Performance and characterization of a non-sintered zeolite porous filter for the simultaneous removal of nitrogen and phosphorus in a biological aerated filter (BAF)

Abstract

A novel non-sintered zeolite porous filter (ZPF) and commercially available ceramsite (CAC) are used to investigate the simultaneous removal of nitrogen and phosphorus from city wastewater treated by biological aerated filter (BAF) reactors. The chemical and physical characteristics of ZPF and CAC are measured. ZPF has a higher porosity and larger total surface area than CAC. In addition, the interconnected porous structure obtained for ZPF is suitable for microbial biofilm growth. In the present study, the influence of the hydraulic retention time on the removal of phosphorus (PO₄³⁻), total nitrogen (TN), ammonia nitrogen (NH₃–N), and total organic carbon (TOC) are studied. The results show that the ZPF BAF performs much better than the CAC BAF. Microbial biofilm morphology also shows that more microorganisms are loaded in the ZPF BAF. The polymerase chain reaction and denaturing gradient gel electrophoresis and sequence analysis of 16S RNA gene fragments show that Comamonas testosteroni, uncultured Comamonas sp., and uncultured Nitrospira sp. are primarily detected in the ZPF BAF and this attached growth benefits the simultaneous nitrification and denitrification performance of the ZPF BAF. Thus, ZPF has potential use as novel material for the simultaneous removal of nitrogen and phosphorus of BAF in wastewater treatment.

---

1. Introduction

Eutrophication has become one of the most serious environmental problems throughout the world. Its leading cause is usually the accumulation of nitrogen and phosphorus in relatively stagnant water. Thus, the removal of nitrogen and phosphorus from wastewater prior to discharge and from eutrophicated natural water is essential.^1,2

The biological aerated filter (BAF) is one of the main biological treatment technologies. It was designed with the principal objective of removing easily or moderately biodegradable compounds.^3,4 The filter medium has a significant role in the BAF.^3,4 Recently, the application of efficient solid materials, involving solid wastes, natural materials, and synthesized materials, in the removal of nitrogen and phosphorus from wastewater has been widely investigated.^5–9 Synthesized materials and natural clays, especially fly ash, waste cement, waste construction, and zeolites, have been proven to be efficient in phosphorus and nitrogen removal.^5–9 Both phosphorus and nitrogen have to be removed from wastewater, and their simultaneous removal using one material has been rarely reported hitherto.^5–9 The merit of using only one material to simultaneously remove phosphorus and nitrogen from wastewater is obvious.^10,11 Only recently, a few researchers have conducted studies to enhance the efficiency of biological treatment. Cui¹² investigated the combined ozone oxidation and BAF processes in the treatment of cyanide containing electroplating wastewater. Zhang¹³ reported the removal of 2,4,6-trinitrotoluene (TNT) red water. These filter media can efficiently remove NH₃–N and TNT. However, studies conducted on the simultaneously removal of nitrogen and phosphorus from wastewater remains limited. In China, commercially available ceramsite (CAC) is currently used for wastewater treatment in city wastewater treatment plants. Raw materials found in CAC generally consist of gangue, river sediment, and raw clay. These materials are produced by the development of CAC calcined at high temperatures (>1200 °C). The glazed glass surface of CAC is ineffectual for the formation of microorganism biofilm, so that phosphorus and nitrogen removal is inefficient.³ It is important to find suitable materials that can replace CAC as soon as possible.

Zeolite is a natural porous mineral described as crystalline hydrated aluminosilicates. Inside the structural framework of zeolite, alkali or alkaline-earth cations are reversibly fixed in the cavities and can easily be exchanged by surrounding positive ions. It has a high storage capacity, low price, and strong adsorption ability. The advantages of a zeolite BAF are improved microorganism growth and reproduction and ammonia nitrogen shock resistance.^10,14 Zhang¹⁴ reported that the number of nitrobacteria growing on the surface of zeolite is 3.5 times higher than the amount of growing on sandy filtering media, resulting in the 70–90% removal of NH₃–N and NO₃–N. Waste cement is a binder, which pertains to a substance that sets, hardens, and fastens materials together. The word “cement” derives from the Latin “caementicium” which describes masonry that resembles modern concrete and that is made from crushed rock and burnt lime, which serves as binder.^15,16 It has been used successfully for the removal of phosphate in wastewater treatment.^15,16

In the present study, the ZPF is prepared by steam curing the mixture of zeolite, waste cement, and aluminum powder. The required air pores for the production of ZPF can be obtained using expanded lightweight aggregates, or the formation of air pores in waste cement paste can be facilitated through the addition of gas-generating agents.^17,18 The formation of interior porosity improves microbial growth. In other words, the creation of pores ensures the formation of an interior pore structure, which improves the microbial breeding.^12–14 This study introduces a new promising method of filter media development for future biofilter applications using zeolite, waste cement, and aluminum powder. To the best of our knowledge, only few studies focus on the use of aluminum powder as a pore-creating material to develop non-sintered porous filter media. This study investigates the porosity characteristics of filter media using X-ray microtomography (micro-CT). Its popularity can be attributed to its ability to provide non-destructive, precise quantitative and qualitative information on the 3D morphology of porous materials. Therefore, micro-CT has an important advantage over other techniques, such as mercury and flow porosimetry.^19–21 However, few researchers use this method involving the application of porous filter media.

This research has the following five objectives: (i) to develop a new non-sintered zeolite porous filter (ZPF), (ii) to investigate the effect of hydraulic retention time (HRT) on the removal of NH₃–N, TN, PO₄³⁻, NO_x–N and TOC in the ZPF and CAC BAFs, (iii) to investigate the physical characteristics of ZPF such as porosity, (iv) to evaluate filter media samples from ZPF BAF, which are covered with microbial biofilm, and (v) to characterize the microbial community structures in the ZPF BAF and further reveal the main contributors to TN removal using polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) coupled with the sequence analysis of 16S rRNA gene fragments.

2. Materials and methods

2.1. Materials

The zeolite powder was obtained from Xuan Cheng, Anhui province, China. Aluminum powder was obtained from Hefei Chemistry Limited Making Company, Hefei City, Anhui province, China. Cement was obtained from Anhui Conch cement Company Limited, Wuhu City, Anhui province, China. CAC was obtained from the city of Ma'an Shan, Anhui Province, China. The chemical analyses of aluminum powder, CAC, zeolite powder, and cement are presented in Table S1.†

2.2. Preparation of ZPF

Zeolite powder was used as the main material; waste cement and aluminum powder (as pore-forming material) served as additives and were mixed with zeolite powder to produce ZPF. Five factors were taken into account with regard to the ZPF preparation: the weight ratio of zeolite powder, waste cement, water content, aluminum powder, and the steam curing temperature. The influences of these five factors on the properties of ZPF were discussed. Previous studies by our group showed that the optimal parameters for the production of ZPF are as follows: correspondence to aluminum powder: 0.12%, waste cement: 32%, and water content: 52% material. The optimal steam temperature and time for ZPF are 123 °C and 480 min, respectively. Photographs of ZPF and CAC are shown in Fig. S1(a) and (b).† The resulting ZPF is found to meet the corresponding regulatory levels, as shown in Table 1.

Table 1 The regulatory levels of ceramics in several of filter media. (i) grain diameter, d/mm; (ii) silt carrying capacity, C_s/%; (iii) void fraction, υ/%; (iv) specific surface area, S_w/cm² g⁻¹; (v) piled density, ρ_p/g cm⁻³; (vi) apparent density, ρ_ap/g cm⁻³; (vii) compression strength, N; (viii) porosity, P/%^a

Filter media	i	ii	iii	iv	v	vi	vii	viii	Reference
a Zeolite porous filter – ZPF; commercially available ceramsite – CAC; red mud particle electrodes – RMPE; sludge ceramsite – SC; sorption functional media – SFM; sludge fly ash ceramic particles – SFCP; blast furnace dust clay sodium silicate ceramic particles – BCSCP; natural zeolite – NZ; ceramic granular – CG; Guang zhou ceramsite – GZC; Jiang xi ceramsite – JXC; Shan xi activated carbon – SXAC.
ZPF	20–25	2.18	74.7	5.953 × 10^5	0.329	1.30	41–47	29.55	This study
CAC	4–6	≤1	≥42	≥2 × 10^4	≤1.0	1.4–1.8	≥87	3.46	This study
RMPE	3–5	—	—	—	0.621	1.058	—	38.17	28
SC	3.5–7	—	—	9.37 × 10^5	0.668	1.251	—	42.52	29
SFM	3–5	—	—	3.617 × 10^5	0.674	1.115	—	42.17	30
SFCP	3–6	—	—	0.899 × 10^5	1.32	2.11	—	37.7	31
BCSCP	—	—	—	0.3843 × 10^5	0.859	2.098	—	54.50	32
NZ	3–5	—	—	0.684 × 10^5	1.015	2.316	—	43.83	33
CG	3–5	—	—	0.411 × 10^5	1.56	0.95	—	>39.0	34
GZC	3.5–7	—	—	3.59 × 10^5	0.76	1.272	—	37.6	34
JXC	3.5–7	—	—	5.62 × 10^5	0.603	0.931	—	36.1	34
SXAC	3–4.5	—	—	68.69 × 10^5	0.374	0.741	—	50.2	34

---

The preparation of ZPF includes the following steps:

(1) The mixture of zeolite powder, waste cement, and aluminum powder with a designed mass ratio was introduced in a small coating machine for the rolling granulation with a diameter of 20–50 mm particles.

(2) The compound was steam cured at 123 °C for 480 min.

(3) The ZPF was cooled down to room temperature for over 24 h.

2.3. Physical characterization of ZPF and CAC

X-ray photoelectron spectroscopy (XPS, Thermo Escalab 250 XI, Thermo Fisher Inc., Waltham, MA, USA) was used to analyze the surface elements. The chemical composition of CAC, zeolite powder, aluminum powder, and waste cement was measured on a Shimadzu XRF-1800 (Shimadzu Corporation, Japan) with Rh radiation. The physical characteristics of ZPF were evaluated in accordance with the sandstone pore structure method of image analysis.²² For the analysis of biological structures, ZPF and CAC were gilt with pores, and their surface morphologies were examined using a scanning electron microscope (SEM, Philips XL30 ESEM). The microscopic observation of the morphology of the microbial biofilm was conducted using a U-RFL-T Olympus biological microscope (Olympus Corporation, Japan). The porosities of the ZPF specimens were determined using micro-CT (μCT 40, Scanco Medical, Brüttisellen, Switzerland). The ZPF specimens were scanned in air at 70 kV and 114 μA, with an isotropic voxel size of 30 μm. A region of interest (ROI) was defined for each gray-value image, in accordance with the exact circumference of the sample. A binary global threshold was applied to each sample, and Scanco built-in reconstruction algorithms were used to derive the porosity and to construct the 3D image.^19–21 The growth of microbial biofilm was determined according methods available in the literature.²³ The microbial populations were measured in accordance with previous literature.^24,25

2.4. Experimental setup

A schematic of the two pilot-scale BAFs used in the experiments is given in Fig. S2.† BAF was constructed with a polyvinyl chloride pipe 60 mm in diameter and 1500 mm in media depth. The two BAFs were separately packed with ZPF and CAC. Wastewater was pumped into the bottom of ZPF–BAF and CAC–BAF with peristaltic pumps. ZPF–BAF and CAC–BAF were monitored for 1.5 years. The operating conditions of ZPF–BAF and CAC–BAF were identical (Table 2). The air–water ratio (A/W) was 3 : 1 (dissolved oxygen [DO] > 4.00 mg L⁻¹). Air was introduced into ZPF–BAF and CAC–BAF with an air diffuser, and the air flow rate was monitored with an air flow meter. Four HRTs of 0.5, 1.75, 3.5, and 7 h were employed. Wastewater samples were collected from the inlet and outlet pipes of ZPF–BAF and CAC–BAF daily for a 1.5 year observation period. All wastewater samples were stored in a refrigerator at 0 ± 1 °C for less than 24 h before analysis. Analytical methods were employed to measure NH₃–N, NO_x–N, and PO₄³⁻ in accordance with Chinese EPA standards.²⁶ A TOC/TN (Jena Multi N/C 2100) analyzer was used to measure TOC and TN.

Table 2 Operating conditions

	Operating conditions			Water quality indexes^a
	pH	T (°C)	HRT (h)	TOC	NH3–N	P
a TOC, NH3–N, and PO4^3− values are given as influent concentrations in mg L^−1.
Stage one	6.5(±0.5)	25–30 °C	7(±0.5)	26–31	9.91–10.05	0.51–0.58
Stage two	6.1(±0.5)	25–30 °C	3.5(±0.8)	27–33	9.82–10.32	0.49–0.60
Stage three	6.7(±0.5)	20–25 °C	1.75(±0.5)	25–30	9.67–10.23	0.46–0.53
Stage four	6.9(±0.5)	18–23 °C	0.5(±0.1)	24–38	9.65–10.45	0.53–0.58

---

2.5. Wastewater quality analysis

The performance efficiencies of ZPF–BAF and CAC–BAF in simultaneous nitrification and denitrification (SND) were calculated as follows (1):[NH₃–N]_Influ – influent concentration (mg L⁻¹), [NH₃–N]_Efflu – effluent concentration (mg L⁻¹), [NO₂–N]_Efflu – effluent concentration (mg L⁻¹), [NO₃–N]_Efflu – effluent concentration (mg L⁻¹).²⁷

3. Results and discussion

3.1. Characterization of ZPF and several filter media

Table 1 shows the physical properties of ZPF and several filter media available in the literature.^28–34 As the core component of BAF, filter media are used for microbial biofilm attachment, which is of great importance for wastewater treatment. Two reasons underlie the use of BAFs. First, microbial biofilms are difficult to load on many types of filter media. Second, stoppage and clogging of BAF easily occur.³⁵ However, compared with several filter media, ZPF exhibits a lower bulk density, higher specific surface area, higher compression strength, higher porosity for easy backwash process, and rougher surface for microbial biofilm growth. All these physical properties are associated with the chemical composition of the material. Aluminum powder is present at 0.12%, which is propitious for manufacturing porous light ZPF; aluminum powder sets off a chemical reaction under alkaline conditions to release hydrogen during the autoclaving test.^36,37 Thus, the use of ZPF would enable BAF to achieve easy start up and superior substrate removal compared with conventional filter media.

3.2. Micro-CT based pore characterization of ZPF and CAC

Porosity is a very important factor for ensuring successful SND in a given ZPF BAF process because of the uneven distribution of DO and nutrient substance inside the ZPF's pores, which allows the simultaneous proliferation of nitrifying and denitrifying bacteria.^34,38 In other words, the structure of ZPF, including the dimension and morphology of the pores, is critical in facilitating the nutrition, oxygen conduction, and microbial biofilm growth in the pores.^34,38 Fig. 1(A) shows the 3D micro-CT reconstruction of ZPF. The silver cube particles in the ZPF sections denote the mold of ZPF. It can be seen that ZPF has a rough surface and is suitable to microbial biofilm growth. Aluminum powder is required to make the ZPF porous and rough, which can produce tiny apertures inside the ZPF.³⁴ Fig. 1(B) and (C) show a 0.6 mm-thick cross-section (i.e., stack of 20 scan slices) of ZPF. It is also observed that the pores are large, with diameters of approximately 500–2000 μm, and the yellow arrows represent the pores of ZPF. Micro-CT analysis of ZPF shows that the ZPF has a porosity of 29.55%, with many unevenly distributed pores, which are suitable for the attached growth of microbial biofilm. The analysis also shows that ZPF has cube-shaped pores that are interconnected with one another by window-like openings (Fig. 1(D)), which results in a large surface area, leads to high biofilm biomass, and induces high bioactivity.^34,39,40 Fig. 1(E) and (F) show a 3D reconstruction and 0.24 mm-thick cross-section (i.e., stack of 20 scan slices, CAC was scanned with 12 μm isotropic voxel size) of CAC, respectively. The porosity was determined to be only 3.46%.

Fig. 1 Micro-CT of ZPF and CAC. (A) 3 dimensional reconstruction of ZPF; (B) 20 slices (0.6 mm) cross-section of ZPF; (C) 3 dimensional reconstruction of ZPF; (D) 20 slices (0.6 mm) cross-section of ZPF; (E) 3 dimensional reconstruction of CAC; (F) 20 slices (0.24 mm) cross-section of CAC.

3.3. Cast thin section of ZPF and CAC

The open porosity of the ZPF surface is beneficial for microbial biofilm fixed on the surface. The microbial biofilm attached to the ZPF surface is a highly hydrophilic substance under conditions of constant flow of wastewater. Microbial biofilm can attach to the surface of the ZPF and be transferred through the ZPF into the interior pores (Fig. 2(A)). Wastewater flow can be divided into three layers: the aqueous adhesion layer, the aerobic layer, and the anoxic layer (Fig. 2(B)). Microbial biofilm leads to this zonation and creates the conditions for SND. SND requires an aerobic zone in the microbial biofilm or floc for nitrification and an anoxic substrate-rich interior for denitrification.^34,38 CAC has low and open porosity. Microbial biofilm cannot be transported through the CAC into the interior of closed pores (Fig. 2(C) and (D)). The wastewater flow through CAC can be separated into two layers: the aqueous adhesion layer and the aerobic layer (Fig. 2(C) and (D)). The CAC's smooth surfaces demonstrate no effect on the biofilm biomass of grown cells when used during the early stages of BAF filtration, so that CAC has poor efficiency.

Fig. 2 Internal structures of ZPF and CAC. (A) Internal porosity of ZPF; (B) diluted oxygen gradient within ZPF; (C) internal porosity of CAC; (D) diluted oxygen gradient within CAC; (flowing wastewater layer (part-1); aqueous adhesion layer (part-2); aerobic layer (part-3); and anoxic layer (part-4)).

In a previous report, stained epoxy resin is used to impregnate the pore space of rock samples. However, few researchers use this method by applying porous filter media.⁴¹ To obtain direct evidence of microbial biofilm in the porous structure of the ZPF, rubber casting experiments were used to generate quantitative size and shape data from the pores in the thin section.⁴¹ Fig. 3(A) show the intergranular pore textures observed in the thin section. The block sections denote the molded composite particles (i.e., zeolite powder, waste cement, and aluminum powder), while light yellow arrows in Fig. 3(A) represent the intergranular pores of ZPF. This demonstrates that ZPF has a large volume of internal porosity, in which microbes can be accommodated. The size of these interconnected pores is approximately 500–2000 μm, and most bacteria are about 0.5 μm in diameter.³ Thus, the microbial biofilm can achieve a sustained growth of population in the open porosity of ZPF. When impregnated with blue-dyed epoxy (shown with red arrows in Fig. 3(B)), the fully interconnected porosity with a diameter of 500 μm can be determined from the gray section (i.e., red arrows in Fig. 3(B)). In Fig. 3(C), CAC also has a low porosity, and the gray sections denote CAC (i.e., light yellow arrows in Fig. 3(C)). Some closed pores, where microbial biofilm growth is not allowed, are shown (i.e., cream white arrows in Fig. 3(D)). Thus, simultaneous removal of nitrogen and phosphorus also shows poor efficiency. In fact, the blue-dyed epoxy in the porosity system must overcome the capillary resistance of the channel size before they enter the porous space, indicating the narrow composition of the pores.⁴¹

Fig. 3 Porosity of ZPF and CAC. (A) Internal surface of the ZPF, (B) filling of ZPF (red arrow – open pores), (C) internal porosity of CAC, (D) filling of CAC (cream white arrow – close pores) 250 μm.

3.4. Performance of TOC and NH₃–N removal in the ZPF and CAC BAFs

HRT is a crucial parameter in biological wastewater treatment.⁴² The effect of HRT (7–0.5 h) on TOC and NH₃–N removal rate is shown in Fig. 4(A) and (B) and Tables 2 and 3. First, the TOC removal rate decreases rapidly when HRT decreases from 7 h to 0.5 h. It is likely that heterotrophic microorganisms in the exterior layer of the microbial biofilm and suspension are highly shocked by the fast flow rate of air and wastewater, which strongly shear presses with short HRT. The outer layer of the microbial biofilm is easily detached, and the heterotrophic microorganisms in the suspension are easily washed out from the BAF because of the high water velocity. Thus, the organic substance in the wastewater can not be degraded effectively.^42,43 As shown in Fig. 4(A), the TOC removal in the ZPF BAF is greater than CAC BAF. The main reason is that the surface and internal pore structure of ZPF is uniform and well developed, as a result of soluble organic matter, nutrient substances, and tiny suspended particles that reach the deep holes and makes the best use of ZPF bodies, which enhance the mass transfer efficiencies. The available space for microorganism growth, with the mass transfer rate of dissolved oxygen and the probability of the dispersion of biofilm biomass, is also increased by the extension of soluble pollutants deep into the holes in ZPF, which results in a more reasonable application of the ZPF and improves the effectiveness of biofiltration.^34,44,45

Fig. 4 Influent and effluent concentrations and removal efficiency of TOC, NH₃–N, TN, and PO₄³⁻ over time.

Table 3 Average rate removals for ZPF and CAC in BAF at different HRTs

	TOC^a				NH3–N^a
a All values are given as average removal rates in %.
HRT (h)	0.5	1.75	3.5	7	0.5	1.75	3.5	7
Material
ZPF	42.1	50.6	76	77.9	78.5	87.6	89.9	96.3
CAC	27.2	50.1	68	63.9	50.5	53.9	49.4	91.2

---

	TN^a				PO4^3−^a
HRT (h)	0.5	1.75	3.5	7	0.5	1.75	3.5	7
Material
ZPF	45	47.9	52.5	67	58.5	72.9	78.2	86.9
CAC	24.4	23.5	25.6	39.3	13.1	33	42.8	44.3

---

As shown in Fig. 4(B) and Tables 2 and 3, both BAFs have excellent NH₃–N removal when HRT is 7 h. When HRT is 0.5 h, NH₃–N is not fully nitrified before being discharged from the BAFs, compared with longer HRTs. With an HRT of 0.5 h, the nitrobacteria do not have sufficient time to nitrify NH₃–N. Moreover, the dissolved oxygen in the effluent at 0.5 h is slightly lower than an HRT of 1.75 h. Less dissolved oxygen in the effluent can cause a slight decrease of NH₃–N removal efficiency. Finally, the possible competition between heterotrophic and autotrophic bacteria in BAFs for the substrates, dissolved oxygen, and inhabitation area can decrease its efficiency. A higher organic loading induced by the increase in hydraulic loading can favor heterotrophic bacteria over autotrophic bacteria.^34,42,43 Thus, nitrification is inhibited, and NH₃–N removal decreases rapidly. Fig. 4(B) shows that ZPF BAF has higher NH₃–N removal than CAC BAF. These findings show the high specific surface area (59.53 m² g⁻¹) and higher porosity of ZPF (29.55%), which in turn promotes greater active microbial biofilm formation than CAC. Thus, the porosity of the exposed ZPF surface and the microbial biofilm adhesion on the open surface become fixed. Another reason is that biological methods do not respond well to shock loads of NH₃–N, which cannot withstand peaks over the discharging levels, which may regularly appear in the effluent NH₃–N concentrations. ZPF has a high cation exchange capacity and is applied in the removal of NH₃–N from wastewater.¹⁰

3.5. Influence of HRTs on TN removal

The time course of TN concentrations at HRTs is shown in Fig. 4(C) and Tables 2 and 3 For TN removal, different efficiencies are observed from Fig. 4(C), when HRTs are increased from 7 h to 0.5 h. At HRTs of 3.5 and 7 h, the average TN removal efficiencies are 52.5% and 67%. These results reveal that, under longer HRT, the ecological structure of the microbial system keeps a dynamic balance in terms of composition and spatial distribution in the microbial biofilm, which accordingly result in high TN removal efficiencies.^34,42,43 Another reason is that the existing grads of DO and substrate concentrations result in the formation of different microenvironments in microbial biofilms, which facilitate SND in the ZPF BAF. SND is relevant to the amounts and activities of anaerobic microorganisms; thus, SND is indirectly controlled by the thickness of anaerobic layers in microbial biofilms. The TN removal rate decreases rapidly as HRT decreases from 1.75 h to 0.5 h because a decrease in HRT leads to a stronger scour for media surfaces, which weakens the thickness of anaerobic layers. All this contributes to the decrease of TN removal efficiencies. As can be seen in Fig. 4(C), the TN removal performance of the ZPF BAF is higher than that of the CAC BAF. Under the same conditions and different permeable capacities of the microbial biofilm layers of the filter media, which are greatly affected by the porous structure of the ZPF, the contact-reaction potential of the nitrifying and denitrifying bacteria and pollutants under the anoxic condition can influence the nitrification and denitrification removal.^34,42,43

3.6. Performance of PO₄³⁻ reduction in the ZPF and CAC BAFs

In terms of PO₄³⁻ removal, the rates at different HRTs can be seen in Fig. 4(D) and Tables 2 and 3, where the ZPF BAF displays higher PO₄³⁻ removal than the CAC BAF. Given that ZPF is rich in calcium oxides (Table S1†), the crystallization may serve as promising wastewater for phosphate removal.^44,45 The mechanism of crystallization involves the reaction of calcium, phosphate, and hydroxyl ions with one another to form alkaline hydroxyapatite (HAP-Ca₅(OH)(PO₄)₃). The PO₄³⁻ in the aqueous solution phase can be continuously removed from the solution by bonding with the calcium (Ca²⁺) and hydroxyl ion (OH⁻). HAP once formed is not easily released in a short time because of its low solubility, which can attain the goal of phosphate removal.^44,45 SiO₂ and calcium oxides constitute 60.24% and 0.41% of CAC composition, respectively (Table S1†). The CAC is high in SiO₂; however, SiO₂ has been reported to show low efficiency for phosphate removal.^44,45

3.7. ZPF–BAF and CAC–BAF efficiencies of simultaneous nitrification and denitrification at a HRT of 7 h

Fig. 4(B) and 5 show the influent and effluent concentrations of NH₃–N and NO_x–N, and the efficiency of SND in ZPF–BAF and CAC–BAF at 7 h HRT. Greater nitrite and nitrate amounts accumulated in CAC–BAF than in ZPF–BAF (Fig. 5(A)). The efficiency of the SND of ZPF–BAF was also much higher than that of CAC–BAF (Fig. 5(B)). Ni (ref. 38 and 46) previously reported that DO importantly affects the distribution of the microbial population in microbial biofilm. DO penetration depth in microbial biofilm influences the conversion rates of different components and therefore the overall nutrient removal efficiency. The competition between autotrophs and heterotrophs for DO and space in microbial biofilm occurs when DO levels are insufficient. Organic substrates can penetrate into the microbial biofilm of the pores (filter media) because of the high substrate concentration in wastewater. However, DO is present only on the outer layers of the microbial biofilm. Thus, DO action involves SND in ZPF. In this research, the biofilm biomass was 60.2 mg TN per g for ZPF and 2.2 mg TN per g for CAC. High biofilm biomass denotes that high bacterial amounts are available to achieve SND of high efficiency in ZPF–BAF. In addition, the morphological and structural characteristics of ZPF (Fig. 1, 3, and 7) indicate that the special characteristics of ZPF are advantageous to microbial biofilm growth.

Fig. 5 ZPF–BAF and CAC–BAF the efficiency of simultaneous nitrification and denitrification at HRT of 7 h.

3.8. X-ray photoelectron spectroscopy (XPS)

A possible mechanism called surface precipitation has been proposed to explain the synergistic adsorption of metal ions, oxyanions, or metal oxides.⁴⁷ Given that XPS is widely used to asses the interaction of inorganic ions with solid surfaces, it is used to find supporting evidence for this hypothesis. Fig. 6(A) shows the results of XPS analysis on the surface of ZPF after use in the BAF. A binding energy peak located at 133.6 eV is designated as P 2p, and the species of phosphorus is HPO₄²⁻, which corresponds to the handbook of XPS.⁴⁷ Previous reports^48–50 show that the Ca–P precipitation has two routes:

(1) Ca²⁺ + HPO₄²⁻ + 2H₂O → CaHPO₄·2H₂O

(2) 10Ca²⁺ + 6HPO₄²⁻ + 8OH⁻ → Ca₁₀(PO₄)₆(OH)₂↓ + H₂O

Fig. 6 X-ray photoelectron spectroscopy (XPS).

The peak of P 2p proves the existence of HPO₄²⁻, but the dominant route remains uncertain. Fig. 6(B) exhibits the Ca 2p doublets peaks (Ca 2p_3/2 at 347.5 and 2p_1/2 at 352.0 eV), which first support the assumption that Ca–P precipitate is the dominant component in the corresponding collected ZPF.^48–50 Second, the binding energy of Ca 2p_3/2 shows that calcium exists as a divalent ion.^48–50 However, the appearance of Ca 2p doublets peaks indicates that CaHPO₄ is not the sole Ca–P precipitation. The area ratio of Ca 2p_3/2 and 2p_1/2 shows that CaHPO₄ is the dominant component, and HAP is another component. Their ratio is about 2.25, according to the peak area in Fig. 6(B).

3.9. SEM analysis

Several SEM images for the ordered structure and microbial biofilm biomass growth of ZPF and CAC are shown in Fig. 7. In Fig. 7(A), the appearance of the rough surface on ZPF is described as a structure with a coral-like porosity that provides shelter from the wastewater shear forces and microstructure of the internal cross-section of the ZPF. It also indicates that the prepared ZPF has a high porosity with rectangular pore openings. The high porosity of ZPF is suitable to serve as a biomedium in BAF. ZPF after use in the BAF are illustrated in Fig. 7(B). The microbial biofilm is found overlain on the surface of ZPF. Two kinds of biological bacteria can be observed: chain-shaped and filamentous bacteria.⁵¹ The micrograph of CAC clearly shows pores with a diameter range of 6.0–10.0 μm that are irregularly distributed on the surface (Fig. 7(C)). The microstructure in Fig. 7(D) suggests that a small amount of microorganisms are immobilized on the inner and outer surfaces of the pores of CAC.

4. Characterization of microbial community in the ZPF BAF

The biological microscope shows a thick microorganism biofilm in the ZPF BAF (Fig. 7). The abundance of biofilm biomass (e.g., coccoid-shaped, bacillus, and filamentous bacteria) and extracellular polymeric substances supports the high removal efficiency of pollutants in the wastewater.⁵¹ To understand the microbial community in the ZPF BAF, PCR-DGGE is used to identify the structure of the bacterial community in the ZPF BAF.^52,53 Fig. 8 and 9 show the DGGE profiles of amplified 16S rRNA fragments from the ZPF samples. Each of the distinguishable bands in the separation pattern represents an individual bacterial species.⁵⁴ In Fig. 8, some bands (i.e., NO 1, NO 2, NO 3, NO 4, NO 5, NO 6, NO 7, NO 8, NO 9, NO 10, NO 11, and NO 12) are found in Lane 1, Lane 2, Lane 3, demonstrating that some dominant bacterial groups are contained for the treatment of wastewater. To identify the dominant species in the bacterial community, the bands were excised from the gel, sequenced, and compared with GenBank. Fig. 9 shows the phylogenetic trees for the partial bacterial 16S rRNA sequences. Sequence NO 3 shows a similarity with uncultured Comamonas sp. 16S rRNA (JF808871.1). Sequence NO 4 shows a similarity with Tychonema sp. 16S rRNA (KM019964.1). In addition, sequence NO 5 (NO 6) is most closely related to Comamonas testosteroni 16S rRNA (KP943129.1). Sequence NO 8 shows a similarity with Ideonella sp. 16S rRNA (KF556698.1). Sequence NO 9 shows a similarity with uncultured Nitrospira sp. 16S rRNA (KJ480931.1). Sequence NO 12 shows a similarity with Gemmatimonas sp. 16S rRNA (KF481682.1). The Comamonas are a genus of Proteobacteria, and like all Proteobacteria, they are Gram-negative bacteria. The Comamonas are aerobic organisms that are motile via bipolar or polar tufts of flagella.⁵⁵ The high abundance of Comamonas is also found in nitrate-removing microbial communities presented in river wetlands and others.^56,57 Comamonas testosteroni is a Gram-negative soil bacterium with heterotrophic nitrification-aerobic denitrification and dephosphorization functions for wastewater treatment.⁵⁸ Ideonella is a genus of Comamonadaceae bacteria. Comamonadaceae are a family of the Betaproteobacteria, and like all Proteobacteria, they are Gram-negative. They are also aerobic, and most of the species are motile via flagella.⁵⁸ Gemmatimonadetes are a family of bacteria with their own phylum (Gemmatimonadetes). This bacterium makes up about 2% of soil bacterial communities and has been identified as one of the top nine phyla found in soils.⁵⁹ Nitrospira sp. is part of a nitrification process which is important in the biogeochemical nitrogen cycle. It has been proved that nitrification involves the oxidation of ammonia into nitrite by autotrophic bacteria of the genus Nitrosomonas and the oxidation of nitrite into nitrate by bacteria in the genus Nitrospira.⁶⁰

Fig. 7 SEM images of ZPF/CAC before and after microbial biofilm loading. (A) Raw external surface of ZPF, (B) microbial biofilm load on the external surface of ZPF, (C) raw external surface of CAC, (D) microbial biofilm load on the external surface of CAC.

Fig. 8 DGGE profiles of bacterial and archea 16S rRNA genes from the ZPF BAF.

Fig. 9 Neighbor-joining tree showing the phylogenetic identities of the 16S rRNA of DGGE sequences obtained from the DGGE bands.

4.1. Economic analysis for the development of ZPF and CAC

As discussed above, using BAF loaded with ZPF is more highly efficient in nitrogen and phosphorus removal than using CAC. This advantage is helpful for reducing operational cost, mitigating the eutrophication problem, and improving operational stability. For further engineering expansion, the benefits of ZPF must be evaluated from an economic viewpoint. In China, CAC cost was determined to be approximately 1200 RMB per t. Such value was particularly based on the use of CAC as filter media in wastewater treatment plants. However, developing CAC calcined at high temperatures (>1200 °C) is a resource-wasting method. Meanwhile, the corresponding ZPF investment cost was estimated to be approximately 197.4 RMB per t in China. The main reason behind these findings is that waste cement is a construction waste material. Previous statistics showed that construction waste materials in China have occupied 30–40% of the whole city garbage in the last few years. These waste materials greatly contribute to city pollution, but they are easily available and cost effective.⁶¹ In China, zeolite exhibits lower cost, which is approximately 180 RMB per t. Aluminum powder cost has also been evaluated to be approximately 14 500 RMB per t. ZPF was prepared using pore-forming material-aluminum powder (0.12%). The corresponding investment cost of CAC was six times higher than that of ZPF. We believe that the proposed advantage of utilizing substantial quantities of ZPF is the low cost and the potential environmental implications that can revolutionize the mitigation of the eutrophication problem.

5. Conclusions

ZPF has a larger specific surface area, higher porosity, and rougher surface, which not only provides a larger inhabitation area for the microorganism biofilm but also has a shielding effect on the attachment of the microorganism biofilms, thereby preventing them from being scoured by shearing forces. The microorganism biofilms were successfully immobilized on the surface of the ZPF. PCR-DGGE analysis also reveals that Comamonas testosteroni bacteria and uncultured Nitrospira sp. are found in ZPF BAF during nitrogen and phosphorus removal. In addition, the micro-CT of the 3D pore structure also shows more details of the pore structure and is more accurate in characterizing porosity. ZPF BAF exhibits the highest phosphate removal efficiency. The removal mechanism of phosphate using ZPF includes the crystalline precipitation of HAP, as confirmed by XPS analyses. ZPF, with a high content of calcium, has the potential to be a novel highly efficient and cost-effective material for the advanced treatment of nitrogen and phosphorus.

Acknowledgements

We gratefully acknowledge the support by the National Natural Science Foundation of China (41130206, 41472047, 41402030), and Anhui Provincial Education Natural Science Foundation (KJ2014ZD23).

References

1. X. Zheng, Y. L. Su, Y. G. Chen, Y. Y. Wei, M. Li and H. N. Huang, RSC Adv., 2014, 4, 45953–45959.
2. Z. C. Kadirova, M. Hojamberdiev, L. Bo, R. Hojiyev and K. Okada, Journal of Water Process Engineering, 2015, 8, 151–159.
3. F. Osorioand and E. Hontoria, J. Environ. Manage., 2002, 65, 79–84.
4. F. Sun and W. L. Sun, Chem. Eng. J., 2012, 210, 263–270.
5. B. H. Zhang, D. Y. Wu, C. Wang, S. B. He, Z. J. Zhang and H. N. Kong, J. Environ. Sci., 2007, 19, 540–545.
6. A. Chen, Y. Chen, C. Ding, H. Liang and B. Yang, RSC Adv., 2015, 5, 59326–59334.
7. F. Ni, J. S. He, Y. B. Wang and Z. K. Luan, Journal of Water Process Engineering, 2015, 6, 158–165.
8. S. S. Yang, W. Q. Guo, Y. D. Chen, X. J. Zhou, H. S. Zheng, X. C. Feng, R. L. Yin and N. Q. Ren, RSC Adv., 2014, 4, 52892–52897.
9. N. M. Agyeia, C. A. Strydom and J. H. Potgieter, Cem. Concr. Res., 2002, 32, 1889–1897.
10. J. G. Chen, H. N. Kong, D. Y. Wu, Z. B. Hu, Z. S. Wang and Y. H. Wang, J. Colloid Interface Sci., 2006, 300, 491–497.
11. J. Werner, B. Besser, C. Brandes, S. Kroll and K. Rezwan, Journal of Water Process Engineering, 2014, 4, 201–211.
12. J. Q. Cui, X. J. Wang, Y. L. Yuan, X. W. Guo, X. Y. Gu and L. Jian, Chem. Eng. J., 2014, 241, 184–189.
13. M. H. Zhang, G. H. Liu, K. Song, Z. Y. Wang, Q. L. Zhao, S. J. Li and Z. F. Ye, Chem. Eng. J., 2015, 259, 876–884.
14. X. L. Zhang, S. Zhang, F. He, S. P. Cheng, W. Liang and Z. B. Wu, Fresenius Environ. Bull., 2007, 16, 1468–1473.
15. J. Y. Park, H. J. Byun, W. H. Choi and W. H. Kang, Chemosphere, 2008, 70, 1429–1437.
16. L. H. Li, C. W. Hu, X. L. Dai, W. J. Jin, C. Hu and F. Ma, Environ. Technol., 2015, 36, 2024–2034.
17. H. Kus and T. Carlsson, Cem. Concr. Res., 2003, 33, 1423–1432.
18. C. Karakurt, H. Kurama and I. B. Topcu, Cem. Concr. Compos., 2010, 32, 1–8.
19. T. Hildebrand and P. Rüegsegger, J. Microsc., 1995, 185, 67–75.
20. T. Hildebrand, A. Laib, R. Mueller, J. Dequeker and P. Ruegsegger, J. Bone Miner. Res., 1999, 14, 1167–1174.
21. T. Hildebrand and P. Rüegsegger, Comput. Methods Biomech. Biomed. Engin., 1997, 1(1), 15–23.
22. Sandstone pore structure method of image analysis (Industry standard CJ/T) (in Chinese).
23. Y. Z. Yu, Y. Feng, L. P. Qiu, W. W. Han and L. P. Guan, Bioresour. Technol., 2008, 99, 4120–4123.
24. J. Tan, J. Wang, J. Xue, S. Y. Liu, S. C. Peng, D. Ma, T. H. Chen and Z. B. Yue, Fuel, 2015, 145, 196–201.
25. X. Q. Zhao, L. Y. Yang, Z. Y. Yu, N. Y. Peng, L. Xiao, D. Q. Yin and B. Q. Qin, J. Environ. Sci., 2008, 20(2), 224–230.
26. Chinese EPA, Methods for Water and Wastewater Analysis, Environmental Science Publishing House of China, Beijing, China, fourth edn, 2002.
27. R. J. Zeng, R. Lemaire, Z. G. Yuan and J. Keller, Biotechnol. Bioeng., 2003, 2, 84.
28. Y. Feng, X. W. Wang, J. Y. Qi, Z. Y. Wang and X. Li, Ind. Eng. Chem. Res., 2015, 54, 6641–6648.
29. Y. Feng, Y. Z. Yu, L. P. Qiu, Y. W. Yang, Z. G. Li, M. W. Li, L. S. Fan and Y. Z. Guo, J. Ind. Eng. Chem., 2015, 21, 704–710.
30. S. X. Han, Q. Y. Yue, M. Yue, B. Y. Gao, Q. Li, H. Yu, Y. Q. Zhao and Y. F. Qi, J. Hazard. Mater., 2009, 171(1–3), 809–814.
31. S. P. Li, J. J. Cui, Q. L. Zhang, J. Fu, J. F. Lian and C. Li, Desalination, 2010, 258, 12–18.
32. S. B. He, G. Xue and H. N. Kong, J. Hazard. Mater., 2007, 143, 291–295.
33. Z. M. Fu, Y. G. Zhang and X. J. Wang, Bioresour. Technol., 2011, 102, 3748–3753.
34. J. L. Zou, G. R. Xu, K. Pan, W. Zhou, Y. Dai, X. Wang, D. Zhang, Y. C. Hu and M. Ma, Sep. Purif. Technol., 2012, 94, 9–15.
35. K. L. Yang, Q. Y. Yue, W. Han, J. J. Kong, B. Y. Gao, P. Zhao and L. Duan, Chem. Eng. J., 2015, 277, 130–139.
36. H. L. Kus and T. Carlsson, Cem. Concr. Res., 2003, 33, 1423–1432.
37. K. Ramamurthy and K. Ramamurthy, Cem. Concr. Compos., 2000, 5(22), 321–329.
38. B. J. Ni, H. Q. Yu and Y. J. Sun, Water Res., 2008, 42, 1583–1594.
39. M. Ding, C. C. Danielsen, I. Hvid and S. Overgaard, Bone, 2012, 51, 953–960.
40. T. D. Kock, M. A. Boone, T. D. Schryver, J. V. Stappen, H. Derluyn and B. Masschaele, Environ. Sci. Technol., 2015, 49, 2867–2874.
41. K. Ruzyla and D. Jezek, J. Sediment. Res., 1987, 57, 777–778.
42. F. Liu, C. C. Zhao, D. F. Zhao and G. H. Liu, J. Hazard. Mater., 2008, 160, 161–167.
43. S. Q. Wu, B. Y. Gao, Y. Gao, C. Z. Fan and S. B. He, J. Hazard. Mater., 2015, 283, 608–616.
44. W. J. Li, L. X. Zeng, Y. Kang, Q. Y. Zhang, J. W. Luo and X. M. Guo, Desalin. Water Treat., 2016, 57, 14169–14177.
45. Y. Feng, Y. Z. Yu, L. P. Qiu, J. B. Wang and J. W. Zhang, Desalin. Water Treat., 2012, 47, 258–265.
46. D. Sándor, G. Zajzon, E. Fleit and A. Szabó, 6th IWA International Conference for Young Water Professionals, Budapest, Hungary, 2012, pp. 1–10.
47. T. Hanawa and M. Ota, Appl. Surf. Sci., 1992, 55, 269–276.
48. J. F. Moulder, W. F. Stickle and P. E. Sobol, Handbook of X-ray Photoelectron Spectroscopy, Perkin-Elmer Corporation Publisher, Montreal, 2004.
49. W. A. House, Environ. Technol., 1999, 20, 727–733.
50. X. B. Chen, N. Birbilis and T. B. Abbott, Corros. Sci., 2012, 55, 226–232.
51. J. H. Guo, S. Y. Wang, Z. W. Wang and Y. Z. Peng, Journal of Water Process Engineering, 2014, 1, 108–114.
52. K. L. Yang, Q. Y. Yue, J. J. Kong, P. Zhao, Y. Gao, K. F. Fu and B. Y. Gao, Chem. Eng. J., 2016, 285, 319–330.
53. E. Pozzi, C. Regina Megda, V. E. A. Caldas, M. Damianovic and E. C. Pires, Journal of Water Process Engineering, 2014, 4, 74–81.
54. X. X. Li, X. C. Liu, S. H. Wu, A. Rasool, J. N. Zuo, C. Li and G. Y. Liu, Chem. Eng. J., 2014, 257, 74–81.
55. N. Boon, J. Goris, P. D. Vos, W. Verstraete and E. M. Top, Appl. Environ. Microbiol., 2000, 66, 2906–2913.
56. Y. C. Wu, S. Shukal, M. Mukherjee and B. Cao, Environ. Sci. Technol., 2015, 49, 11551–11559.
57. S. Shams, C. Capelli, L. Cerasino, A. Ballot, D. R. Dietrich, K. Sivonen and N. Salmaso, Water Res., 2015, 69, 68–79.
58. A. Willems, J. D. Ley, M. Gillis and K. Kersters, Int. J. Syst. Bacteriol., 1991, 41, 445–450.
59. F. Mariam, Master's thesis, University of Tennessee, 2013.
60. E. Spieck, C. Hartwig, I. McCormack, F. Maixner, M. Wagner, A. Lipski and H. Daims, Environ. Microbiol., 2006, 8, 405–415.
61. C. S. Poon, T. Ann, W. Yu and L. H. Ng, Resour., Conserv. Recycl., 2001, 32, 157–172.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra05417j

---