Stimulating Nitrogen Biokinetics with the Addition of Hydrogen Peroxide to Secondary Effluent Biofiltration

Abstract

Tertiary wastewater treatment could provide a reliable source of water for reuse. Amongst these types of wastewater treatment, deep-bed filtration of secondary effluents can effectively remove particles and organic matter; however, NH₄⁺ and NO₂⁻ are not easily removed. This study examined the feasibility of stimulating microbial activity using hydrogen peroxide (H₂O₂) as a bio-specific and clean oxygen source that leaves no residuals in the water and is advantageous upon aeration due to the solubility limitations of the oxygen. The performance of a pilot bio-filtration system at a filtration velocity of 5–6 m/h, was enhanced by the addition of H₂O₂ for particle, organic matter, NH₄⁺, and NO₂⁻ removal. Hydrogen peroxide provided the oxygen demand for full nitrification. As a result, influent concentrations of 4.2 ± 2.5 mg/L N-NH₄⁺ and 0.65 ± 0.4 mg/L N-NO₂ were removed during the short hydraulic residence time (HRT). In comparison, filtration without H₂O₂ addition only removed up to 0.6 mg/L N-NH₄⁺ and almost no N-NO₂⁻. A DNA metagenome analysis of the functional genes of the media biomass reflected a significant potential for simultaneous nitrification and denitrification activity. It is hypothesized that the low biodegradability of the organic carbon and H₂O₂ addition stimulated oxygen utilization in favor of nitrification, followed by the enhancement of anoxic activity.

1. Introduction

The tertiary and advanced treatment of wastewater improves water quality, enabling us to meet environmental concerns but also find a potential source of water for reuse [1,2]. The filtration of coagulated–flocculated effluent via high-rate deep-bed filters (velocities between 5 and 20 m/h) is widely used for tertiary wastewater treatment [3]. The removal mostly combines particles and some dissolved organic matter (15%–20%) [4]. A hybrid process of filtration and bioactivity, termed biofiltration, allows significant nutrient and organic matter removal in addition to particle filtration [5,6,7].

Molecular methods provide better insight into the processes that are essential for optimal system design. Recent research on the treatment of secondary effluent or groundwater, as sand-filtration and AOP processes, such as UV/H₂O₂, made use of advanced methods as metagenomics, metaproteomics or metatranscriptomics, to analyze the structure of the community attached to the media and determine the related metabolism [8,9,10,11,12].

In biological wastewater systems, dissolved oxygen (DO) is a key parameter in system performance and plays an important role in biotransformation pathways and rates [13,14]. The competition for oxygen among ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and ordinary heterotrophic organisms (OHO) results in oxygen depletion; this, in turn, increases the potential for anoxic (carbon oxidation through reduction of NO₂⁻ or NO₃⁻) pathways, such as full or partial denitrification, depending on substrate availability and biodegradability [15,16,17,18]. Compared to suspended growth systems, nitrification activity can increase significantly in attached biomass systems when oxygen is more available [19]. DO concentration in the attached biomass is limited by the temperature of the solute (~8 mg/L at 25 °C) and decreases along the biofilm depth, resulting in increased denitrification rates [20,21]. To overcome these limitations, a few studies have examined the use of 100% pure oxygen or hydrogen peroxide (hereafter H₂O₂) degradation (Equation (1)) in attached-biomass systems [22,23,24,25].

$$1{\mathrm{H}}_2{\mathrm{O}}_2\underset{Catalase}{\Rightarrow }0.53{\mathrm{H}}_2\mathrm{O}+0.47{\mathrm{O}}_2(K_{cat}={10}^7{\sec }^{-1})$$

(1)

H₂O₂ degradation by the enzyme catalase. Stochiometry is by weight (g); K_cat—catalytic reaction rate [26].

Catalase is a significant component of the cell defense mechanism against oxidative stress, it scavenges two molecules of H₂O₂ into water and molecular oxygen in one of the highest documented enzyme reaction rates [27,28].

H₂O₂ has advantages as a supplemental oxygen source, namely, high solubility in aqueous solution and an improved mass transfer of dissolved H₂O₂ (liquid–liquid) into the permeable biofilm compared to oxygen that depends also on the oxygen–water transfer into the liquid phase. In addition, rapid H₂O₂ degradation is bacterium-specific and leaves no persistent residual traces in the water. The disinfection characteristics of H₂O₂ could limit microbial-based reactors, which depend on concentration and time (C × t). Significantly tolerant degradation rates and oxygen utilization have been observed in biofilms, in comparison to suspended bacteria, in addition to changes in microbial community structure [29,30,31,32,33]. However, the application of H₂O₂ as a supplemental oxygen source via high-rate media filtration for NH₄⁺ removal at a short hydraulic residence time (HRT) has never been examined. In this study, we demonstrate the impact of adding H₂O₂ as a supplemental oxygen source on the operational efficiency of a secondary effluent filtration system with biologically active media. The setup design was aimed at combining the removal of particles, NH₄⁺, and, especially, NO₂⁻ to reduce ozone demand for a subsequent ozonation treatment (detailed in Zucker et al., [34]). In short, the biofilter was part of a multistep pretreatment unit prior to ozonation to allow better soil aquifer treatment (SAT) performance by reducing particle clogging and minimizing oxygen demand during infiltration in the upper-layer SAT. The specific goals of this study were to examine whether significant biotransformation can be achieved with high-rate (relative to standard biofiltration) filtration conditions and H₂O₂ addition.

2. Experiments

2.1. Pretreatment System Setup

The biofilter system was operated in direct-filtration at the pilot site of the Shafdan wastewater treatment plant (WWTP), operated by Mekorot. The biofilter system, detailed in Figure 1, was part of the multistage setup specified herein:

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Feed flow was 6 m³/h (144 m³/day) of the secondary effluent from the WWTP (Figure 1a) following a 500-µm wire-mesh filtration (Figure 1b) to remove coarse particles.

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Coagulation/flocculation was carried out by injecting polyaluminum chloride (PACl, 18% Al₂O₃) by peristaltic pump (Figure 1c) to achieve a final concentration of ~2.7–3.6 mg/L as PACl. Flocculation was performed in a modified flocculator, which consisted of a pressure filter (Figure 1d) with ~15 min hydraulic retention time (HRT). PACl was chosen for this study as it has been shown to be preferable for flocculation and is widely used [35,36].

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Following flocculation, H₂O₂ was added (10%) to the inlet of the high-rate biofilter (Figure 1f) using a peristaltic pump (Figure 1e). The final concentration of 27 mg/L was achieved to provide a surplus of DO for full nitrification of 3.5 mg/L-N NH₄⁺ and 0.5 mg/L-N NO₂⁻. The biofilter tank had a surface area of 1.13 m², 1.2 m diameter, 1.1 m³ media volume, and 36% free headspace (additional characteristics are specified in Zucker et al., [34]).

During the adjustment period with stepwise H₂O₂ addition, an average measured concentration of 4.29 ± 0.56 mg/L DO at the outlet of the filter was obtained with the addition of ~27 mg/L H₂O₂. The theoretical calculation of oxygen mass balance is provided in Appendix A.1. During the research period, the loads of NH₄⁺ in the secondary effluent increased due to changes in the process of the plant, which enabled investigating the system performance for NH₄⁺ oxidation and N removal under higher NH₄⁺ loads and O₂ limitation.

2.2. System Specification and Operational Parameters

The biofiltration was operated with a modified active media filter, operated in downflow mode, with a filtration velocity of 5–6 m/h (~5 min hydraulic residence time (HRT, the low range of high-rate filtration) and a backwash cycle every 12 h. The filtered effluent water was stored in a 10-m³ tank (Figure 1g) and was used for backwash. Adjustments were performed to avoid the clogging of the filter while maintaining a steady and active biomass to avoid the detachment of biofilm from the media (Appendix A.2). The microbial community of the biomass was indigenous and developed over time by feeding the biofilter with the secondary effluent. It was assumed that the accumulation and growth of viable bacteria in the filter, most of which were fixed to the media, was the basis for the bacterial activity [6,37,38,39].

2.3. Solute Sampling and Analyses

After two months of system operation without H₂O₂ addition, samples were further taken over for a period of four months with stable performance. Stabilization after H₂O₂ addition took also about two months and then a sampling campaign was conducted every 2–4 weeks for over two years. At every sampling campaign, samples were collected 6–7 h after the start of the morning filtration cycle (middle of the cycle) from sampling faucet located past the mesh filter, named the secondary effluent (SE), before the biofilter (BF) and after the biofilter (AF). An analysis of water characteristics was conducted at the Shafdan WWTP laboratory specified in the Appendix A.3.

2.4. Particle Sampling and Analysis

Effluent particles and aggregates were analyzed by Micro Flow Imaging (MFI) technology (DPA 4100, Protein Simple Inc., Ottawa, ON, Canada). This apparatus employs a digital camera with an illumination and magnification system to capture in-situ images of suspended particles in a flowing sample. A detailed description of this analysis is published elsewhere [32,36]. An analysis was conducted on particles of between ~2 and 400 µm.

2.5. Media Sampling and Metagenome Analysis

A metagenome analysis was conducted to investigate the functional potential of the microbial community in the media. In the middle of the operation cycle (6 h), the biofilter was emptied and a sample of 50 g anthracite media was taken from a 40-cm depth, thoroughly mixed for homogenization, and immediately stored in dry ice. Sampling was conducted a few days after the sampling campaign designated in star in Figure A1 in the Appendix A. DNA from the media was extracted using an Exgene Soil DNA Kit. The whole-genome (shotgun) libraries were prepared using the TruSeq DNA PCR-Free HT Library Preparation Kit (Illumina, San Diego, CA, USA) and were sequenced using an Illumina MiSeq benchtop sequencer with a target fragment length of 250 bp. After the removal of adapters and low-quality reads, ~8.2 M reads were left with an average length of 225 base pairs. (Appendix A Figure A2). High-quality reads were mapped to N-cycle related genes using BWA [40]. The potential for dominant biotransformation of the microbial community was deduced from the relative abundance of specific functional genes. The genes for nitrification (amoA, nxrB, cyt c), denitrification (narG, nirK, nirS, norB, nosZ), and other various metabolic pathways, such as methane (pmoA), hydrogen sulfide (drsEFH), and hydrogen (HiFe) oxidation were analyzed and compared on a log scale [9]. The gene catalog was extracted from NCBI’s nucleotide database. Genes aligning to the reads were sorted according to their respective identified organisms, which varied between 6%–19% among all the reads related to each gene (Figure A2 in the Appendix A).

3. Results

The goal of this study was to design a biofilter with short HRT to support nitrification activity, by surplus oxygen, for the removal of NO₂⁻ and NH₄⁺. The system showed a significant removal of NO₂⁻ and NH₄⁺ when H₂O₂ was added in comparison to the control. The biofilter was optimized in the matter of H₂O₂ addition and evaluated as a single step in an integrated treatment with the ultimate goal to produce a streamwith lower oxygen demand for following SAT or non-potable reuse purposes.

3.1. Particle Distribution

To determine biofilter performance in removing particles as a function of equivalent circular diameter (ECD), and the particles’ size and the size distribution in the secondary effluent, a dynamic image analysis was made. The particle-size distribution (PSD) of secondary effluents based on 13 campaigns is presented in Figure A1 in the Appendix A, taken at the SE sampling point. In general, the PSD was similar in comparison to the high variation in total particle number. Almost all (99%) of the particles were analyzed (<50 µm), with the highest variation in the fraction with ECD = 2–3 µm. The total particle removal performance was found to be highly efficient (95 ± 2%) for all sampling campaigns. No difference was observed in particle removal without H₂O₂. A typical PSD analysis of the secondary effluent after 500 µm, post flocculation and after the biofilter, is presented in Figure A1 in the Appendix A. Flocculation before biofiltration increased particle counts due to the flocculation of the macromolecules and colloid particles (<2 µm), which were not analyzed. As expected, particle concentration decreased dramatically after biofiltration for all ECDs.

3.2. H₂O₂ Decomposition

The concentration of DO before filtration varied between 3–5 mg/L. No H₂O₂ was detected at the inlet or outlet during an operation without H₂O₂ addition, while the DO values at the outlet were <1 mg/L, which clearly indicated maximum oxygen utilization and reflected anoxic conditions [20]. The preliminary lab-bench experiments revealed no decomposition of H₂O₂ when mixed with effluents within the interval of the HRT (20 min), as also documented by Lakretz et al. [32]. When H₂O₂ was added, the measured H₂O₂ concentration at the inlet was 27 ± 2 mg/L, and <1 mg/L at the outlet, which affirms full H₂O₂ degradation. Furthermore, DO values after filtration were similar or slightly higher than before filtration. Thus, we conclude that H₂O₂ decomposition in the system occurs almost solely via bacterial activity and functions as a source of available oxygen.

3.3. Reference Measurements—No Addition of H₂O₂

Sampling campaigns were conducted without the addition of H₂O₂ as a reference: DO values at the outlet were <1 mg/L, which reflects maximum oxygen utilization and anaerobic conditions inside the reactor [20]. The dissolved organic carbon (DOC)- and chemical oxygen demand (COD)-removal rates were similar (13% ± 0.3%, and 11% ± 2%, respectively) and were mainly caused by flocculation/filtration. Data were insufficient for the ultra violet absorption (UVA) measurements. The average NH₄⁺, NO₂⁻ and NO₃⁻ concentrations at the BF sampling point were 3.1 ± 0.60 mg/L-N, 0.37 ± 0.07 mg/L-N, and 0.34 ± 0.05 mg/L-N, respectively. While NH₄⁺ removal was constant at 0.6 ± 0.08 mg/L-N, NO₂⁻, removal was not constant, and in some samples no removal of NO₂⁻ was observed. The average increase in NO₃⁻ concentration was 0.73 ± 0.21 mg/L-N. The calculated nitrogen loss was fairly low and did not exceed 4 ± 1%, which suggests removal by filtration rather than via denitrification.

3.4. Organic Carbon Removal with H₂O₂ Addition

The biofilter removal rates of DOC and UVA were stable at 22% ± 5% and 20% ± 3%, respectively, regardless of the variation in DOC (10.4 ± 1.4 mg/L) and UVA (217 ± 13 m⁻¹) at the inlet. Without H₂O₂, the ΔDOC, ΔUVA₂₅₄ and ΔCOD was 1.6 ± 0.4 mg/L, 7 ± 1 m⁻¹ and 3.3 ± 1 mg/L, respectively. With addition of H₂O₂, the ΔDOC, ΔUVA₂₅₄ and ΔCOD increased to 2.3 ± 0.7 mg/L, 44 ± 7 1/m and 14.6 ± 4 mg/L, respectively, indicating additional organic carbon utilization by bacteria (Appendix A Figure A5: concentrations and the removal of DOC and UVA₂₅₄ over sampling campaigns before the biofilter and after the biofilter). The oxygen utilization, in favor of nitrification rather than carbon oxidation, reflects the low biodegradability of organic matter, which is typical for effluent post-secondary treatment with flocculation–filtration removal [41,42,43,44]. This is also supported by no removal of most trace organic compounds in the biofilter, besides Acesulfame and Iopromide that were removed by 60% and 30%, respectively [34]. These results show that the biofilter was efficient and robust at removing particles, DOC, and UVA.

3.5. NH₄⁺ and NO₃⁻ Transformation, Variation, and Nitrogen Mass Balance

To better illustrate the potential nitrogen tranformations in the biofilter due to H₂O₂ addition, and especially the NH₄⁺ removal (mg/L-N) ability of the system, the parameters at all the figures were sorted by NH₄⁺ concentrations at the inlet (BF) sampling point, as illustrated in Figure 2. It can be clearly observed that the concentration of NO₃⁻ that increased at the outlet of the biofilter is in parallel to the decrease in NH₄⁺ concentration (Figure 2). Full NH₄⁺ removal with H₂O₂ addition was obtained when the BF concentration was lower than 4 mg/L-N, whereas above 4 mg/L-N residual NH₄⁺ was detected at the AF sampling point. The average NO₂⁻ concentration of 0.64 ± 0.4 mg/L-N was fully removed after the biofilter (AF) for all samples (Supporting data Figure A6: removal of NO₂⁻), indicating rapid NO₂⁻ oxidation to NO₃⁻ (nitratation). Thus, H₂O₂ addition enabled a significantly higher nitrification rate. Given that the nitrification rate in the biofilter was stable and was solely dependent on H₂O₂ addition, regardless of particle numbers and removal, it is suggested that most of the microbial activity occurred in the biomass attached to the media, perhaps independent of the filtration cake and its size. The ammonium oxidation rate (AOR) (the ratio of formed NO₃⁻ to the transformed NH₄⁺ and NO₂⁻), is calculated according to Equation (2) and shown for all sampling points

$$\mathrm{AOR}=-\frac{\mathsf{\Delta }[{\mathrm{NO}}_3^{-}-\mathrm{N}]}{\{\mathsf{\Delta }\left[{\mathrm{NH}}_4^{+}-\mathrm{N}\right]+\mathsf{\Delta }[{\mathrm{NO}}_2^{-}-\mathrm{N}]\}}$$

(2)

ONT = Ammonification, organic nitrogen transformed to NH₄⁺

Nit = Nitrification

NX = Anoxic/AMX (anaerobic ammonia oxidation) nitrogen transformation to dinitrogen

Inorganic nitrogen balance rate to indicate dominant pathway:

• When AOR > 100%, ONT + Nit >> NX
• When AOR < 100%, significant NX
• When AOR ≈ 100%, Nit >> ONT, NX

In most samples, AOR revealed values of less than 100% (Figure 2, green dashed line), indicating the loss of ammoniacal nitrogen as dinitrogen, which will be further detailed.

Moreover, the calculated AOR was less than ~70% when NH₄⁺ values at the BF sampling point were higher than 4 mg/L-N. Values over 100% can be explained by the contribution of NH₄⁺ via a higher rate of organic nitrogen (amino acids) decomposition than nitritation. The relatively low removal rates of organic compounds by the biofilter when H₂O₂ was added, in comparison to NH₄⁺ and NO₂⁻, suggests that the level of organic carbon degradability was a limiting factor in favoring oxygen utilization for nitrification on the one hand and carbon utilization for denitrification on the other.

3.6. Mass Balance

Calculations of the total nitrogen mass balance are presented in Figure 3, considering organic nitrogen values, revealed an average nitrogen loss of 2.24 ± 1 mg-N/L with H₂O₂ and 0.25 ± 0.06 mg-N/L at the control. In addition, a higher nitrogen loss was calculated in parallel to the increase in NH₄⁺ concentration at the inlet. Calculations to evaluate the nitrogen loss in favor of assimilation with H₂O₂ addition revealed that the assimilation rate was around 50% from all nitrogen removed (

Table A1. Nitrogen mass balance of the biofilter system with and without H₂O₂.

OHO Yield = 0.6	# Campaign	N Loss (mg-N/L)	OHO Assimilation (mg-N/L)	N Loss for Denitrification (mg-N/L)	Denitrification Credit (COD mg/L Utilized for Denitrification)
	0	1.52	1.04	0.48	1.3
	6	1.32	0.82	0.50	1.4
	15	1.43	1.04	0.39	1.1
	17	1.97	1.26	0.71	2.0
	20	3.71	1.64	2.07	5.8
	21	3.47	1.12	2.36	6.6
	Average H2O2	2.24	1.15	1.09	3.0
	STD dev. H2O2	0.98	0.25	0.81	2.27
	28	0.32	0.30	0.02	0.1
	29	0.25	0.15	0.10	0.3
	30	0.18	0.30	0.00	0.0
	Average Control	0.25	0.25	0.04	0.1
	STD dev Control	0.06	0.07	0.04	0.12
OHO Yield = 0.4	# Campaign	N Loss (mg-N/L)	OHO Assimilation (mg-N/L)	N Loss for Deni (mg-N/L)	Denitrification Credit (COD mg/L Utilized for Deni)
	0	1.52	0.69	0.83	2.3
	6	1.32	0.74	0.58	1.6
	15	1.43	0.69	0.74	2.1
	17	1.97	0.84	1.13	3.2
	20	3.71	1.09	2.62	7.3
	21	3.47	0.74	2.73	7.6
	Average H2O2	2.24	0.80	1.44	4.0
	STD H2O2	0.98	0.14	0.89	2.50
	28	0.32	0.20	0.12	0.3
	29	0.25	0.10	0.15	0.4
	30	0.18	0.20	0.00	0.0
	Average Control	0.25	0.17	0.09	0.3
	STD dev. Control	0.06	0.05	0.07	0.18

Notes: denitrification refers to denitrification.

). This indicates that significant nitrogen loss was due to anoxic activity in the biofilter, which occurred simultaneously with nitrification. In addition, denitrification credit, the calculated oxygen that was credited due to utilization of NO₃⁻ as electron acceptor, was significant and varied between 1–7 mg/L of COD and also increased in parallel to the increase in NH₄⁺ concentrations at the inlet. To explain the higher nitrogen loss under aerobic conditions that was enhanced by the oxygen surpluss as H₂O₂, we hypothesized that H₂O₂, due to a lack of solubility limitations, better penetrates into the depth of the biofilm, decomposes, and stimulates activity.

3.7. Functional Potential of the Biomass

The Metagenomics preformed on the sample yielded 8.2 M high-quality reads, out of which, 3.6 M were aligned to ~1250 organisms, when >98% are bacteria, both in the abundance and number of strains. The resulting outcome indicated a highly diverse bacterial community, with a calculated Shannon diversity index value of 5.6 as opposed to the theoretical value of 7.13 for a sample with equal diversity. The relative abundance of functional genes in the biofilter biomass is presented in Figure 4. The relative abundance of genes involved in nitrification (AmoA and NxrB) was higher, by 3 to 4 orders of magnitude, than the genes involved in other electron donor pathways (as drsEFH or aprA). In addition, the log relative abundance of the genes involved in denitrification and oxygen utilization (cyt c oxidases) was also significantly higher in comparison to the other various metabolic pathways examined.

Considering that cyt c is harbored by AOB (nitritation) [45], as well as denitrifying organisms (denitrification) [46], the relative high abundance of this gene is consistent with the experimental results of simultaneous nitrification and denitrification activity (Figure 2). The higher relative abundance obtained for the gene of NO₂⁻ oxidation (nxrB) over NH₄⁺ oxidation, might suggest the possible occurrence of a “ping pong” mechanism of cyclic NO₂⁻ oxidation and reduction, resulting in a higher NO₂⁻ oxidation rate than NH₄⁺ [47]. In parallel, the composition of identified genera of each of the N related genes (Figure 4b) revealed a variety of heterotrophs capable of conducting denitrification, when the genera belonging to the family Comamondaceae was the most dominant one. Interestingly, the identified genus related to NH₄⁺ oxidation was related to AOB and NOB, while the identified genus related to NO₂⁻ oxidation was of heterotrophs. This might suggest that the involvement of commamox and heterotrophs in nitrification [48,49,50].

The comparison of the functional potential of the microbial community reflects clear dominance for N biotransformation by simultaneous nitrification and denitrification. The significant anoxic potential reflected from the relative abundance of functional genes can be explained by the denitrifying community capable of being active at the presence of oxygen (low half-saturation rate of oxygen inhibition, K_OI = 0.1–0.2 mg/L) [43].

Moreover, favoring oxygen utilization for nitrification, with a C/N ratio similar to wastewater, can be explained by the selection of organisms that are more competitive and characterized with a low substrate half-saturation rate coefficient (K_ONitrospira = 0.1–0.2 mg/L, K_{OAOB =} 0.26–0.15 mg/L), which is more typical to nitrifiers in comparison to heterotrophs [51,52]. Jiang et al. (2013) also observed that the increase in oxygen availability in the upflow biofilter resulted in utilization by nitrifiers rather than heterotrophic biomass [19].

We suggest that the typical low biodegradability of the organic carbon at the secondary effluent, which was also reflected in the low removal rate of the COD, BOD, and UVA₂₅₄, mentioned earlier (Section 3.4, Figure A5a,b in the Appendix A) was a key parameter on the rates of heterotrophic activity [44].

The environmental conditions of a low degradability rate of the organic matter and the addition of oxygen via H₂O_2, could be a possible explanation for favoring NH₄⁺ rather than organic material oxidation. Such conditions, may promote simultaneous nitrification/nitrogen-removal activity. The significant relative abundance of genes with the potential for NO₂⁻ oxidation on one hand and NO₂⁻ reduction on the other hand strongly suggest that competition for NO₂⁻ is a key in the dynamics of the community structure and function.

To address the paradox of excess oxygen availability in the reactor, due to H₂O₂ addition and degradation, while the relative abundance of genes related to anoxic activity reflects conditions of limited oxygen, we hypothesize that this community structure reflects inner localization in the biofilm: Whereas AOB and NOB are localized at the outer layer of the biofilm, degrade the H₂O₂, and utilize the supplemental oxygen for nitrification, in parallel with the depletion of oxygen along the depth of the biofilm, NO₂⁻ and NO₃⁻ are reduced. It is also possible that the nitrifiers are located in the pockets inside the biofilm and the better mass transfer of H₂O₂ enables utilization in a deeper location. We also suggest that the bacterial community that was investigated in this study indicates maximized thermodynamic utilization of electron acceptors and donors, with significant rates of anoxic activity.

The limitation of the process performance under increasing loads of NH₄⁺ might be due to a lack of oxygen, limited HRT, or reactor volume and should be further investigated. As mentioned in previous research [34] the biofilter system succeeded in decreasing the oxygen demand and the drop in the ORP in following SAT. We suggest that the design presented in this study could be beneficial for treating secondary effluents containing residual levels of NH₄⁺ (few mg/L-N), which is required to be removed under discharge or reuse restrictions.

4. Discussion

We suggest a model to describe the biokinetics of tertiary wastewater biofiltration systems with the addition of H₂O₂. The model focuses on the soluble (not particulate) content, carbon content similar to the secondary effluent (low biodegradability, nitrogen content), and the significance of metabolism over assimilation (Appendix A.1 and Table A1). Generally, although some full reduction of NO₃⁻ to NH₄⁺ may occur simultaneously, it is neglected in the model.

The model suggest that the addition of H₂O₂ (Figure 5) promoted the rates of the following pathways: (i) nitritation and (ii) nitratation, which occurs at high rates, and (iii) biodegradable COD oxidation, which occurs at minor rates. In parallel, (v) NO₂⁻ and (vi) NO₃⁻ reduction via denitrification and (vii) are also stimulated.

5. Conclusions

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The addition of H₂O₂ to stimulate aerobic activity within a bed filtration process, practiced under the low range of the high rate bed filtration (5–6 m/h), enabled a combined effect of particle filtration and nitrification.The H₂O₂ was fully degraded, limiting the nitrification rate.

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The trends in the concentration of NH₄⁺, NO₂⁻, and NO₃⁻ with addition of H₂O₂ demonstrated significant nitirification activity at the bed filter.

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Metagenome analysis results were in line with the performance obtained and reflected significant potential for the simultanious nitrification denitrification activity of the attached biomass.

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The process presented herin may have a techno-economical potential, especially with the development of new and novel methods for the direct production of H₂O₂ [53,54] and the efficient application of the high-rate filtration.

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In cases of residual NH₄⁺ concentration (<5 mg N-NH₄⁺/L) in secondary WWTP effluents, the presented technology shows potential for either managing ammonia concentration in the effluent or for reducing oxygen demand in following processes, such as SAT or direct reuse, under local regulation.