Organic Matter Removal in a Simultaneous Nitrification-Denitrification Process Using Fixed-Film System.

doi:10.21203/rs.3.rs-959611/v1

This study focused on evaluating different support media for COD and nitrogen compound removal from an Upflow Anaerobic Sludge Blanket (UASB) reactor fed with swine wastewater. Maximum specific nitrification (MSNA) and denitrification (MSDA) activity tests were performed in two fixed-film systems with (1) polyurethane foam (R₁) and (2) polyethylene rings (R₂). The results showed that the R₂ system performed more efficiently than R₁, reaching organic matter removal of 77 ± 8% and nitrogen of 98 ± 4%, attributed to higher specific denitrifying activity recorded (5.3 ± 0.34 g NO₃^--N/g VTS ∙h). In this sense, MSDA tests indicated that the suspended biomass was responsible for at least 70% of nitrogen removal in the form of ammonium compared with 20% attributed to biomass in the form of biofilm. On the other hand, 40 ± 5% of initial nitrogen could not be quantified in the system effluents, but 10 ± 1% was attributed to loss by volatilization. According to the analyses, the previous information infers the development of simultaneous nitrification-denitrification (SND) routes. Respect to the analyses of microbial diversity and abundance in the biofilm of R₂ rings_,the presence of the genus Pseudomonas dominated the prokaryotic community of the system in 54.4%.

Molecular Biology

Biotechnology and Bioengineering

SND

support media

nitrogen removal

nitrifying activity

denitrifying activity

Nitrogen is an essential biological growth nutrient and one of the main constituents of all living organisms. However, its excessive presence should be avoided due to several reasons: (a) Nitrogen in reduced forms exerts oxygen demand in the receiving water body¹. (b) Ammonia and nitrite are toxic for fish in concentrations higher than 0.045 and 120 mg/L, respectively². (c) Wastewater with high nitrogen concentrations requires a great amount of chlorine for its disinfection³. and (d) Nitrite and nitrate in concentrations greater than 0.2 and 1.5 mg/L, respectively, jointly with phosphorus in concentrations greater than 0.10 mg/L may cause eutrophication of lakes and water bodies, which results in an uncontrolled growth of algae and other aquatic plants ^{4, 5}.

Nitrogen appears in swine wastewater in different ionized forms, such as ammonia or ammonium (NH₃⁺ and NH₄⁺, respectively), nitrate (NO₃⁻) and nitrite (NO₂⁻) as well as organic compounds measured as total nitrogen⁶. Thus, the nitrogen found in swine wastewater must be removed, since it can reach a concentration of up to 2000 mg/L⁷. Nitrogen removal may be carried out by means of processes, such as biological nitrification and denitrification, so understanding the microbial structure that conforms the biological system is needed for its proper function and operation¹.

Nitrification is a biological aerobic process that oxidizes NH₄⁺ to NO₂⁻ with the help of ammonia oxidizing bacteria (AOB) followed by the conversion of nitrite to nitrate by oxidizing bacteria (NOB), both bacterial groups are called chemoautotrophic nitrifying bacteria ⁸. Nitrosomonas, Nitrosococcus and Nitrosospira are the main bacteria reported for ammonia oxidation whereas Nitrobacter, Nitrocystis and Nitrospira have been reported for nitrite oxidation ⁹.

On the other hand, denitrification is a non-assimilative reduction process of oxidized nitrogen forms (NO₂⁻ and NO₃⁻) to molecular nitrogen under anoxic conditions. This process is carried out by heterotrophic bacteria using organic carbon source for their metabolism⁹. Denitrification may be carried out by diverse groups of bacteria, but they are generally heterotrophic microorganisms (Phylum, Proteobacteria, Firmicutes, Thiobacillus versutus, etc.) and less frequently by autotroph organisms (Thiobacillus denitrificans and Mocrococus denitrificans)¹⁰ Additionally, other biological processes exist for nitrogen removal, for example, the (i) single reactor system for high ammonium removal over nitrite – best know as the SHARON process. This process is based on ammonium oxidation to nitrites of 50%, in low oxygen conditions (< 0.7 mg of O₂/L) ¹¹, (ii) Anaerobic ammonium oxidation (ANAMMOX), where ammonium functions as electron donor and nitrite oxygen as electron receptor to obtain gaseous nitrogen; and (iii) Simultaneous nitrification-denitrification (SND), which is given by forming anoxic microzones in the interior of the bacterial consortia found in the aerobic reactor¹¹. Additionally, SND may be carried out by heterotrophic bacteria using an organic carbon source for metabolism^{12, 13}.

In the last years, some aerobic systems have been improved by installing support materials like polyurethane (PUR), polyvinylchloride (PVC) and polyethylene (PE) within the system for biomass growth in biofilm form that may favor SND. According to Ruiz et al.¹⁴, this type of systems may be classified as hybrids. Hybrid support systems have a great diversity of microorganisms mainly due to the anoxic microzones that allow different types of bacterial growth, both aerobic and facultative¹⁵. Examples of the different bacterial population diversity, both in suspended biomass and biofilms, can be Pseudomonas stutzeri, Enterobacter cloacae HNR strain, Vibrio diabolicus and Bacillus sp. This bacteria have been reported to favor consumption of different contaminants that cannot be degraded totally in conventional systems¹⁶.

In this context, the objective of this study is to assess nitrogen and organic matter removal from two aerobic fixed-film systems with polyurethane foam (R₁) and polyethylene rings (R₂). Additionally, a microbiological analysis of the most efficient pack-bed biofilm system is performed to determine the main microorganisms involved in nitrogen removal process.

Inoculum

The biomass used for the experiments was obtained from an aerated lagoon as part of the process of a wastewater treatment plant located southward from Cd. Obregón, Sonora, in northwest Mexico. The reactors were inoculated with the support media previously placed in contact for one week with 1 L of aerobic biomass (20.4 g/L of total solids (TS) and 7.66 g/L of Total Volatile Solids (TVS).

Operation of the aerobic systems

The two aerobic fixed-film reactors, R₁ (filled with polyethylene rings) and R₂ (filled with polyurethane foam) had a capacity of 0.9 L each one and operated continuously for 330 days with hydraulic retention time (HRT) from 0.4-0.5 days and an average dissolved oxygen (DO) concentration of 3.35 mg/L. Buttom up aeration was performed in the reactors through the mammoth pump. Figure 1 shows the schematic diagram of the systems. Table 1 shows the characteristics of the support materials.

Table 1

Packing media characteristics
Parameter	R₁	R₂
Material	Polyurethane	Polyethylene
Dimensions	Width: 0.5 cm; Height: 0.5 cm	Diameter: 3 cm; Height: 3 cm
Shape	Rectangular	Cylindrical
Percentage in system (%)	20	20
Support mass (unit)	1.2	6
Density (g/L)	7.3	77.8
Superficial Area (m²/g)	23.5*	6.1**
Sahariah et al.¹⁷ *Silva et al.¹⁸

Influent characteristics

The fixed-film reactors were fed with the effluent of an UASB with swine wastewater coming from a farm with a maternity production process. Table 2 shows the physicochemical characteristics of the two fixed-film system feed evaluated, which maintained an organic load rate (OLR) of 0.6 ± 0.3 kg of chemical oxygen demand (COD)/m³day.

Table 2

Physicochemical characteristics of packed systems influent.
Parameter*	Concentration (mg/L)
Chemical oxygen demand (COD)	300 ± 100
Nitrate (NO₃⁻-N)	5 ± 4
Nitrite (NO₂⁻ -N)	5 ± 4
Ammonium (NH₄⁺ -N)	100 ± 20
Total solids (TS)	650 ± 150
Total volatile solids (TVS)	200 ± 100
* The parameters were quantified according to the techniques described in the analytical methods section.

Analytical methods

In the influent and effluent of the fixed-film reactors, the following analytical techniques were performed: organic matter chemical oxygen demand (COD), total solids (TS), Total Volatile Solids (TVS), nitrates, nitrites and ammonium according to that established in the American Public Health Association¹⁹.

Nitrifying and denitrifying activities

To know the nitrifying and denitrifying activities of the systems evaluated, discontinuous assays were performed based on the proposed methodology by Bassin et al.²⁰. In the nitrifying activity assay, R₁ and R₂ were evaluated (1) with suspended biomass + biofilm in the support material and (2) only with suspended biomass (without support material). The nitrifying activity was performed discontinuously, stopping system feed, followed by injecting a stock solution of 100 mg NH₄⁺-N /L. The samples were collected from the supernatant of each reactor every hour for 10 h and subsequently every two hours with a total of 36 h. To know the nitrifying activity, soluble nitrogen forms (NH₄⁺-N, NO₂⁻-N and NO₃⁻ -N) were determined in each established time. After determining NH₄⁺-N removal at the final sampling time, its volumetric removal rate (VRR) was calculated according to Equation 1:

\(\text{V}\text{R}\text{R} (\text{m}\text{g} {NH}_{4}^{+}-\text{N}/\text{L}\bullet \text{h})\) = \(\frac{{{NH}_{4}^{+}-\text{N} }_{\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l} }- \text{N}-{NH}_{4 \text{f}\text{i}\text{n}\text{a}\text{l}}^{+} (\text{m}\text{g}/\text{L})}{Time \left(h\right)}\) (Equation 1)

The result of the maximum specific nitrifying activity (MSNA) in milligrams NH₄⁺-N/g TVS∙h was obtained from dividing the volumetric removal rate (VRR) of nitrogen in the form of ammonium by the TVS of suspended and fixed biomass concentration (Equation 2):

\(MSNA (mg {NH}_{4}^{+}-\text{N}/g VTS\bullet h)\) =\(\frac{\text{V}\text{R}\text{R} (\text{m}\text{g} {NH}_{4}^{+ }-\text{N}/\text{L}\bullet \text{h})}{ (\text{g} /\text{L}) }\) (Equation 2)

With respect to the denitrifying activity, it was developed similarly to the nitrifying one, also performed in the two aerobic systems discontinuously stopping their feed. During the maximum specific denitrifying activity (MSDA) the R₁ and R₂ systems were injected with a concentrated stock solution of NaNO₃, and C₂H₃NaO₂. As a result, concentrations at time zero 82 ± 0.17 and 77.71 ± 2.02 mg of NO₃⁻ -N /L and 261.42 ± 8.75 and 250±5.41 mg COD/L were obtained for R₁ and R₂, respectively, with the purpose of reaching a theoretical value of 80 mg of NO₃⁻ -N/L and 250 mg of COD/L, simulating the conditions of the influent in the continuous systems.

Times for sampling in this experiment were equal to those of the nitrifying activity. After determining nitrogen concentrations in nitrates in the final sampling time, the VRR of NO₃⁻ -N was calculated and obtained by dividing nitrogen concentration in the form of removed nitrate in mg/L by the time in hours (Equation 3):

\(\text{V}\text{R}\text{R} (\text{m}\text{g} {NO}_{3}^{-}-\text{N}/\text{L}\bullet \text{h})\) = \(\frac{ {{NO}_{3}^{-}-\text{N} }_{initial }- {{NO}_{3}^{-}-\text{N}}_{final}(\text{m}\text{g}/\text{L})}{Time \left(h\right)}\) (Equation 3)

Subsequently, the MSDA was obtained from dividing nitrogen VRR in nitrates by the TVS of biomass concentration in the systems (Equation 4):

\(MSDA (mg {NO}_{3}^{- }-\text{N}/g VTS\bullet h)\) =\(\frac{\text{V}\text{R}\text{R} (\text{m}\text{g} {NO}_{3}^{-} -\text{N}/\text{L}\bullet \text{h})}{ (\text{g}/\text{L}) }\) (Equation 4)

Extraction, amplification and DNA analyses in supports

Biofilm volumes of 1.5 mL were taken in sterile conical microtubes and centrifuged at 5000 rpm for 15 min. The supernatant of each microtube was discarded, and the final pellet was used in the nucleic acid extraction. The total DNA extraction was performed starting from 0.1-0.12 g of pellets with the DNeasy PowerSoil Kit (Qiagen, Hiden, DE) according to the manufacturer’s instructions. Subsequently to the extraction, DNA was quaintified in a fluorometer Qubit 3.0 (ThermoFisher Scientific, Waltham, MA, USA), preserving the samples at -20°C up to their subsequent processing. The diversity of bacteria and archaea was determined by amplifying the V4 16S rRNA region with the specific primers 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The endpoint polymerase chain reactions (PCR) were performed in a total volume of 25 µL, whose concentration for each reaction consisted of: ~10 ng DNA, 1X PCR buffer (free of Mg²⁺), 0.4 µM of each oligonucleotide primer, 800 µM of deoxynucleoside triphosphate (dNTP) mix (dATP, dCTP, dTTP and dGTP), 5% of dimethylsulfoxide (DMSO), 1.5 mM of MgCl₂ and 1 U of DNA Taq polymerase ExTaKaRa Taq (Takara Bio Inc. Kusatsu, Shiga, JP). The amplification protocole consisted of an initial DNA denaturalization at 95°C for 3 min, followed by 35 denaturalization cycles (95°C, 30 s), hybridation (52°C, 40 s) and extension (72°C, 90 s), with a final extension at 72°C for 10 min. The amplicons obtained were purified with magnetic pearls using the Agencourt AMPure XP PCR Purification System (Beckman Coulter, Brea, CA, USA) kit and shipped for sequencing in the plataform Illumina MiSeq (Yale University, USA). The sequences obtained were analyzed with the platform QIIME2 (https://qiime2.org)²¹. The amplicon sequence variant (ASV) were taxonomically classified using Silva (https://www.arb-silva.de/) database. The charts and relative abundance of the main microbial groups were performed with the program ‘phyloseq’²² in the RStudio (1.2.5042) environment of the R (The R Core Team 2012) platform.

Statistical analyses

The results are expressed as average standard deviation (SD) +/-. Data were analyzed using a two-way analysis of variance (ANOVA) with the Minitab (Version 17.0) software. When ANOVA identified the differences among groups, multiple comparison of means were performed using Tukey’s honestly significant difference (HSD) test with a level of significance of P < 0.05.

Aerobic fixed-film system performance

Organic matter removal was evaluated by measuring organic load rate both in the influent and effluent of the systems (Fig. 2). The influent concentration was maintained in 300 ± 100 mg COD/L and both R₁ and R₂ indicated a similar COD removal of organic matter of 72 ± 7 and 77 ± 8%, respectively. It is worth to mention that the system feed was stopped to perform the nitrifying activity tests with suspended biomass + biofilm (day 150) only with suspended biomass (day 200) and denitrifying activity (day 250). Additionally, suspended biomass and biofilm samples were taken subsequent to each assay to quantify the volatile suspended solids (VSS). Thus, destabilization in R₁ and R₂ was observed in these days causing decreases in removal efficiency (Fig. 2). The statistical analyses showed that no significant difference was observed in organic matter removal in R₁ and R₂ (P ≤ 0.05), reaching a stationary state after the first 50 days of operation.

Similar results have been reached by mobile packed-bed bioreactors coupled to membrane bioreactors (MBBR-MBR) fed with domestic wastewater with a COD concentration of 185.80 ± 45.8 mg/L and operating with a hydraulic retention time (HRT) of 0.5 days. These packed-bed systems with a commercial Kaldnes type “K₁”, in a filling ratio of 35% of total volume, showed global COD removal efficiency of 83 ± 2.11%²³. Mazioti et al.²⁴ reported COD removal efficiency of 86.6% when they operated a MBBR system of 4.5 L useful volume with domestic wastewater and influent concentration of 270 mg COD/L, operated with HRT of 1.1 day, packed with the commercial AnoxKaldnes type “k3” support material in a filling volume ratio of 30%. Boutet et al. ²⁵ obtained COD removal efficiencies of 47%, operating packed-bed systems with inert material “BIONEST” using municipal wastewater with an average concentration of 457 mg/L COD and HRT of 0.5 day.

In general, the systems assessed in this study reached similar or higher efficiency to those reported by other authors, which not only depended on variables, such as filling material percentage, COD concentration in the influent or type of supporting material but also on biomass concentration developed in the system interior. The biomass developed on the material surface was 18 ± 5 and 21 ± 3 g/L of R₁ and R₂, respectively. This biofilm possibly gives rise to different oxygen concentration gradients towards the interior of these materials. The differences in oxygen concentration in the system could favor the appearance of anoxic zones in the deepest areas of the biofilm where oxygen cannot easily penetrate. The previous allows heterotroph organisms to assimilate organic carbon for their metabolism and growth, giving rise to denitrification processes and favoring removal of organic matter in the form of COD ^{11, 16, 26}.

Figure 3 shows the fixed-film system performance for NH₄⁺-N removal. It is worth to point out that this performance represents the system behavior when fed with swine wastewater, while the nitrifying and denitrifying activities were performed with synthetic solutions as the methodology indicated. The influent concentration was maintained in 100 ± 35 mg NH₄⁺-N /L for 330 days of operation of the systems R₁ and R₂ showing a complete removal efficiency of NH₄⁺ -N (99.9%).

Lo et al.²⁷ concluded that high efficiency is possible thanks to the nitrifying activity carried out in aerobic systems with support. The nitrifying activity is performed both because of the suspended biomass within the systems and fixed biomass adhered to the supports. However, Bassin et al.²⁰ demonstrated that suspended biomass plays the most important role in ammonium removal process, showing a nitrifying activity relatively greater compared to biofilm, which plays the main role in the denitrifying process. Additionally, total nitrogen removal may be achieved indirectly as a result of both SND process and nitrogen assimilation by the heterotroph organisms for new cell formation. Lo et al.²⁷ reported that approximately 34% of total initial nitrogen was used for biomass formation where a SND process was performed in a hybrid biofilm system with an HRT of eight hours. This result implies a process where the greatest part of nitrogen is removed by a SND process, but with a yield in biomass formation. Coupled to this process, bacteria involved in SND, such as Pseudomonas, have low replication times of up to 30 min²⁸ which is why nitrogen assimilation through these bacteria possibly plays an important role in R₁ and R₂ performance.

Matsumoto et al.²⁹ and Wu et al.³⁰ observed SND process in biofilm systems with inert materials, such as plastic and ceramic membranes, evidencing these processes by the presence of AOB and NOB bacteria in the internal zone of the biofilm and heterotroph bacteria in the same biofilm surface. In this sense, according to Bassin et al.²⁰, up to 20% of NH₄⁺-N removal might be attributed to biofilm while the suspended biomass contributed up to 70% of this process. Lastly, Figure 3 also shows that NH₄⁺-N removal efficiency reached by each system were stable and without significant differences between them, according to statistical analyses (P < 0.05).

Sahariah et al.¹⁷ operated a sequential mobile packed-bed bioreactor with polymer foam support with a filling Volume of 15.7% and fed with a concentration of 125 mg NH₄⁺-N /L. The reported systems showed removal efficiency of 68% of NH₄⁺-N, a lower value than that obtained in this study. On the other hand, Bassin et al.²⁰ operarated two mobile packed-bed bioreactors of 1 L of useful Volume, one packed with the commercial synthetic support “Kaldnes K1” and the other one with “MutagBiochip” with the filling Volume of 50%, operated with HRT of 0.5 days and fed with a concentration of 100 mg NH₄⁺-N/L. The authors achieved NH₄⁺-N removal efficiency higher than 90%. It should be mentioned that the support materials evaluated in this study occupied a filling Volume with a range from 15-50%, reaching NH₄⁺-N removal efficiency of >99%. In this sense, the systems evaluated in this study demonstrated much higher efficiency with respect to similar systems. As mentioned previously, this excellent performance could have been due to biofilm presence (18 ± 5 and 21 ± 3 g TVS/L for R₁ and R₂, respectively), which was measured at the end of the assays. Ødegaard et al. ³¹ and Bassin et al. ²⁰ suggested that the quantity of adhered biomass to a support medium not onl1y depends on the superficial area but also its form or material configuration. These findings indicate that supports, as Mutag Biochip that has the form of a satellite dish, are frequently subjected to attrition forces due to the intense contact with the surrounding liquid, favoring biofilm detachment and the quantity of adhered solids. Whereas the types of support with cylindrical shape or rings favor biofilm accumulation.

Nitrifying and denitrifying activity

The nitrifying and denitrifying activity of the systems were assessed in a period of 36 h for R₁ and R₂. The systems operated continuously and for these assays they were set in batch mode, stopping feed flux. Figure 4 shows ammonium removal during the MSNA assays. The NH₄⁺ removal was 20 ± 5% for both systems at hour 10 of the assay; five hours later the systems reached two-fold removal. Starting from hour 10, removal started to increase significantly until it reached 90 ± 6 and 98 ± 4% of NH₄⁺-N removal for R₁ and R₂, respectively (Figure 4). This result could indicate an adaptation process of the microorganisms in the systems when they went from operating continuously to discontinously. Although it did not inhibit the nitrifying process, it made it slower.

Despite the behavior was similar for both assays (R₁ and R₂), the R₂ system reached greater NH₄⁺-N removal, whereas R₁ showed a slightly lower removal though not significant according to the statistical analyses performed (P > 0.05). Additionally, at the end of the assay, loss of nitrogen was evident in both systems, but it was not found in any of the determined soluble forms, approximately 60% and 65% for R₁ and R₂, respectively. Presumably, these non-quantified nitrogen percentages have been converted to molecular nitrogen by means of SND. Garzón-Zuñiga et al. ¹¹ explained that aeration systems with fixed biomass in support materials are capable of developing denitrifying processes starting from heterotroph bacteria that achieve growing in anoxic environments. On their part, Lo et al. ²⁷ studied nitrogen transformation in the form of ammonium to nitrogen, gas in a hybrid biofilm system. The results showed that approximately 60% of soluble nitrogen was converted to nitrogen gas by a SND process. On the other hand, some Pseudomonas species have been reported capable of reducing nitrate to nitrite, among which P. stutzeri perform denitrification processes²⁸.

Table 3 shows the results of MSNA, as well as those reported by different authors, where the obtained values are found within the range bibliographically reported for systems operated under similar conditions. The results allow observing the importance of suspended biomass in MSNA: 3.13 and 2.05 mg NH₄⁺-N /gTVS∙h for R₁ and R₂ respectively, even higher including that reached by the systems with both biomass types (suspended and fixed): 0.352 and 0.253 mg NH₄⁺-N /g TVS∙h for R₁ and R₂, respectively. Lo et al. ²⁷ observed that in a hybrid biofilm system, nitrification was produced mainly in suspended biomass while biofilm played the main role in denitrification. In this manner, biofilm and suspended sludge interaction in the same reactor gave as a result a better general yield in nitrogen removal by a SND. The previous information may be observed in the nitrifying assays (Table 3). On the other hand, Mašić and Eberl ³² found evidence through mathematical models that suspended biomass contributes in a more important manner to ammonium removal in biofilm systems. However, nitrifying activity is not considered frequently in the suspended biomass, assuming that nitrification only takes place in the biofilm ³³.

Table 3

Specific Nitrifying Activity obtained in different laboratory-scale studies.
	Bassin et al. ²⁰	Salvetti et al. ³⁴		Bassin et al. ³⁵	Reif-Lopez ³⁶	Lu et al. ³⁷	This study
Type system	MBBR	MBBR		SBR	MBR	SBBR	Packed-bed
Material	Kaldnes K1 y MutagBiochip		KMT	NR	Membrane Zenon ZW-10	Polyurethane	Polyurethane	Polyethylene
Ammonium	0.2	1.96		0.2	0.04-0.08	NR	0.18
OLR	0.82-3.2	1.28		0.9	0.45-0.9	NR	0.75
HRT	12-3.1	0.3-0.6		5.2	24-12.1	12	12
TVS	4.9-5.52	6.67		10.0-14.2	0.5-2	NR	15.79	20.44
MSNA	1.2-5.6^a	15-44^a		1.2-5.6^a 9.5-18^b	0.12-0.16^a	7.0-22^a	0.35^a 3.13^b	0.25^a 2.05^b
^a Fixed biomass, ^b Suspended biomass. Where: Maximum specific nitrifying activity (MSNA) is expressed in mg NH₄⁺-N/g TVS∙h; Volumetric organic load rate (OLR): kg Chemical oxygen demand (COD) m³/d; Ammonium load: kg NH₄⁺-N /m³; Hydraulic retention time (HRT): h; Total volatile solids (TVS): g/L. NR = No reported

On the one hand, MSDA was 4.64 ± 0.13 and 5.3 ± 0.34 mg of NO₃⁻ -N/g TVS∙h, for R₁ and R₂, respectively, results that are found within the range reported for biological nitrogen removal (BNR) systems. On the other hand, the determined MSDA agreed with that reported for the SND processes (1.6-30 mg of NO₃⁻ -N/g TVS h) for BNR systems inoculated with aerobic biomass and fed with real wastewater. Whereas lower MSDA values were reported for conventional denitrification routes and ANAMMOX (0.5-1.56 mg of NO₃⁻ -N/g TVS h)¹². In the case of R₁ and R₂, nitrification and denitrification metabolism activation is performed in the same system simultaneously SND and not in different or sequential reactors as conventionally reported¹⁰. The previous is due to the presence of anoxic microzones in the aerobic system, given as a result dissolved oxygen gradients that limit their diffusion through the systems¹².

In this sense, the main explanation for SND is because denitrification organisms may exist both in the biofilm and suspended biomass of the system. Additionally, the existence of facultative microorganisms has been proven that use NH₄⁺-N as electron donor and NO₂⁻-N as electron receptor, producing N₂ and NO_X in SND¹¹.

In the case of MSDA, polyethylene rings showed superiority over polyurethane foam, which was directly related to the amount of developed biofilm. Thus, polyethylene rings were selected as the most efficient support material in NH₄⁺-N removal. Additionally, the statistical analyses indicated a significant and higher difference (P ≤ 0.05) in the denitrifying activity, where the rings could favor SDN alternate routes because of factors, such as configuration and material type that could create better conditions to form anoxic zones where the denitrification process mainly takes place.

Figure 5 shows the results obtained from monitoring NO₃⁻ -N behavior, organic matter in the form of COD and removal efficiency for the MSDA assays. Differently from MSNA, R₁ and R₂ had a different behavior, of which system R₂ was the most efficient by removing 91 ± 2.24% of NO₃⁻ -N and 67.86 ± 0.4% of COD, whereas system R₁ removed 52.32 ± 0.6% of NO₃⁻-N and 57.42 ± 1.24% of COD. The results indicated that 2.54 g COD/g of NO₃⁻-N reduced were used, which correspond to the organic requirements reported by Chatterjee et al. ³⁸ for heterotrophic denitrification (2.86 g COD/g of NO₃⁻ -N removed), and more specifically 2.08 g COD/g of NO₃⁻-N reduced when using C₂H₃NaO₂ as a carbon source¹².

Balance of nitrogen species

The result of the nitrogen forms measured in the effluents R₁ and R_2, evidenced a nitrogen concentration that could not be quantified (~ 40 ± 5%) with respect to NH₄⁺-N measured in the influent. The material balance indicated that it was quantified in the effluents of R₁ and R₂: 55 ± 11% and 54 ± 10% in the form of NO₃⁻ -N; 2.58 ± 2 and 3.4 ± 2.5% in NO₂⁻-N and 3.03 ± 4.02% and 5.07 ± 6.84% as NH₄⁺-N. Based on these results, the operated systems might have shown a SND process.

According to Matsumoto et al.²⁹ SND is associated to reactors that have suspended biomass and biofilm and show nitrogen loss in the effluent. Anoxia conditions activate denitrifying metabolism, which are given by the anoxic microzones in the interior of the biofilm bacterial consorptia. In these microzones, oxygen cannot penetrate, but the NOx generated by the nitrifying bacteria can. According to Garzón-Zúñiga, ¹¹ the nitrates produced by the nitrifying bacteria in the superficial layers of the biofilm may penetrate toward the deepest layers by one concentration gradient. When they penetrate toward these deepest layers where oxygen concentration is very low or null, the denitrifying bacteria use nitrites and nitrates as receptor and transform them into molecular nitrogen (N₂), which escapes from the system with gaseous effluent, making it possible to be counted in soluble form.

The previous information agrees with the volatilization assays that were performed additionally where a loss of NH₄⁺-N to the environment in gaseous form was determined 10 ± 1%. It is worth to mention that some authors also reported loss by Stripping of 8-15%^{38, 11}. In this sense, Garzón-Zúñiga et al.¹¹ found that in a packed-bed biofilter with organic material, nitrogen loss was performed by biologic sorption, filtration and assimilation mechanisms. These authors reported that from a total NH₄⁺-N found in the influent, 10% oxidized NO₂⁻-N and another 10% to NO₃⁻ N, 40% was lost during the SND processes, 10% volatilized, 6% was retained in the system and 3.5% was found as residual NH₄⁺-N. Zhao et al. ³⁹ also reported SND processes in packed-bed systems, examining the combination of different support media, such as grapefruit skin and several conventional plastics as polyurethane, SPR-1 suspension and elastic filling TA-II. The results showed that by combining these materials efficient SND processes could be achieved with total ammonium and nitrogen removal of 96.8 ± 4.0% and 78.9 ± 9.5%, respectively. Additionally, the microbial analysis evidenced dominant genera of Thiothrix, Gemmata and Comanonadaceae, which indicated a heterotroph nitrification –same which favored the SND process. Furthermore, Walters et al. ⁴⁰ operarated a batch system with suspended biomass and biofilm adhered to a biodegradable support medium. The results and experiments of these authors clearly indicated that nitrification may be achieved in suspended biomass while denitrification is performed at the interior of the support structure pores.

Microbial community analyses

The microbial community found in the biofilms of polyethylene rings was analyzed. This system was selected for analysis to show better performace as to the capacity of nitrogen and organic matter removal besides a larger concentration with respect to polyurethane foam. This study was performed through the analysis of the 16S rRNA fragments. A taxonomic classification of the total microbial community diversity was performed, which highlighted the microorganisms obtained at the level of phylum and genus. The bacterial abundance obtained from the sample was 99%. Table 4 shows that Proteobacteria was the dominant phylum that biofilm rings conformed, followed by Bacteroidetes and Firmicutes that are common in swine wastewater ⁴¹. This result agrees with that reported by Alzate ⁴², who mentioned that typical microbiology of aerobic systems with activated sludge are composed approximately of 95% bacteria. On the other hand, a certain abundance of archaea was observed, which was not significant (~1%).

Table 4

Relative abundance of different edges identified in biofilm of polyethylene rings
Dominium	Phylum	Relative abundance (%)
Bacteria	Proteobacteria	56.10
	Bacteroidetes	24.54
	Firmicutes	9.59
	Tenericutes	3.54
	Spirochaetes	2.43
	Fibrobacteres	0.73
	Kiritimatiellaeota	0.50
	Verrucomicrobia	0.36
	Epsilonbacteraeota	0.34
	Cloacimonetes	0.32
	Actinobacteria	0.20
	Otros	0.28
Archaea	Euryarchaeota	0.91
	Nanoarchaeaeota	0.17

Within these phyla, the presence of Pseudomonas was detected in the biofilm rings. This bacterial genus is associated to denitrification processes in the presence of aeration^{43, 44}. Zhang et al. ⁴⁵ identified P. stutzeri in swine wastewater. These authors concluded that this type of Pseudomonas may transform not only nitrate and nitrite but also ammonium with the capacity of a complete removal up to 200 mg/L of NO₃⁻-N and 170 mg/L of NO₂⁻-N in aerobic conditions. They also observed NH₄⁺-N removal of approximately 95% through a denitrification process and from this one 39% of NH₄⁺-N removed was oxidized completely to gaseous nitrogen in a total of 18 h. This result indicated that the strain has capacities for heterotrophic nitrification and aerobic denitrification with the notable capacity of removing nitrogen efficiently in the form of ammonium. This percentage agrees including with 40% ot the nitrogen not found in the effluent systems of this study in any of its soluble forms.

On the other hand, the presence of Clostridium (2.43%) in the biofilm rings would indicate nitrification processes¹⁰. Interestingly, bacteria of the type Nitrosomonas and Nitrobacter -responsible for nitrification in aerobic conditions were not found in the taxonomic analysis despite having obtained a removal efficiency of NH₄⁺ greater than 95% in system R₂. It should be highlighted that according to the MSNA assays, only may 20% of NH₄⁺-N removal be attributed to biofilm, whereas 80% of this process would have been performed by suspended biomass, which was not microbiologically analyzed.

Figure 6 shows a phylogenetic tree of the 50 most abundant bacteria found in biofilm polyethylene rings. The circles of different size correspond to abundance in readings of each microorganism, while the color indicates the order to which the genus represented in the tree belong. Finally, the bacteria not classified at the level of order and/or genus are indicated.

The following bacteria at the level of order and by abundance are: Pseudomonadales (54.81%), Bacteroidales (24.17%), Clostridiales (8.59%), Acholeplasmatales (3.01%), Spirochaetales (2.01%). The rest of the organisms that appear in Figure 6 were found in a percentage lower than 1%. In contrast with the results mentioned, Nascimento et al. ⁴⁶ reported that bacteria of the order Clostridiales are usually the most abundant in aerobic biomass. However, Pseudomonales have the capacity of growing in limited media. In other words, this phylum which showed greater proportion at genus level (56.10%) could suppress the development of taxa as Clostridium including bacteria in charge of ammonium oxidation in nitrifying conditions as Nitrosomonas spp.

After 330 days of operation of the fixed-film systems with polyethylene rings (R₁) or polyurethane foam (R₂), removal efficiency of organic matter and nitrogen were greater than 70 and 95%, respectively. However, the results of the maximum specific nitrifying activity (MSNA) kinetics were higher in the polyethylene rings (5.3 ± 0.34 mg of NO₃⁻-N/g VTS ∙h) than in polyurethane foam (4.64 ± 0.13 mg of NO₃⁻-N/g VTS ∙h), associated to the depth of the developed biofilm. Furthermore, starting from the MSNA determination, the suspended biomass contribution was estimated at 70% compared with the biomass in the form of biofilm (20%).

Finally, evidence of SND process was observed estimating that (i) approximately 40 ± 5% of nitrogen in the effluents of R₁ and R₂ that could not be quantified in any of its oxidized forms may have been transformed to molecular nitrogen; (ii) the obtained MSDA values agree with processes where nitrogen is transformed to N₂; and (iii) the analyses of the 16S rRNA fragments of the biofilm rings showed an abundance of Pseudomonas higher than 50%, bacterial genus associated to the denitrifying process in the presence of oxygen.

Acknowledgments

This work was supported by CONACYT fund Projects of Basic Science CB-2017–2018 [Grant number A1-S-43472] also by PROFAPI _2021_0047.

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No competing interests reported.

Organic Matter Removal in a Simultaneous Nitrification-Denitrification Process Using Fixed-Film System.

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Abstract

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Introduction

Materials And Methods