized particles are repeatedly impacted by the solution during the oscillation process, and after multiple cycles of use, the properties of the particles are weakened and cell leakage occurs (Chen et al. 2008; Wu et al. 2009). Therefore, to ensure the removal effect of NH4+-N, the number of reuse times of SAEMZ should be limited to no more than three.
Storage stability test
Storage stability is an important factor in the practical application of immobilized cell systems (Wu et al. 2009). For free cells, prolonged storage reduces their biological activity, and their ability to degrade pollutants may also significantly decrease, while the storage stability of immobilized cells is better than that of free cells, which is more advantageous in practical applications (Lin et al. 2013a, 2013b). Experiments were conducted on the NH4+-N removal effect of SAEMZ with different storage times, and the results are shown in Figure 7(d).
As can be seen from Figure 7(d), the SAEMZ maintained a good NH4+-N removal effect when the storage time did not exceed 60 days, and the NH4+-N's removal rate only dropped by 7.4%. When the SAEMZ was stored at 4 °C for more than 90 days, the NH4+-N removal efficiency decreased, but to a lesser extent, while the SAEMZ stored for 120 days still had 63.39% of the NH4+-N removal rate. Besides, it can be found that the elasticity and mechanical strength of the SAEMZ did not changed significantly at different storage periods and maintained their original physical and chemical properties. The immobilization of bacteria reduced cell leakage and enhanced resistance to environmental changes. The experimental results show that the SAEMZ still maintains its physiological stability and high mechanical strength within a certain storage time, and has good NH4+-N removal performance.
In this paper, the strains with high-efficiency NH4+-N degradation performance were screened from black-odorous water. The effect factors of the NH4+-N removal performance, reusability, and storage stability of the SAEMZ were investigated. We specifically obtained that the strain S1, whose removal rate of NH4+-N can reach 88.06%, decreased the NH4+-N concentration from 61.83 mg/L to 7.80 mg/L, is a Gram-positive bacterium, belonging to the genus Achromobacter sp, with the optimal growth conditions of pH 7–8, rotation speed 150–210 r/min, temperature 25–35 °C; the appropriate concentration for NH4+-N removal by SAEMZ is 8–20 mg/L; The NH4+-N's removal rate improved with increasing SAEMZ dosing; Ca2+ in the solution can boost the removal of NH4+-N, while Mg2+ and Mn2+ inhibit it; the SAEMZ still has a 63.39% removal efficiency of NH4+-N after 120 days of storage at 4 °C, its recycling times should be limited to no more than three in practical applications.
Based on this study, the following recommendations are made for future research:
Considering the synergistic effects between different microorganisms, strain compounding studies can be carried out to treat the target pollutants using a combination of bacterial agents to improve their ability to resist adverse living conditions, while enhancing the practical application of immobilized microbial agents to complex water bodies (for example: black smelly water bodies).
When immobilized microbial agents are examined for reusability in the future, the activation methods of immobilized microbial agents can be explored to improve their reusability.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.