Preparation and Characterization of Temperature/pH Dual-Responsive Gel Spheres for Immobilizing Nitro Bacteria.

The temperature/pH dual-responsive gel spheres were prepared by orthogonal experiments and response surface methodology, and finally, the optimal synthesis conditions were obtained by a composite score, including swelling, mechanical properties, mass transfer properties, and so forth. The results showed that a sodium alginate concentration of 3% (w/v), CaCl2 concentration of 2% (w/v), gelling time of 40 h, drop height of 14 cm, NaCl concentration of 0.6% (w/v), N-isopropylacrylamide concentration of 0.03% (w/v), and acrylic acid concentration of 4.06% (w/v) were optimal synthesis conditions. The environmental change tolerance experiments showed that the nitrogen removal of the dual-response nitrifying gel spheres was better than the domesticated sludge at low temperatures (4 °C) and in alkaline (pH 9 and 10) conditions. The as-obtained gel spheres can respond intelligently to the changes in ambient temperature and pH. It is hoped that this study will provide technical parameters for the development and application of microbial immobilization carriers.

Introduction

Microbial immobilization is a vital way to enhance the functionality of microorganisms, where the target microorganisms are confined to a limited area (carrier material) to maintain high biomass and biological activity.[1] Compared to free bacteria, immobilized microorganisms have the advantages of high biodegradation rates, good environmental tolerance, and easy solid–liquid separation.[2] Simultaneously, the immobilization technology is suitable for various functional microorganisms, including anaerobic ammonia-oxidizing bacteria, aerobic denitrifying bacteria, and hydrocarbon-degrading bacteria.[3−6] Therefore, the technology has gradually shown great potential in different areas such as bio-hydrogen production, soil remediation, and wastewater treatment.[7−9]

The choice of the carrier material is crucial to the diffusion of microbial immobilization techniques. A good carrier material should have unique features, including good stability, high mass transfer capacity, cheap and easy raw materials availability, and good biocompatibility.[10,11] Particularly prominent among these is the natural organic carrier sodium alginate (SA), a linear anionic polysaccharide composed of β-d-mannuronic acid and α-l-guluronic acid.[12−14] It is enriched with −OH and −COOH, can form stable hydrogels with multivalent cations such as Ca2+ (except Mg2+ cations), and is currently one of the most demanded carrier materials.[15,16] Since the integrated adsorption—biodegradation—assimilation process is the only mechanism behind the removal of targeted pollutants by immobilized microorganisms,[2] changes in the external environment such as temperature, pH, magnetic field, ionic strength, and so forth can inhibit the growth and metabolism of free microorganisms, resulting in lower pollutant removal rates. Therefore, the development and promotion of gel spheres resistant to external environmental changes are highly recommended.

Poly-N-isopropyl acrylamide (PNIPAAm) and polyacrylic acid (PAA) are typical sensitive polymers utilized to prepare responsive gel spheres. PNIPAAm is thermally responsive due to its inherently lower critical solution temperature (LCST), approximately 32 °C.[17−19] This is demonstrated by the fact that when the ambient temperature is below LCST, PNIPAAm appears to swell hydrophilically, and conversely, it shrinks in volume and is hydrophobic.[20] PAA can respond to pH value changes by protonation or deprotonation due to the abundance of ionizable −COOH moieties in its structure.[21] Numerous papers have shown that gel spheres modified by PNIPAAm or PAA show a satisfactory response to temperature/pH changes, thus resisting the inhibition of functional microorganisms’ activity by external environmental changes.[22−25]

In this study, the optimum calcium alginate (CA) gel spheres were successfully prepared using orthogonal experiments; on this behalf, the pore structure was optimized by the modification effect of NaCl solution. The biocompatible CA gel spheres were used as the core substrate and interacted with NIPAAm, acrylic acid (AA), and N,N-methylenebisacrylamide (MBA) reagents to produce a temperature/pH responsive layer and result in the formation of optimizing environmentally dual-responsive gel spheres. It is proposed that this study will provide a new platform for the development and application of microbial immobilization carriers.

Results and Discussion

Preparation and Optimization of CA Gel Spheres

SA is the prime material required for the preparation of CA gel spheres, and its mass concentration affects the sphericity, mechanical strength, and ease of preparation of gel spheres. Lower concentration of SA results in poor sphericity and poor strength, while higher concentration of SA provides higher strength CA spheres due to its high viscosity, but it still suffers from severe trailing and complicated preparation. Besides, previous studies have suggested that CaCl2 concentration, gel time, and drop height also have a dominant effect on CA gel sphere properties (such as sphericity, mechanical strength, and swelling).[26−28] Therefore, in this study, the above four factors were tested orthogonally to determine the optimal synthesis conditions for CA gel spheres, and the results are shown in Table . The relationship between factors and scores was also plotted using the value of each factor level as the horizontal coordinate and the mean value of the corresponding composite score for each factor level as the vertical coordinate (Figure ). As depicted in Figure , the composite score of CA gel spheres tends to increase first and then decrease with increasing SA concentration, with a maximum value achieved at a SA concentration of 3% (w/v). Similarly, the CA gel spheres achieved a maximum composite score at a CaCl2 concentration of 2% (w/v), a gelling time of 40 h, and a drop height of 14 cm. It was deduced that the optimal synthesis conditions for CA gel spheres are A4B1C4D5 (A4, B1, C4, and D5 indicate SA concentration of 3% (w/v), CaCl2 concentration of 2% (w/v), gelling time of 40 h, and drop height of 14 cm, respectively). Based on the magnitude of the R-value of the extreme difference derived from the orthogonal experiment and the results of the ANOVA presented in Table , it could be seen that the ranking of the factors affecting the composite score was A(20.4) > C(4.8) > B(4) = D(4). SA concentration significantly affected the composite score.

Table 1

Orthogonal Test Resultsa

	factors
sample	A (%)	B (%)	C (h)	D (cm)	composite score
1	0.5	2	10	6	22
2	0.5	3	20	8	24
3	0.5	4	30	10	15
4	0.5	5	40	12	22
5	0.5	6	50	14	14
6	1	2	20	10	30
7	1	3	30	12	27
8	1	4	40	14	32
9	1	5	50	6	28
10	1	6	10	8	25
11	2	2	30	12	37
12	2	3	40	14	39
13	2	4	50	6	32
14	2	5	10	8	40
15	2	6	20	10	31
16	3	2	40	14	43
17	3	3	50	6	40
18	3	4	10	8	40
19	3	5	20	10	36
20	3	6	30	12	40
21	4	2	50	6	38
22	4	3	10	8	38
23	4	4	20	10	36
24	4	5	30	12	38
25	4	6	40	14	40

Note: A, B, C, and D indicate SA concentration, CaCl2 concentration, gelling time, and drop height, respectively.

Figure 1

Relationship diagram between factors and score.

Table 2

Analysis of Variance and Significance Testa

source	sum of squares of deviations	freedom	mean square	F-value	F0.05 (4,4)	F0.01 (4,4)	significance
A	1413.04	4	353.26	23.93	6.39	15.98	**
B	59.04	4	14.76	1.00	6.39	15.98
C	70.64	4	17.66	1.20	6.39	15.98
D	52.64	4	13.16	0.89	6.39	15.98
error	59.04	4	14.76
sum	1654.4	20

Note: A, B, C, and D indicate SA concentration, CaCl2 concentration, gelling time, and drop height, respectively, ** indicates highly significant correlation.

Preparation and Optimization of NaCl-Modified Gel Spheres

High concentrations of Ca2+ ions can cause changes in the structure of the resulting CA gel spheres to make them too dense, which negatively impacts their mass transfer properties.[29] It has been shown that immersing CA gel spheres in a suitable concentration of NaCl solution can effectively improve this defect, and some of the chelated carboxylate moieties are replaced by Ca2+ to free state by Na+, making the internal structure of the gel spheres more loose and porous.[30] In this study, the optimum CA gel spheres produced in Section were placed in NaCl solutions of different concentrations (0–0.9%, w/v) and scored in four aspects: swelling, mechanical strength, oscillatory breakage rate, and mass transfer performance. As can be seen from the results presented in Figure , the highest composite score was achieved at 0.6% (w/v) NaCl-modified gel spheres. Therefore, 0.6% (w/v) NaCl solution was chosen as the optimal synthesis condition for the NaCl-modified gel spheres.

Figure 2

Relationship diagram between NaCl concentration and score.

Preparation and Optimization of Temperature/pH Dual-Response Gel Spheres

Response surface methodology (RSM) is one of the ideal methods to reduce the number of experiments and is used for modeling, data analysis, and optimization purposes.[31]Table shows the results of the trials of the effect of the independent variables NIPAAm, MBA1, AA, and MBA2 concentration on the composite score. Figure shows a response surface plot of the effect of the independent variables on the composite score. Figure a,c shows that NIPAAm concentration was inversely proportional to the composite score if MBA1 concentration was certain. The predicted maximum composite score of 38.13 was achieved at NIPAAm and MBA1 concentration of 0.03 and 0.02%, respectively. From Figure b,d, it could be seen that if the MBA2 concentration was certain, the composite score tends to increase and then decrease with the increase of AA concentration. The maximum composite score of 38.13 was achieved at AA and MBA2 concentration of 4.06 and 0.26%, respectively. Therefore, the optimal synthesis conditions for the dual-response gel spheres were selected as 0.03 and 0.02% of NIPAAm and MBA1 in the temperature-modified solution, 4.06, and 0.26% of AA and MBA2 in the pH-modified solution.

Table 3

Box-Behnken Design and Effect of Independent Variables on Composite Score as a Response

runs	NIPAAm (%)	MBA1 (%)	AA (%)	MBA2 (%)	composite score
1	0.93	0.01	0.1	0.23	28
2	1.83	0.02	5	0.23	36
3	0.93	0.02	2.55	0.23	37
4	0.93	0.03	2.55	0.01	36
5	0.03	0.02	0.1	0.23	29
6	0.93	0.02	2.55	0.23	37
7	0.93	0.01	2.55	0.45	35
8	0.03	0.01	2.55	0.23	37
9	0.93	0.02	0.1	0.01	28
10	0.93	0.02	2.55	0.23	37
11	0.93	0.02	5	0.45	36
12	1.83	0.02	2.55	0.01	34
13	1.83	0.02	2.55	0.45	36
14	0.93	0.03	5	0.23	35
15	0.03	0.02	5	0.23	37
16	1.83	0.01	2.55	0.23	36
17	0.93	0.02	2.55	0.23	37
18	0.93	0.03	2.55	0.45	37
19	0.93	0.02	2.55	0.23	36
20	0.93	0.03	0.1	0.23	29
21	0.03	0.03	2.55	0.23	36
22	0.03	0.02	2.55	0.01	36
23	1.83	0.03	2.55	0.23	35
24	0.93	0.01	2.55	0.01	33
25	0.03	0.02	2.55	0.45	35
26	0.93	0.02	0.1	0.45	31
27	1.83	0.02	0.1	0.23	33
28	0.93	0.02	5	0.01	32
29	0.93	0.01	5	0.23	37

Figure 3

Surface plot for composite score with respect to the three-dimension (3D) of NIPAAm and MBA1(a); 3D of AA and MBA2(b); 2D of NIPAAm and MBA1(c); and 2D of AA and MBA2(d).

Gel Sphere Characterization

SEM Analysis

The optimum CA gel spheres, NaCl-modified gel spheres, and dual-response gel spheres were not significantly different, all being spheres of uniform particle size (approximately 4 mm in diameter) and opalescent and translucent throughout. After dehydration-liquid nitrogen-freeze dried operations, all types of gel spheres had exhibited an undulating surface (Figure a–c) and a complete network structure (Figure d–f). The surface of the optimum CA gel spheres modified by NaCl and dual response, in turn, showed a trend of greater fold (Figure a), insignificant fold (Figure b), greater, uniform, and lesser fold (Figure c). This phenomenon may be related to the pore distribution and structure of the various types of gel spheres. From the monitored profile, it can be stated that the NaCl-modified gel spheres (Figure e) showed evident delamination from the outside to the inside compared to the optimum CA gel spheres (Figure d), and this phenomenon may be related to the length of the NaCl modification time. Meanwhile, the shell–core structure (dense outside and sparse inside) shown in Figure f confirms the successful development of optimal dual-response gel spheres, forming a composite gel sphere with a CA as the core and a temperature/pH responsive layer as the shell layer. Besides, this structure effectively traps the microorganisms embedded in the gel spheres, providing sufficient space for them to grow and multiply. Moreover, it can provide a stable living environment for the proper growth of microorganisms. When the temperature/pH of the environment fluctuates, the outer temperature/pH response layer can achieve a real-time response and maintain a relatively stable and favorable microenvironment for microorganisms to grow.

Figure 4

SEM surface image of the optimum CA gel spheres (a), NaCl-modified gel spheres (b), and dual-response gel spheres (c); SEM interior image of the optimum CA gel spheres (d), NaCl-modified gel spheres (e), and dual-response gel spheres (f).

BET Analysis

From the summary table of gel sphere properties (Table ), it could be observed that the average particle size of the three optimum gel spheres was 3.8 mm, and there was no significant difference found in density (all close to the density of water). Regarding specific surface area and pore size, the optimum CA gel spheres had a significant specific surface area value of 1.455 m2/g with an average pore size of 15.10 nm. The specific surface area of the optimum NaCl-modified gel spheres decreased abruptly up to 0.052 m2/g, probably due to the full replacement of Ca2+ in the CA gel spheres by Na+, resulting in a lower cross-link density and increased macropore content (average pore size of 62.10 nm).[30] The optimum dual-response gel spheres were based on the optimum NaCl-modified gel spheres modified with polymers PNIPAAm and PAA, whose 3D network structure intertwines with the CA gel spheres, a 3D grid structure, and formed a temperature/pH responsive layer on the surface.[32] The final mesoporous material with a specific surface area of 0.113 m2/g and an average pore size of 26.88 nm was produced.

Table 4

Summary of the Three Types of Gel Sphere Properties

type	diameter mm	density g/cm3	BET specific surface area m2/g	average pore size nm
the optimum CA gel spheres	3.8	0.98	1.455	15.10
the optimum NaCl-modified gel spheres		1.00	0.052	62.10
the optimum dual-response gel spheres		0.99	0.113	26.88

FT-IR Analysis

The optimum CA gel spheres (Figure S1a) and the optimum NaCl modified gel spheres (Figure S1 b) showed the same trend in infrared (IR) spectra, both having −OH stretching vibration peaks (positioned at 3339 cm–1), −COO– antisymmetric (located at 1592 cm–1), and symmetric (peaked at 1416 cm–1) stretching vibration peaks.[33,34] However, the latter individual characteristic peaks emerged with greater intensity and were more pronounced, indicating that the arrangement of the functional groups was sparser after modification with NaCl. Compared to Figure S1b, a broad peak detected at 3398 cm–1 was assigned to N–H conjugation, peak monitored at 1243 cm–1 was referred to C–N bond stretching vibration, isopropyl C–H bond out-of-plane wobble vibration at 672 cm–1, and C=O stretching vibration at 1719 cm–1 (characteristic peaks of carboxyl groups) in Figure S1c, all endorsed the successful preparation of the optimum dual-response gel spheres.

Gel Sphere Response Experiment

Figure exhibits the variations in swelling for three types of optimum gel spheres (made in Sections 2.1–2.3, respectively) for different temperatures 5–50 °C (at pH 7) or different pH 2–12 (at temperature 20 °C). In Figure a, it could be seen that the swelling of the three types of optimum gel spheres tends to increase first and then decrease with the increase in temperature, and the maximum swelling is achieved at 30 °C. The optimum dual-response gel spheres swelling increased for the temperature range 5–30 °C, probably due to the increased hydration of the gel sphere network at higher temperatures.[35] As in numerous previous studies, the dual-response gel spheres did not achieve high swelling at high temperatures (40–50 °C).[36] This may be due to the fact that under high temperature conditions, the intramolecular hydrogen bonds were broken, the hydrophobicity of the gel spheres was increased, and the water molecules within the structure were released, reducing the degree of swelling.[37,38] The optimum NaCl-modified gel spheres had significantly lower mechanical properties (6–9 N) compared to the optimum CA gel spheres (>20 N), although they had a large swelling at all temperatures. The optimum dual-response gel spheres were temperature-sensitive and had a high mechanical strength (11–14 N), making them more beneficial for encapsulating microorganisms for biological treatment of wastewater.

Figure 5

Effect of response on the swelling of gel spheres: (a) temperature and (b) pH.

Figure b shows that the swelling of the three types of gel spheres increased with an increase in the pH value. The change in swelling was relatively flat for the pH range 2–8 and 10–12 and increased significantly for the pH range 8–10. At pH 10, the swelling of the optimum CA gel spheres, the optimum NaCl-modified gel spheres, and the optimum dual-response gel spheres increased to 51.19, 48.01, and 53.14%, respectively. Overall, a minimum of 14.67% (at pH 2) and maximum of 53.99% (at pH 12) swelling of the gel spheres occurred on the dual-response gel spheres, achieving a dual-response gel sphere response characteristic to pH. This was closely related to the formation of a −COOH– rich temperature/pH-responsive layer on its surface (consistent with the results in Figure S1c). Moreover, −COOH was negatively charged (−COO–) at higher pH due to deprotonation, with an increase in electrostatic repulsion, changes in the pore structure of the gel spheres, and a consequent increase in swelling monitored.[21,39,40]

Figure shows that the morphology of the optimum dual-response gel spheres at different temperatures and pH conditions. At the same temperature (T = 5 °C), the higher the pH, the larger the size of the gel spheres (increasing by about 0.8 mm). Similarly, at the same pH (pH = 10), the higher the T, the larger the size of the gel spheres (increasing by about 0.7 mm).

Figure 6

Morphology of dual-response gel spheres: (a) T = 5 °C, pH = 7; (b) T = 5 °C, pH = 10; and (c) T = 30 °C, pH = 10.

Environmental Change Tolerance Experiment

Figure exhibits denitrification performance of the optimum dual-response nitrifying bacteria gel spheres for different temperatures 4–50 °C (at pH 7) or pH 4–10 (at temperature 20 °C). The NH4+–N removal rate by the optimum dual-response nitrifying bacteria gel spheres increased first and then decreased with increasing temperature (Figure a). The maximum NH4+–N removal rate (45.21%) was achieved at 30 °C. The reason may be that the gel spheres had a temperature/pH responsive layer, when the temperature was lower than LCST, the gel spheres appear to swell hydrophilically and the surface micropores open up, allowing the nitrobacteria embedded in the inner layer of the gel spheres to come into full contact with the NH4+–N in solution.[37] However, compared to domesticated sludge, it did not show nitrogen removal advantages at medium temperatures (20 and 30 °C) due to mass transfer limitations. In contrast, the optimum dual-response nitrifying bacteria gel spheres showed superior denitrification performance at low temperatures (4 °C) with 31.07% removal of NH4+–N, 6.87% higher than the domesticated sludge. The reason may be that the gel slowed down the inhibition of microbial growth at low temperatures and provided a good living space for microorganisms, thus offering the possibility of effective degradation of ammonia nitrogen.[14,41,42]

Figure 7

Tolerance to environmental changes of the optimum dual-response nitrifying bacteria gel spheres (A) and acclimated sludge (B): (a) temperature and (b) pH.

Overall, the effect of pH on the domesticated sludge and the optimum dual-response nitrifying bacteria gel spheres showed similar trends: NH4+–N removal rate increased and then decreased with increasing pH (Figure b). Due to the intrinsic characteristics of the nitrifying bacteria [optimum pH range for AOB and NOB (7.5–8.5),[43] the maximum NH4+–N removal rate (52.21%) was achieved at pH 8 for the domesticated sludge. The optimum dual-response nitrifying bacteria gel spheres did not show nitrogen removal benefits at this pH due to mass transfer limitations. On the contrary, at pH 9 and 10, there was a high ammonia removal (45.10 and 31.63%, respectively), which was 2.89 and 3.69% higher than that of the domesticated sludge, respectively. The reason was that at high pH, the pore structure of the optimum dual-response nitrifying bacteria gel spheres changes, with a more porous and sparse surface and increased removal of ammonia nitrogen.[39]

In summary, the optimum dual-response gel spheres had the ability to enhance the tolerance of microorganisms to unfavorable external environments and was expected to provide a reference for developing and applying microbial immobilization carriers.

Conclusions

In the study, we explored the optimal synthesis conditions for the dual-response gel spheres. The main conclusions can be summed up as follows

By constructing an orthogonal experiment with the composite score as the evaluation index, it was concluded that the optimum synthesis conditions for CA gel spheres were SA concentration of 3% (w/v), CaCl2 concentration of 2% (w/v), gelling time of 40 h, and drop height of 14 cm. In addition, the factors affecting the composite score of the gel spheres were ranked as follows: SA concentration > gelling time > CaCl2 concentration = drop height.

The swelling and mass transfer properties of the CA gel spheres modified with 0.6% (w/v) NaCl were significantly improved than those of the pristine CA gel spheres.

The optimal synthesis conditions for the dual-response gel spheres were explored using the RSM: NIPAAm, MBA1, AA, and MBA2 concentrations of 0.03% (w/v), 0.02% (w/v), 4.06% (w/v), and 0.26% (w/v), respectively.

The results of the environmental change tolerance experiment, the response experiment, and various characterization methods (SEM, BET, and FT-IR) showed that the optimum temperature/pH dual-response gel spheres were successfully prepared. It is hoped that it will provide a valuable reference for the development and application of microbial immobilization carriers, and consideration can be given to exploring the implementation of LCST and pH control of the material in the future.

Materials and Methods

Reagents and Instruments

SA(AR), CaCl2(AR), NaCl(AR), ammonium persulfate (APS) (AR), and tetramethylethylenediamine (TMEDA) (BR) were purchased from Sinopharm Chemical Reagent Co. MBA(AR) was purchased from Sas Chemical Technology (Shanghai) Co. NIPAAm(BR) and AA(BR) were purchased from Tricia (Shanghai) Chemical Industry Development Co. Sodium bisulfite (SBS) (ACS) was purchased from Beijing Bailingway Technology Co.

A digital push–pull meter (HP-50, HANDPI, China) was used to measure the mechanical strength of the gel spheres. The structure and specific surface area of the gel spheres were observed and analyzed by using scanning electron microscopy (SEM) (JSM-6460LV, JEOL, Japan) and a specific surface and porosimetry instrument (ASAP 2020, Micromertics, USA), respectively. The changes in the gel spheres’ characteristic peaks after modification were analyzed by Fourier transform IR (FTIR) (Nicolet 6700, Thermo Scientific, USA) to cross-check the changes noticed in the material structure.

Preparation of Gel Spheres

The overall process of preparing the gel spheres is presented in Figure . The specific steps are as follows:

Figure 8

Preparation of gel spheres.

CA Gel Spheres

The SA solution was first cooled to room temperature after complete dissolution (50–60 °C water bath) and then added drop by drop to the CaCl2 solution at a fixed height using a rubber-tipped dropper. It was cross-linked at 4 °C for a specific time and rinsed 2–3 times with deionized water. To obtain the optimum CA gel spheres, a 4-factor [A: SA concentration (%), B: CaCl2 concentration (%), C: gelling time (h), and D: drop height (h)] 5-level orthogonal experiment was used in this study. The ultimate use of the gel spheres is to encapsulate microorganisms for the biological treatment of wastewater. As there is no unified evaluation standard, in order to evaluate the gel spheres quantitatively, a preliminary study was conducted by equally assigning weights to five aspects (sphericity,[33,44,45] swelling,[46−48] mechanical properties,[49−51] oscillatory breakage rate,[44,52,53] and mass transfer properties[42,44,54]) on the basis of previous studies by other scholars (Table ). The orthogonal experimental design factors and scoring criteria are given in Tables and 7.

Table 5

Summary of Gel Sphere Performance

carrier	performance	remarks	references
PVA–SA	sphericity	scored for sphericity, size, and shape	(44)
CA		shape characterization of hydrogels using spherical factors	(33)
CA		SA concentrations were selected based on whether spherical and uniformly sized CA gel spheres were produced	(45)
SA/PEI	swelling	characterization using the swelling rate	(46)
semi-IPN superabsorbent nanocomposite			(47)
PVA/alginate			(48)
PAC–SA	mechanical properties	characterization using compressive strength	(49)
3D PVA gel beads			(50)
methyl cellulose/CA beads			(51)
PVA/SA	oscillatory breakage rate	agitation of gel beads at 600 rpm and final recording of the breakage rate	(52)
PVA/PPG hydrogel		agitation of gel beads at 2000 rpm and final recording of the breakage rate	(53)
PVA–SA		the pellets were placed in an isometric shaker for 48 h and the breakage was recorded	(44)
PVA–SA–diatomite	mass transfer properties	characterization using mass transfer rates	(42)
PAC–SA			(54)
PVA–SA		the gel spheres were immersed in ink for 20 min and characterized by the color shade of the central section and the radius of immersion	(44)

Table 6

Orthogonal Experimental Table

	factorsa
levels	A (%)	B (%)	C (h)	D (cm)
1	0.5	2	10	6
2	1	3	20	8
3	2	4	30	10
4	3	5	40	12
5	4	6	50	14

Note: A, B, C, and D indicate SA concentration, CaCl2 concentration, gelling time, and drop height, respectively.

Table 7

Gel Spheres Scoring Criteria

performance		score
sphericity	90–100% uniformly spherical in size (diameter = 4 mm), 0–20% adhesion	9–10
	50–90% uniformly spherical in size (diameter = 4 mm), the rest is ellipsoidal and olive shapes, 0–20% adhesion	5–8
	more than 50% non-spherical, heavily (more than 50%) adherent	0–4
swelling	swelling of 15–30	9–10
	swelling of 5–15 and 30–40	5–8
	swelling of 0–5 or >40	0–4
mechanical properties	mechanical strength of 20–25 N	9–10
	mechanical strength of 10–20 N	5–8
	mechanical strength of 0–1 N	0–4
oscillatory breakage rate	breakage rate of 0–10%	9–10
	breakage rate of 10–50%	5–8
	breakage rate >50%	0–4
mass transfer properties	actual nitrite nitrogen concentration/theoretical nitrite nitrogen concentration in gel spheres of 90–100%	9–10
	actual nitrite nitrogen concentration/theoretical nitrite nitrogen concentration in gel spheres of 70–90%	5–8
	actual nitrite nitrogen concentration/theoretical nitrite nitrogen concentration in gel spheres <70%	0–4

NaCl-Modified CA Gel Spheres

The optimum CA gel spheres were cast into different concentrations (0–0.9%) of NaCl solution, left to react for 2 h, and rinsed 2–3 times with deionized water to obtain the optimum NaCl-modified CA gel spheres.

Temperature/pH Dual-Response Gel Spheres

5 g of drained optimized NaCl-modified CA gel spheres was cast into the temperature-modified solution [a mixture(15 mL) of NIPAAm, MBA, APS (6.0 mg), and TMEDA (17.0 μL)], left to react for 2 h, and rinsed 2–3 times with deionized water to produce temperature-responsive gel spheres. It was continued to be cast into a pH-modified solution [a mixture (10 mL) of AA, MBA, APS (6.0 mg), and SBS (6.0 mg)], left to react for 30 min, and rinsed 2–3 times with deionized water to finally produce a temperature/pH dual-response gel sphere. Design-expert 8.0.5 software with the Box-Behnken RSM was used to determine the relationship between the variables and the responses. NIPAAm (X1), MBA1 (X2), AA (X3), and MBA2 (X4) concentrations were used as independent variables and the composite score (including swelling, mechanical properties, oscillatory breakage rate, and mass transfer properties) as the dependent variable to finally obtain the optimum temperature/pH dual-response gel spheres. The levels and ranges of the experimental independent variables are given in Table . Here, MBA1 and MBA2 were the cross linker MBA added to the temperature-modified solution and the pH-modified solution, respectively.

Table 8

Design Factor Level Table

			factor coding level
number	variables	unit	–1	0	1
X1	NIPAAm	(%)	0.03	0.93	1.83
X2	MBA1		0.01	0.02	0.03
X3	AA		0.1	2.55	5
X4	MBA2		0.01	0.23	0.45

Optimum Dual-Response Nitrification Gel Spheres

The sludge (from the aeration tank of a university wastewater plant in Xi’an), which had been domesticated for 23 days, was made into a bacterial suspension (100 g/L) by centrifugation (2000 rpm, 10 min). The mixture was added to the SA solution in a certain ratio (VSA/Vbacteria suspension = 4:1) and mixed well. The subsequent preparation steps were the same as those for the final preferred optimum dual-response gel spheres.

Performance Characterization

Swelling

20 dried gel spheres of uniform particle size were immersed in a 0.9% NaCl solution and left to swell at 25 °C until equilibrium reached, at which point the gel sphere mass (We) was constant. The formula is as followswhere We is the weight of gel sphere after swelling equilibrium in g; Wd is the weight of gel spheres after vacuum drying in g.

Mechanical properties

A digital push–pull gauge was used to measure the maximum pressure borne by the gel spheres. A uniform size gel sphere was placed on the lower platen, and the handwheel was shaken to move the upper platen downwards until the gel sphere was broken, and then the pressure was recorded at this point. The average of the three measurements was the maximum pressure to which the gel sphere was subjected.

Oscillatory breakage rate

50 intact gel spheres were placed in equal amounts of 0.9% NaCl solution (30 mL), then put in a shaking incubator, and shaken at a constant temperature (25 °C) at an equal frequency (200 rpm for 3 days). The gel spheres were observed under a light microscope, and the breakage was analyzed. The formula for oscillatory breakage rate is as follows:where S0 is number of unbroken gel spheres before oscillation = 50; S1 is number of unbroken gel spheres after oscillation.

Mass Transfer Properties

10 g gel spheres were added to 20 mL of nitrite solution (10 mg/L), placed in an incubator, and shaken at constant temperature (25 °C) and at equal frequency (200 rpm) for 2 h. The concentration of nitrite in the solution was measured by the formula given belowwhere V1 is initial nitrite nitrogen solution volume, 20 mL; V2 is total volume of gel spheres dosed, 10 mL; C1 is initial nitrite nitrogen solution concentration, 10 mg/L; and C2 is nitrite nitrogen solution concentration after 2 h, mg/L.

SEM analysis

The gel spheres were dehydrated in a graded ethanol series [10 min in each of 20, 40, 50, 60, 70, 80, 90% (w/v) ethanol and then twice in anhydrous ethanol to remove the final traces of water]. The dehydrated beads were then placed in liquid nitrogen (5 min)—freeze dried (24 h) to obtain dry gel sphere particles. It was then cut in half with a knife, sprayed with gold, and placed on a conductive gel, and the surface structure and profile of the gel sphere were observed using SEM with a secondary electron detector, a scanning voltage of 20 kv, and a working distance of 7.0 mm.

BET analysis

The gel spheres were first treated with liquid nitrogen (5 min)—freeze dried (24 h) and then ground and sieved (80 mesh) to obtain the final dried gel sphere powder. 0.2 g was put into an ASAP-2020 pore structure surface area analyzer, and the sample was tested by a N2 adsorption–desorption technique. The Brunauer–Emmett–Teller (BET) equation was used to calculate the specific surface area of the sample, and the BJH model was used for pore size analysis.

FTIR Analysis

FTIR spectra were recorded in the wavenumber range of 400–4000 cm using the KBr pellet method.

Temperature/pH Response Experiments

Three types of optimum gel spheres (optimum CA gel spheres, optimum NaCl modified gel spheres, and optimum dual-response gel spheres) were investigated for swelling. Thirty vacuum-dried spheres of the three optimum gel types were put into solutions of different temperatures (5, 20, 30, 40, and 50 °C) or pH (2, 4, 6, 8, 10, and 12) to investigate the variation of swelling with temperature and pH, respectively. The temperature values of the dispersions were adjusted with a refrigerator and a water bath thermostatic oscillator. The pH values were adjusted with a 0.1 M HCl solution and a 0.1 M NaOH solution.

The optimum dual-response nitrifying bacteria gel spheres (20% dosing rate) after three days of activation and the domesticated sludge with the same amount of bacteria were put into the synthetic wastewater (Table for specific components) at different temperatures (4–50 °C) or pH (4–10). Ammonia removal rate within 5 h was used as a performance indicator to assess tolerance to external environmental changes.

Table 9

Composition of Synthetic Wastewater

components		concentration (mg/L)
NH4+–N	50
NaHCO3	600
trace elements (0.25 mL/L)	ZnSO4·7H2O	0.11
	Na2MoO4·2H2O	0.11
	CoCl2·6H2O	0.12
	MnSO4·H2O	0.117
	NiCl2·6H2O	0.104
	Na2HPO4	0.57
	FeCl3·6H2O	0.124