Next Article in Journal
Synergistic Effects of PDO and IOD on Water Vapor Transport in the Preflood Season over South China
Next Article in Special Issue
Salt Removal by Chemically Modified Graphene in Capacitive Deionization (CDI)
Previous Article in Journal
Application of BiVO4–Microalgae Combined Treatment to Remove High Concentration Mixture of Sulfamethazine and Sulfadiazine
Previous Article in Special Issue
Effect of Radio-Frequency Treatment on the Changes of Dissolved Organic Matter in Rainwater
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Efficacies of Nitrogen Removal and Comparisons of Microbial Communities in Full-Scale (Pre-Anoxic Systems) Municipal Water Resource Recovery Facilities at Low and High COD:TN Ratios

1
Department of Knowledge of The Land for Sustainable, School of Integrated Science, Kasetsart University, Bangkok 10900, Thailand
2
Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand
3
Department of Water Resources and Environmental Engineering, Tamkang University, New Taipei City 25137, Taiwan
4
Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-gu, Daegu 702-701, Korea
*
Author to whom correspondence should be addressed.
Water 2022, 14(5), 720; https://doi.org/10.3390/w14050720
Submission received: 1 January 2022 / Revised: 14 February 2022 / Accepted: 18 February 2022 / Published: 24 February 2022

Abstract

:
At a low COD:TN ratio (≤5) in influent, maintaining a longer HRT (≥9 h) and longer SRT (≥30 d) are suggested to improve higher N removal efficiency in case of operation at low DO (Dissolved oxygen) level (0.9 ± 0.2 mg-O2/L). However, in case of operation at high DO level (4.0 ± 0.5 mg-O2/L), short HRT (1 h) and typical SRT (17 d) make it possible to achieve nitrogen removal. On the other hand, at a high COD:TN ratio (≥8.4), a typical HRT (9–15 h), SRT (12–19 d), and DO level (1.3–2.6 mg-O2/L) would be applied. Microbial distribution analysis showed an abundance of AOA (Ammonia-oxidizing archaea) under conditions of low DO (≤0.9 mg-O2/L). Nitrosomonas sp. are mostly found in the all investigated water resource recovery facilities (WRRFs). Nitrosospira sp. are only found under operating conditions of longer SRT for WRRFs with a low COD:TN ratio. In comparison between abundances of Nitrobacter sp. and Nitrospira sp., abundances of Nitrobacter sp. are proportional to low DO concentration rather than abundance of Nitrospira sp. A predominance of nosZ-type denitrifiers were found at low DO level. Abundance of denitrifiers by using nirS genes showed an over-abundance of denitrifiers by using nirK genes at low and high COD:TN ratios.

1. Introduction

Organic matter and inorganic nutrients (nitrogen, N and phosphorus, P) are the main contaminants to be treated in municipal wastewaters. Discharge of inorganic nutrients into the environment is responsible for eutrophication or algal blooms and toxic effects to aquatic life. For this reason, organic matter and inorganic nutrients from municipal wastewaters need to be removed before being discharged to our environment. A biological treatment process is often recommended because of its high removal efficiency and inexpensive operational costs compared to physical and chemical treatment processes. Pre-anoxic systems, which include Modified Ludzack–Ettinger (MLE), step feed, and sequencing batch reactor (SBR), are popular. These systems consist of an anoxic tank (first zone) followed by an aerobic tank (second zone) and are specifically designed for N removal. An anaerobic system prior to anoxic and aerobic systems is designed and operated biologically, in which there is an abundance of microorganisms responsible for both N and P removals. With alkalinity provided for the nitrification step (aerobic zone) and produced denitrification step (anoxic zone), N can be removed efficiently and a good settling sludge can be produced. The energy cost of this system is low, and operation is relatively simple. Moreover, internal nitrate recycling through proper control and return of activated sludge (RAS) from the aerobic zone to the anoxic zone is the key to operating the process successfully [1]. When designing an anaerobic system prior to an anoxic and an aerobic system (or w/- and w/o anaerobic at front), wastewater characteristics, such as chemical oxygen demand (COD), total nitrogen (TN), and operational parameters including contact time in the anaerobic tank, the solids retention time (SRT), the hydraulic retention time (HRT), and the DO concentration must be taken into consideration. The proper COD:TN ratio in influent wastewater is an important parameter for biological N removal. In municipal wastewater with a low COD:TN ratio, there is insufficient carbon for the denitrification process, resulting in low N removal [2]. External carbon source addition is a significant approach to improve biological N removal (BNR) performance for wastewater with a low COD:TN ratio [3]. However, adding an external carbon source could be expensive in the case of a full-scale WRRF, where there is high capacity. To save costs, operating with longer SRT might be a potential approach to improve biological N removal performance for wastewater with a low COD:TN ratio. Phanwilai et al. [4] achieved significant N removal with a step feed treatment process operated at an SRT >60 d. Liu et al. [5] reported that a system with an SRT at 40 d outperformed systems with shorter SRTs (5, 10, and 20 d).
Maintaining low DO level in the aerobic tank could be another operating parameter to increase BNR performance. In instances with very low DO levels, such as 0–0.5 mg-O2/L, ammonia-oxidizing archaea (AOA) would be the dominant microorganism group responsible for N removal [6]. Increasing the abundance of ammonia-oxidizing bacteria (AOB) was reported with a high DO level of 1.9–3.5 mg-O2/L [6]. The domination of Nitrospira was observed at DO below 1.0 mg-O2/L [7].
Temperature and free ammonia (FA) are also important factors affecting the microbial community. A range of temperature of 10–20 °C was reported to be optimal for Nitrospira [8] and a temperature of 24–25 °C is favorable for Nitrobacter [7]. FA was an inhibitor of nitrite-oxidizing bacteria (NOB) activity [9]. Furthermore, Nitrobacter is more sensitive to FA than Nitrospira [10].
Total nitrogen removal evidence for full-scale (pre-anoxic systems) municipal WRRFs, especially for low and high COD:TN ratios, longer and typical SRTs, and various DO concentrations and temperatures is not available. For this reason, this research focused on a comparison of N removal performance, and identification and quantification of microbial communities from anaerobic, anoxic, and aerobic tanks in four full-scale municipal WRRFs having low (≤5) to high (≥8.4) COD:TN ratios. In this work, only N removal efficiencies with operational parameters (HRT, SRT, and DO level) were discussed by using results from microbial abundance and communities of bacteria related to N removal, such as AOA, AOB, NOB, and denitrifying bacteria (DNB). In addition, the results from this work could be applied to increase N removal efficiencies of other pre-anoxic w/- and w/o anaerobic WRRFs that have low and high COD:TN ratios in influent.

2. Materials and Methods

2.1. Full Scale Descriptions

A total of four full-scale municipal wastewater treatment plants were investigated: two pre-anoxic without (w/o) anaerobic process, located at the Dindaeng water resource recovery facility (WRRF), Bangkok, Thailand (L1) and the Metro Wastewater Reclamation District (MWRD), Denver, USA (H1); and two pre-anoxic with (w/-) anaerobic processes, located at the Dalseocheon WRRFs, Daegu, South Korea (L2) and the Suvarnabhumi Airport WRRF, Samutprakarn, Thailand (H2). The full-scale WRRFs were mainly designed for biological nutrient removal (BNR), especially for removal of both N and P. The influent COD:TN mass ratios at L1, L2, H1, and H2 were 3.7, 4.2, 10.9, and 8.4, respectively. Low and high COD:TN ratios of WRRFs are ≤5 and ≥8.4, respectively. At H2, the wastewaters were mainly generated from aircrafts and business and commercial buildings, such as hotels and airlines’ offices, in the area surrounding the Suvarnabhumi airport.
The schematic layouts of the full-scale pre-anoxic without (w/o) anaerobic processes at the L1 and H1 plants are shown in Figure 1A,B, respectively, and the full-scale pre-anoxic with (w/-) anaerobic processes at L2 and H2 are shown in Figure 1C,D, respectively. No primary clarifier was designed for L1 or H2 in Thailand. A total of two internal recycles are designed in these plants: the first is from an aerobic zone to an anoxic zone and the second is for the return activated sludge (RAS) that is recycled from the 2nd clarifier back to the anaerobic system. All wastewater samples (n = 12 samples) from these four full-scale WRRFs were collected every month from each sampling point (anaerobic, anoxic, and aerobic zones) twice between 2018 and 2019 (before the COVID-19 pandemic).

2.2. Wastewater Quality Analysis

BOD, COD, NH4+-N, NO2-N, NO3-N, organic-N, TKN, TN, TP, TSS, and SS from all wastewater samples were analyzed by following the standard method [11]. Only two effluent wastewater samples from L2 and H1 were measured for E. coli.

2.3. Microbiological Analysis

Molecular analysis of microbes was conducted on selected sludge samples from anaerobic, anoxic, and aerobic zones. Before the DNA extraction step, the sludge from each zone was harvested and kept on ice. A total of one mL of sludge was used for DNA extraction according to the procedures of Zhou et al. [12].
Focusing on microbial abundance by quantitative polymerase chain reaction (qPCR) analysis, a 20-μL sample was mixed with 1 μL of template DNA and 20 pmol of each primer. All qPCR reactions were performed by using a CFX96 TouchTM Real-Time PCR and CFX Manager version 3.1.1517.0823 (Bio Rad Laboratories, Inc., Hercules, CA, USA). Efficiency, slope, and r2 values of individual real-time PCR assays are 98.3–106.1%, (−3.1)–(−3.4), and 0.993–0.997, respectively, and the linearity range is 101–108, see Table S1. Total bacteria were identified via 16S-rRNA EUB gene. Ammonium-oxidizing bacteria and archaea were identified through AOB-and AOA-amoA genes. NOB were identified as Nitrospira and Nitrobacter via 16S rRNA NSR and Nitro genes, respectively. DNB were identified via nirS, nirK, and nosZ genes. Pair oligonucleotide primers of EUB gene were performed with 338F/518R (5’-ACT CCT ACG GGA GGC AGC-3’ [13]/5’-TAC CGC GGC TGC TGG CAC-3’ [14]), amoA gene of AOB with amoA-1F/2R (5’-GGG GTT TCT ACT GGT GGT-3’/5’-CCC CTC KGS AAA GCC TTC TTC–3’) [15], AOA with Arch-amoAF/AR (5’-STA ATG GTC TGG CTT AGA CG-3’/5’-GCG GCC ATC CAT CTG TAT GT-3’) [16], 16S rDNA-Nitrobacter with Nb1000F/1387R (5’-TGC GAC CGG TCA TGG-3’/5’-GGG CGG WGT GTA CAA GGC-3’) [17], 16S rDNA-Nitrospira with NSR1113F/1264R (5’-CCT GCT TTC AGT TGC TAC CG-3’ [17]/5’-GTT TGC AGC GCT TTG TAC CG-3’ [18]), DNB genes of nirS gene with cd3AF/R3cd (5’-GTS AAC GTS AAG GAR ACS GG-3’ [19]/5’-GAS TTC GGR TGS GTC TTG A-3’ [20]), nirK gene with F1aCu/ R3Cu (5’-ATY GGC GGV CAY GGC GA-3’/5’-GCC TCG ATC AGR TTR TGG TT-3’) [21], nosZ gene with nosZ2F/2R (5’-CGC RAC GGC AAS AAG GTS MSS GT-3’/5’-CAK RTG CAK SGC RTG GCA GAA-3’) [22].
Microbial communities responsible for N removal were determined by Denaturing gradient gel electrophoresis (DGGE) analysis. Pair oligonucleotide primers of 16S rRNA AOB gene were performed by nested PCR protocol with 2 steps; 1st step: CTO189fABC/CTO654r (5’-GGA GRA AAG YAG GGG ATC G-3’/5’-CTA GCY TTG TAG TTT CAA ACG C-3’) [23], and 2nd step: 357f-GC/518r (5’-CCT ACG GGA GGC AGC AG-3’/5’-ATT ACC GCG GCT GCT GG-3’) [14]. DNB genes were performed with nirS gene with cd3AF/R3cd-GC (5’-GTS AAC GTS AAG GAR ACS GG-3’/5’-GAS TTC GGR TGS GTC TTG A–3’) [20], and nirK gene with F1aCu/R3Cu-GC (5’-ATY GGC GGV CAY GGC GA-3’/5’-GCC TCG ATC AGR TTR TGG TT-3’) [20]. Each 25-μL reaction mixture was added to 1 μL of template DNA with concentrations of 10–20 ng/µL 10× Ex TaqTM buffer, 5 units/µL TaKaRa Ex TaqTM, 2.5 mM dNTP Mixture, and 10 pmol of each primer, and the mixture was finally diluted with nuclease-free water. All PCR reactions were performed by using a T100TM Thermal cycler (BioRad Laboratories, Hercules, CA, USA). The PCR product of 15 μL was loaded into individual lanes on 8% (w/v) acrylamide gel with 35–55% gradient for EUB target and with 35–50% gradient for AOB target. The electrophoresis step was performed in 1× TAE buffer at 58 °C with a constant voltage of 80 V for 16 h. The shaped DNA band on acrylamide gel was excised by a scalpel. The DNA fragments were eluted by milli-Q water and set aside in a refrigerator overnight, and then amplified by PCR with the same primer without attached CG-camp. Sequencing bases were aligned by using database of the National Center for Biotechnology Information (NCBI).

2.4. Calculations

The removal efficiencies (%) of nutrients and contaminants were calculated using Equation (1), where Cinf and Cout are concentrations (mg/L) of water quality parameters in influent and effluent of a treatment process, respectively.
Removal   efficiency   ( % ) = C inf     C out C inf × 100  
COD loading rate (kg COD/m3·d), BOD loading rate (kg BOD/m3·d), and ammonia loading rate (ALR) (kg NH4+-N/m3·d) were calculated according to Equations (2)–(4), respectively, where TCODinf and BODinf are concentration (mg/L) of total COD of the influent, (kg COD/m3) and BOD concentration of the influent, (kg BOD/m3), respectively. NH4+inf is the ammonia concentration of the influent, (kg NH4+-N/m3), Q is flow rate, (m3/d), and V is volume of the reactor, (m3).
TCOD   ( kg - N / m 3 · d ) =   TCOD inf   ×   Q   V  
BOD   ( kg - N / m 3 · d ) =   BOD inf   ×   Q   V  
ALR   ( kg - N / m 3 · d ) =   NH 4   inf +   ×   Q   V  
Free ammonia (FA) was calculated using Equation (5) according to Anthonisen et al. [24], where NH4+inf is the influent ammonium concentration (mg-N/L) and T is the temperature of the effluent (°C).
FA   mg - N / L = 17 14   ×   NH 4 + inf   ×   10 pH exp 6334 273 + T + 10 pH

2.5. Statistical Analysis for Microbial Abundances

One-way analysis of variance (one-way ANOVA) with Tukey’s honestly significant difference (HSD, at p < 0.05) was performed using Minitab 18.1 for microorganism abundance as copies-DNA. The level for statistical significance was 95%.

3. Results and Discussion

3.1. Major Operational Parameters and Performance of Full-Scale Pre-Anoxic w/o and w/- Anaerobic Process

The operational parameters (SRT, HRT, and DO) of the four WRRFs are presented in Table 1. Comparing these operational parameters at L1, L2, H1, and H2, low DO level in aerobic zone (0.9 ± 0.2 mg-O2/L), longer SRT of 30 d, and HRT (8 h) were found at L1 and high DO level (4.0 ± 0.5 mg-O2/L), and shorter SRT of 17 d and HRT (3.6 h) were found at L2. At H1, a quite low DO level (1.3 ± 0.4 mg-O2/L), SRT of 12 d, and longer HRT (9.5 h) were found, while a quite high DO level (2.6 ± 0.2 mg-O2/L), typical SRT of 19 d, and longer HRT (15.4 h) were found at H2.
The average physical and chemical characteristics of wastewater quality of the four full-scale cases are compared in Table 2. The average flow rates at L1 and L2 were high compared to H1 and H2. BOD (30 mg/L) and (75 mg/L) were found at L1 and L2, respectively. BOD (283 mg/L) and (260 mg/L) were found at H1 and H2, respectively. L1 and L2 received wastewaters with BOD (from 30 mg/L to 75 mg/L) because they treated wastewater collected from a combined sewer system with domestic sewage being diluted by storm water. Infiltration and inflow are able to enter this combined sewer system. Additionally, at L1, the high temperature inside the sewer lines could promote the degradation of BOD, and septic tank installation in the residential houses could remove BOD before wastewater entering the sewer lines. The H1 and H2 WRRFs received wastewaters with high BOD. At these WRRFs, sewage and storm water lines are separated.
BOD, COD, and N removal efficiencies in the four full-scale cases are shown at the bottom of Table 2. At low COD:TN, BOD, COD, NH4+-N, and TN removal efficiencies were 83%, 67%, 95%, and 49%, respectively at L1 and, 96%, 89%, 99%, and 70%, respectively, at L2. At high COD:TN, BOD, COD, NH4+-N, and TN removal efficiencies were 98%, 98%, 91%, and 81%, respectively at H1, and 98%, 92%, 91%, and 86%, respectively, at H2.
The average N concentration and removal efficiencies in each month are shown in Figure 2. The total nitrogen (TN) removal efficiency at L1 was quite low (only 49%) in comparison to the other WRRFs. The low TN removal could be explained by the low COD:TN ratio (≤5) in the wastewater received at L1. Low N removal efficiencies were also reported by Liu et al. [25] for WRRFs treating wastewater of relatively low COD:TN ratios. It was reported that the denitrification process could not significantly occur due to the insufficient carbon source for denitrification in wastewater having relatively low COD:TN ratios. On the contrary, the L2 with COD:TN ratio of 4.2 had an efficient TN removal of 70%. It is postulated that the plant operator has to operate with high DO level (4.0 ± 0.5 mg-O2/L), short HRT (1 h), and typical SRT (15–20 d). Associated with typical SRT, the plant operator really needs to keep significantly low or negligible DO concentration in the anoxic tank for the denitrification process to occur. In this case, it requires skillful operators to control the system correctly.
For WRRFs treating COD:TN ratio (<4) wastewater, maintaining a very long SRT (≥60 d) is recommended to overcome the low TN removal efficiency [4] as the longer SRT would increase the nitrifying bacteria abundance. Meanwhile, a long SRT could also enhance NH4+ removal by increasing nitrification activity [1]. The effluent NH4+ concentration at H2 was 4.8 mg-N/L and this was the highest among the WRRFs studied due to the plant having the highest NH4+ concentration in the raw water (55.4 mg-N/L). The effluent NH4+ concentrations in the activated sludge process were reported for SRTs at 5 d (2.6 ± 2.3 mg-N/L), 10 d (0.04 ± 0.01 mg-N/L), 20 d (0.03 ± 0.007 mg-N/L), and 40 d (0.02 ± 0.003 mg-N/L), corresponding to NH4+-N removal efficiencies of 94.5%, 99.9%, 99.9%, and 99.9%, respectively [5]. To further enhance the removal of NH4+-N, a long SRT of >19 d is recommended because it is assumed that a complete biodegradation of organic matters including readily biodegradable COD (rbCOD) and slow biodegradable COD (sbCOD) and endogenous decay of bacteria could occur due to long SRT conditions, which significantly affects denitrification process.
To solve this carbon limitation at L1 WRRF without external carbon addition implies that a future operator could operate a system with HRT (≥9 h) and long SRT (≥30 d). Associated with longer SRT, a low DO level (0.9 ± 0.2 mg-O2/L) is able to be maintained in aerobic tank. It could be postulated that the reason to remain under all these conditions is that partial nitritation and simultaneous nitrification and denitrification (SND) processes are expected to occur in the aerobic tank; possible evidence to support this statement is in Section 3.2. Furthermore, nitrogen-cycling microbial abundances and communities are related to the various environmental factors such as DO level, SRT, temperature, pH, and ammonium loading rates (ALRs), etc.

3.2. Nitrogen-Cycling Microbial Abundances and Communities

3.2.1. Ammonia-Oxidizing Archaea (AOA) and Ammonia-Oxidizing Bacteria (AOB) Targeting

Autotrophic nitrifying bacteria responsible for ammonia oxidation process were detected at L1 and belonged to two orders: Nitrosomonadales (affiliated with Nitrosomonas sp. Nitrosospira sp., Nitrosococcus sp., and Thiobacillus sp.) and Rhodocyclales (affiliated with Azospira sp., Thauera sp., and Zoogloea sp.) as shown in Table 3. Zhang et al. [26] reported that in full-scale municipal WRRFs, the most important genera of AOB were Nitrosomonas and Nitrosospira. Furthermore, they mentioned that Nitrosomonas were predominant. Consistently, in the full-scale w/- and w/o pre-anaerobic WRRFs, Nitrosomonas sp. are the most dominant AOB in the WRRFs operated at low and high DO levels. The microbial community of Nitrosospira sp. was found at the L1 plant because this WRRF was operated under a long SRT, a favorable condition for the growth of Nitrosospira sp. (see Table 3). Although the abundance of Nitrosospira sp. is less than that of Nitrosomonas sp., the existence of Nitrosospira sp. might be a suitable factor for satisfying an efficient nitrification process when the conditions are not optimal for growth of nitrifying bacteria [27].
Figure 3A shows the abundance of AOA-amoA genes at the L1 and H1 w/o pre-anaerobic systems, which is higher than in the L2 and H2 w/- pre-anaerobic systems. The abundance at L1 is the highest among the full-scale WRRFs and the statistical significance of each zone shows the high mean difference of letter grouping (Table S2, identified a letter of anoxic and aerobic zones but the others show b, c, cd, and d letters, p <0.05).
Both the L1 and H1 w/o pre-anaerobic systems had higher AOA abundance, which was expected because the lower DO level, higher temperature, and longer SRT (>30 d) would significantly promote the growth of AOA. This result is similar to the result by Yin et al. [28]. Gao et al. [6] studied the effects of DO levels on the growth of AOB-amoA and AOA-amoA, showing the former is more abundant under high DO levels of 1.9–3.5 mg-O2/L. Phanwilai et al. [4] analyzed the abundance of microorganisms in the step-feed aerobic tanks of a municipal WRRF, reporting that AOA-amoA were the most abundant genes in the tank with low DO levels (0.9 ± 0.5 mg-O2/L), while AOB-amoA genes were higher than AOA-amoA genes in the tank with high DO level (1.8 ± 0.5 mg-O2/L). In this work, the result of AOB and AOA abundance at L2 and H2 WRRFs, which are operated at high DO levels of 2.4–4.5 mg-O2/L, are in line with the results by Gao et al. [6] and Phanwilai et al. [4] (see Figure 3A). Other factors such as the high NH4+ loading rate could also increase AOB abundance. The predominance of the AOB-amoA gene over the AOA-amoA gene at L2 and H2 compared to L1 and H1 could be attributed to the higher NH4+ loading rates in those plants (see Table 2), and the significance of the gene (p <0.05) shown by the difference of letter grouping (see Table S2). The typical design DO level for a nitrogen-removal process of around 2 mg-O2/L was recommended by [1].
Although an abundance of AOA was not found at the L2 and H2 WRRFs, AOA and AOB would collaborate and offer a possible advantage in ammonia oxidation rates at the lower ammonia concentration at L1 and H1. It is postulated that in the practical operation, it is desired to maintain low DO level in an aerobic tank to reduce energy and sustain SRT range based on characteristics of each full-scale WRRF, and the abundance of AOA might be a possible group of microorganisms to collaborate with AOB for the nitrification process. However, in further research a suitable DO level and SRT range would be investigated to find the optimum conditions of growth of AOA that could collaborate with AOB.

3.2.2. Nitrite-Oxidizing Bacteria (NOB) Targeting

Figure 3B shows that Nitrobacter was more abundant than Nitrospira at L1. The DO levels (0.7 to 1.1 mg-O2/L) at L1 are the lowest among the WRRFs investigated; H1 was 0.9 to 1.7 mg-O2/L and the DO concentration ranged from 2.4 to 4.5 mg-O2/L for the other two WRRFs. The low DO condition is favorable for the growth of Nitrobacter and presented the highest significance in each zone, their grouping showed they were statistically different w/out all the extra text on the letters themselves (p < 0.05, Table S2). However, Huang et al. [7] reported that DO concentration of >1.0 mg-O2/L was a suitable condition for the growth of Nitrobacter, while a DO concentration of <1.0 mg-O2/L was optimum for growth of Nitrospira. Similarly, Park et al. [29] suggested that at the low operational DO concentration of 0.5–0.6 mg-O2/L, Nitrospira was selectively enriched over Nitrobacter in the activated sludge from a small-scale SBR. Furthermore, Liu and Wang [30] investigated the nitrification performance of activated sludge with the long-term effect of low DO concentration, finding a higher abundance of Nitrospira (1012) than Nitrobacter (1010.4) under the condition of 0.16 mg-O2/L.
Longer SRT might be possible to increase abundance of Nitrospira. Roots et al. [31] mentioned that Nitrospira increased from 3.1 to 53% under the DO level of 0.2–1.0 mg-O2/L with a 99 d SRT and NH4+ 0–14 mg-N/L. Qian et al. [32] found Nitrospira decreased from 0.44% to 0.04% with a DO level of 0.8–1.5 mg-O2/L with SRTs between 33 and 56 d and NH4+ 105 mg-N/L. Comparatively, Sun et al. [33] set a short SRT of 15 d with a DO concentration at 1.0 and 2.0 mg-O2/L that Nitrospira increased 1.81 and 2.99%, respectively. Under the longer SRT (30 d) and DO level (0.7–1.1 mg-O2/L) at L1 there was lower abundance of Nitrospira than Nitrobacter, while the three plants with the shorter SRT (17 to 26 d) and higher DO level (2.4–4.5 mg-O2/L) presented higher abundance of Nitrobacter than Nitrospira. At L2 and H2, Nitrospira was more abundant than Nitrobacter. These plants were operated at DO concentrations of 2.4–4.5 mg-O2/L, HRTs of 3.6 to 15.4 h, and SRTs of 17–19 d. These operational parameters along with the ammonium loading rate (ALR) of 0.07 and 0.14 NH4+-N/m3·d, respectively, were important factors affecting Nitrospira growth but had a lesser effect on Nitrobacter growth. However, SRT might not be the sole major effect on Nitrospira but other factors: DO, temperature, NH4+ influent, pH, HRT, FA, and ALR could also be significant factors affecting the competition between Nitrospira and Nitrobacter [9].
During the collection of all samples of this work, the temperature was recorded from 18.5 to 28 °C. For this reason, the optimal temperature ranges for Nitrobacter and Nitrospira growth are not exactly reported. Huang et al. [7] concluded that Nitrobacter was the favorable species under the temperature ranges of 24–25 °C while Nitrospira dominated at a relatively high temperature range of 29–30 °C. On the contrary, Alawi et al. [34] indicated that a lower temperature range of 10–20 °C was the optimum condition for Nitrospira growth.
Meanwhile, Nitrobacter is more sensitive to free ammonia (FA) concentration compared to Nitrospira [10]. Mehrani et al. [9] reported that FA was a major inhibitor of NOB activity. FA concentrations at L2 (0.17 mg-N/L) and H2 (0.28 mg-N/L) were higher than at L1 (0.15 mg-N/L) and H1 (0.15 mg-N/L). It could be postulated that the FA concentration was an inhibitor and decreased the abundance of Nitrobacter in these WRRFs w/- the anaerobic system, which have lower FA concentrations than L1 and H1.
In this work, only the qPCR technique was used to identify both Nitrobacter and Nitrosipra; using the specific primers to detect nitrifying bacteria population for Nitrobacter and Nitrospira are recommended in the further research. This is because Nitrospira are able to complete oxidation of NH4+ direct to NO3 without conversion to NO2 (complete ammonia oxidizer (comammox) process). If the information of Nitrospira in full-scale WRRF is reliable, a new approach for the comammox process would be applied for increasing biological N removal in the future.

3.2.3. Denitrifying Bacteria (DNB) Targeting

A total of three coding genes for nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases were evaluated for the abundance of denitrifying bacteria from the four full-scale WRRFs. As indicated in Figure 3C, a higher abundance of nosZ-type denitrifiers was found at L1 among the WRRFs investigated due to the low COD:TN ratio of ≤3.7 in L1 (see Table S2). The effects of the COD:TN ratio on the abundance of nosZ-type denitrifiers were consistent with the results reported by Yuan et al. [35] who reported that the abundance of nosZ-type denitrifiers was two orders of magnitude higher at an influent COD:TN ratio of 4.6 (1.29 × 108 copies/g-SS) compared to an influent COD:TN ratio of 8.4 (1.31 × 106 copies/g-SS) at the Beijing municipal WRRF in China.
The average number of DNB copies presenting at L1 and H1 shows that nosZ-type denitrifiers were predominant in anoxic and anaerobic zones. Wang et al. [36] found that the abundance of nosZ was a good indicator for rechecking oxygen levels of anoxic and anaerobic tanks. Based on this result, it can be concluded that the DO level in the anoxic and anaerobic tanks of L1 was quite low, and denitrifying bacteria could not use NO3 as the electron acceptor for the denitrification process, resulting in poor denitrification efficiency at L1 in the anoxic condition. As shown in Table 1, the DO level in the anoxic zone at L1 was 0.3 ± 0.1 mg-O2/L and the DO level of anoxic zone at H1 was negligible. It should be noted that the low denitrification efficiency at L1 could also be attributed to the low COD:TN ratio.
Tallec et al. [37] and Jia et al. [38] indicated that a low DO concentration in WRRFs favors nitrous oxide (N2O) production during the nitrification and denitrification process. High abundance of nosZ gene in denitrifiers was also found in the aerobic tanks of L1 and H1 WRRFs. Henry et al. [22] indicated that nosZ-type denitrifiers could be responsible in N2O production. It could be postulated that the BNR process at L1 and H1 could produce higher N2O gas among WRRFs investigated due to the low DO levels of their plants (0.9 ± 0.2 and 1.3 ± 0.4 mg-O2/L, respectively).
On the other hand, a high abundance of nirS-type denitrifiers and lower abundance of nosZ-type denitrifiers were found in the anaerobic and anoxic zones at L2 and H2 due to high DO concentration (2.4–4.5 mg-O2/L) operated by the pre-anoxic process w/- anaerobic system. Meanwhile, nirS-type denitrifiers were more prevalent than the nirK-type denitrifiers at all full-scale WRRFs. Complete denitrification is possible with nirS-type denitrifiers [36]. Che et al. [39] found a predominance of nirS-type over nirK-type in eight full-scale municipal WRRFs in different cities of China. Based on regression analysis, Zhang et al. [40] suggested that the abundance of nirK-type denitrifiers was correlated with temperature and abundance of nirS-type denitrifiers was linearly correlated with both temperature and ammonium concentration.
Both heterotrophic and autotrophic communities of denitrifying bacteria were found, as indicated in Table 3. Heterotrophic denitrifying bacteria (Ilumatobacter sp., Comamonas sp., Rhodoferax sp., Terrimonas sp., Niabella sp., Sediminibacterium sp., Tistrella sp., and Oryzobacter sp.) are normally found in WRRFs [41,42]. Autotrophic denitrifying bacteria belonging to Chloroflexi, Azospira, and Thauera commonly found in wastewater worldwide were also present. Chloroflexi are the filamentous autotrophic denitrifying bacteria, play a role in sludge flocculation, and are more commonly found in WRRFs designed to remove nutrients, and most appear with a long SRT operation and exposure of the biomass to anaerobic conditions [43]. Haliscomenobacter sp. are filamentous bacteria and thrive in phosphorus concentrations [44]. These filamentous bacteria were found and achieved removal of phosphorus in the pre-anoxic process.
Heterotrophic nitrifying bacteria (affiliated to Pseudomonas sp.) were found only at H1. Heterotrophic nitrifying bacteria (HNB) have remarkable potential in wastewater BNR engineering fields, and they can also perform aerobic denitrification reactions, directly converting NH4+ to N2 gas by a single bacterial species [45]. The high ammonium removal efficiency at H1 with the highest COD:TN ratio is likely related to the presence of the HNB.
In this work, DGGE analysis was used for identifying microbial communities responsible for only N removal. However, in further research work, high-throughput sequencing based on 16S rRNA technology or advanced techniques on the next generation sequencing (NGS) with different variable regions would be suggested as the method for microbial analysis instead of DGGE at these full-scale WRRFs.

4. Conclusions

With the low COD:N ratio in the influent of L1 and L2 WRRFs, there is insufficient carbon source for denitrifying bacteria. To solve this carbon limitation without external carbon addition, a plant operator has two options. As a first option, the plant operator is able to operate a system with HRT (≥9 h) and long SRT (≥30 d). With a longer SRT, a low DO level (0.9 ± 0.2 mg-O2/L) is able to be maintained in an aerobic tank. As a second option, the plant operator must operate with high DO level (4.0 ± 0.5 mg-O2/L), short HRT (1 h), and typical SRT (15–20 d). With a typical SRT, a plant operator needs to keep a significantly low or negligible DO concentration in the anoxic tank for denitrification process to occur.
High N removal performances of full-scale pre-anoxic process at H1 and H2 (high COD:TN ratios of ≥8.4) occurred with typical operational parameters: HRT of 9–15 h and SRT of 12–16 d.
A low DO level from 0.7 to 1.7 mg-O2/L at L1 and H1 is responsible for the high abundance of AOA over AOB. Nitrosospira could indicate that the long SRT (>30 d) is maintained at L1. In contrast, a high DO (2.4 to 4.5 mg-O2/L) at L2 and H2 contributed to the abundance of AOB over AOA. Nitrosomonas were the most abundant and other AOB populations Nitrosococcus, Thiobacillu, and Zoogloea were also present. The WRRF with high COD:TN operated with low DO level facilitated heterotrophic nitrifying bacteria as Pseudomonas sp. to high NH4+ removal efficiency. Nitrobacter sp. are more competitive than Nitrospira sp. at the low operational DO concentration of L1 and H1. In contrast, the abundance of Nitrospira could be higher than the abundance of Nitrobacter under the high DO level.
The nirS outnumbered nirK-type denitrifiers under both the low and high COD:TN conditions. A high abundance of gene-type denitrifiers (nosZ) could be found in both WRRFs with low DO concentration. Chloroflexi, Azospira, Thauera, and Haliscomenobacter are representative of the autotrophic denitrifying bacterium and Ilumatobacter, Comamonas, Rhodoferax, Terrimonas, Niabella, Sediminibacterium, Tistrella, and Oryzobacter species would work with the heterotrophic denitrifying bacteria. Maintaining a low DO level during operation of the pre-anoxic process WRRF for saving energy could be possible. However, N2O gas is able to be produced when maintaining low DO concentration in comparison to when operating at a high DO level. For this reason, future research in N2O production should be recommended in the full-scale pre-anoxic WRRF at low COD:TN ratios.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w14050720/s1, Table S1. Efficiency, slope and r2 values of individual real-time PCR assays, Table S2. Overall gene abundance of the with and without pre-anaerobic plants by multiple mean comparisons of one-way ANOVA test.

Author Contributions

Performing research, analyzing data, and writing the first draft, S.P.; intiative idea of project including funding acquisition, designing research, troubleshooting, and analyzing data, P.N.; writing—review and editing, P.N., C.-W.L., and K.-H.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by the NSRF via the Program Management Unit for Human Resources and Institutional Development, Research and Innovation (grant number: B16F630088) and Postdoctoral Fellowship from Kasetsart University Research and Development Institute (KURDI) (received funding support in October 2020 through September 2021).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This research work was supported by the NSRF via the Program Management Unit for Human Resources and Institutional Development, Research and Innovation (grant number: B16F630088) and Postdoctoral Fellowship from Kasetsart University Research and Development Institute (KURDI). The authors also would like to thank the Faculty of Engineering, Kasetsart University for their good support and Nimaradee Boonapatcharoen at Pilot Plant Development and Training Institute King Mongkut’s University of Technology Thonburi (Bangkuntien) for helpful suggestions on molecular techniques, and Barbara A. Butler, Ph.D. at U.S. Environmental Protection Agency) for final editing.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Tchobanoglous, G.; Burton, F.L.; Stensel, H.D. Wastewater Engineering: Treatment and Reuse, 4th ed.; McGraw-Hill Higher Education (Asia), McGraw-Hill: New York, NY, USA, 2004; pp. 810–815. [Google Scholar]
  2. Qian, G.; Li, L.; Hu, X.; Yu, X.; Ye, L. Enhancement of the biodegradability of activated sludge by the electric-coagulation multistage A/O membrane bioreactor treating low C/N industrial wastewater. Int. Biodeterior. Biodegrad. 2017, 125, 1–12. [Google Scholar] [CrossRef]
  3. Huang, X.; Dong, W.; Wang, H.; Jiang, S. Biological nutrient removal and molecular biological characteristics in an anaerobic-multistage anaerobic/oxic (A-MAO) process to treat municipal wastewater. Bioresour. Technol. 2017, 241, 969–978. [Google Scholar] [CrossRef]
  4. Phanwilai, S.; Noophan, P.; Li, C.-W.; Choo, K.-H. Effect of COD:N ratio on biological nitrogen removal using full-scale step-feed in municipal wastewater treatment plants. Sustain. Environ. Res. 2020, 30, 1–9. [Google Scholar] [CrossRef]
  5. Liu, G.; Wang, J. Role of solids retention time in ammonia-based feedback aeration control. J. Environ. Eng. 2016, 142, 1–8. [Google Scholar] [CrossRef]
  6. Gao, J.F.; Luo, X.; Wu, G.X.; Li, T.; Peng, Y.Z. Quantitative analyses of the composition and abundance of ammonia-oxidizing archaea and ammonia-oxidizing bacteria in eight full-scale biological wastewater treatment plants. Bioresour. Technol. 2013, 138, 285–296. [Google Scholar] [CrossRef] [PubMed]
  7. Huang, Z.; Gedalanga, P.B.; Asvapathanagul, P.; Olson, B.H. Influence of physicochemical and operational parameters on Nitrobacter and Nitrospira communities in an aerobic activated sludge bioreactor. Water Res. 2010, 44, 4351–4358. [Google Scholar] [CrossRef] [PubMed]
  8. Chen, C.; Ouyang, W.; Huang, S.; Peng, X. Microbial community composition in a simultaneous nitrification and denitrification bioreactor for domestic wastewater treatment. In IOP Conference Series: Earth and Environmental Science, Proceedings of the 2nd International Conference on Environmental Engineering and Sustainable Development (CEESD 2017) Surat Thani, Thailand, 8–10 December 2017; IOP Publishing: Bristol, UK, 2017; Volume 112, p. 012007. [Google Scholar]
  9. Mehrani, M.J.; Sobotka, D.; Kowal, P.; Ciesielski, S.; Makinia, J. The occurrence and role of Nitrospira in nitrogen removal systems. Bioresour. Technol. 2020, 303, 122936. [Google Scholar] [CrossRef] [PubMed]
  10. Ushiki, N.; Jinno, M.; Fujitani, H.; Suenaga, T.; Terada, A.; Tsuneda, S. Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation. J. Biosci. Bioeng. 2017, 123, 581–589. [Google Scholar] [CrossRef] [PubMed]
  11. APHA. Standard Methods for the Examination of Water and Wastewater, 23rd ed.; American Public Health Association: Washington, DC, USA, 2005; pp. 742–783. [Google Scholar]
  12. Zhou, L.; Bruns, M.A.; Tiedje, J.M. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 1996, 62, 316–322. [Google Scholar] [CrossRef] [Green Version]
  13. Lane, D. 16S/23S rRNA Sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley & Sons: West Sussex, UK, 1991; pp. 115–175. [Google Scholar]
  14. Muyzer, G.; de Waal, E.C.; Uitterlinden, A.G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 1993, 59, 695–700. [Google Scholar] [CrossRef] [Green Version]
  15. Rotthauwe, J.H.; Witzel, K.P.; Liesack, W. The ammonia monooxygenase structural gene amoA as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl. Environ. Microbiol. 1997, 63, 4704–4712. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Francis, C.A.; Roberts, K.J.; Beman, J.M.; Santoro, A.E.; Oakley, B.B. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc. Natl. Acad. Sci USA. 2005, 102, 14683–14688. [Google Scholar] [CrossRef] [Green Version]
  17. Wang, F.; Liu, Y.; Wang, J.H.; Zhang, Y.L.; Yang, H.Z. Influence of growth manner on nitrifying bacterial communities and nitrification kinetics in three lab-scale bioreactors. J. Ind. Microbiol. Biotechnol. 2012, 39, 595–604. [Google Scholar] [CrossRef]
  18. Yapsakli, K.; Aliyazicioglu, C.; Mertoglu, B. Identification and quantitative evaluation of nitrogen-converting organisms in a full-scale leachate treatment plant. J. Environ. Manage. 2011, 92, 714–723. [Google Scholar] [CrossRef] [PubMed]
  19. Michotey, V.; Mejean, V.; Bonin, P. Comparison of methods for quantification of cytochrome cd1-denitrifying bacteria in environmental marine samples. Appl. Environ. Microbiol. 2000, 66, 1564–1571. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Throbäck, I.N.; Enwall, K.; Jarvis, A.; Hallin, S. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol. Ecol. 2004, 49, 401–417. [Google Scholar] [CrossRef]
  21. Hallin, S.; Lidgren, R.E. PCR detection of genes encoding nitrite reductase in denitrifying bacteria. Appl. Environ. Microb. 1999, 65, 1652–1657. [Google Scholar] [CrossRef] [Green Version]
  22. Henry, S.; Bru, D.; Stres, B.; Hallet, S.; Philippot, L. Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ Genes in soils. Appl. Environ. Microbial. 2006, 72, 5181–5189. [Google Scholar] [CrossRef] [Green Version]
  23. Kowalchuk, G.A.; Stephen, R.J.; Boer, W.D.; Prosser, J.I.; Embley, T.M.; Woldendorp, J.W. Analysis of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl. Environ. Microbiol. 1997, 63, 1489–1497. [Google Scholar] [CrossRef] [Green Version]
  24. Anthonisen, A.C.; Loehr, C.; Prakasam, T.B.S. Srinath EG. Inhibition of nitrification and nitrous acid compounds. J. Water Pollut. Control Fed. 1976, 48, 835–852. [Google Scholar]
  25. Liu, H.B.; Yang, C.Z.; Pu, W.H. Removal of nitrogen from wastewater for reusing to boiler feed-water by an anaerobic/aerobic/membrane bioreactor. Chem. Eng. J. 2008, 140, 122–129. [Google Scholar] [CrossRef]
  26. Zhang, T.; Ye, L.; Yan Tong, A.H.; Shao, M.-F.; Lok, S. Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors. Appl. Microbiol. Biotechnol. 2011, 91, 1215–1225. [Google Scholar] [CrossRef] [Green Version]
  27. Siripong, S.; Rittmann, B.E. Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants. Water Res. 2007, 41, 1110–1120. [Google Scholar] [CrossRef] [PubMed]
  28. Yin, Z.; Bi, X.; Xu, C. Ammonia-oxidizing archaea (AOA) play with ammonia-oxidizing bacteria (AOB) in nitrogen removal from wastewater. Archaea. 2018, 4, 8429145. [Google Scholar] [CrossRef] [Green Version]
  29. Park, M.R.; Park, H.; Chandran, K. Molecular and kinetic characterization of planktonic Nitrospira spp. selectively enriched from activated sludge. Environ. Sci. Technol. 2017, 51, 2720–2728. [Google Scholar] [CrossRef]
  30. Liu, G.; Wang, J. Long-term low DO enriches and shifts nitrifier community in activated sludge. Environ. Sci. Technol. 2013, 47, 5109–5117. [Google Scholar] [CrossRef]
  31. Alawi, M.; Lipski, A.; Sanders, T.; Eva Maria, P.; Spieck, E. Cultivation of a novel cold-adapted nitrite oxidizing betaproteobacterium from the Siberian Arctic. Isme J. 2007, 1, 256–264. [Google Scholar] [CrossRef] [Green Version]
  32. Roots, P.; Wang, Y.; Rosenthal, A.; Griffin, J.; Sabba, F.; Petrovich, M.; Yang, F.; Kozak, J.; Zhang, H.; Wells, G. Comammox Nitrospira are the dominant ammonia oxidizers in a mainstream low dissolved oxygen nitrification reactor. Water Res. 2018, 157, 396–405. [Google Scholar] [CrossRef] [PubMed]
  33. Qian, F.; Wang, J.; Shen, Y.; Wang, Y.; Wang, S.; Chen, X. Achieving high performance completely autotrophic nitrogen removal in a continuous granular sludge reactor. Biochem. Eng. J. 2017, 118, 97–104. [Google Scholar] [CrossRef]
  34. Sun, Z.; Liu, C.; Cao, Z.; Chen, W. Study on regeneration effect and mechanism of high-frequency ultrasound on biological activated carbon. Ultrason. Sonochem. 2018, 44, 86–96. [Google Scholar] [CrossRef] [PubMed]
  35. Yuan, Q.; Wang, H.Y.; Chua, Z.-S.; Hang, Q.-Y.; Liu, K.; Li, C.M. Influence of C/N ratio on MBBR denitrification for advanced nitrogen removal of wastewater treatment plant effluent. Desalin. Water. Treat. 2017, 66, 158–165. [Google Scholar] [CrossRef]
  36. Wang, Z.; Zhang, X.X.; Lu, X.; Liu, B.; Li, Y.; Long, C.; Li, A. Abundance and diversity of bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants revealed by high-throughput sequencing. PLoS ONE. 2014, 9, e113603. [Google Scholar] [CrossRef] [PubMed]
  37. Tallec, G.; Garnier, J.; Billen, G.; Gousailles, M. Nitrous oxide emissions from secondary activated sludge in nitrifying conditions of urban wastewater treatment plants: Effect of oxygenation level. Water Res. 2006, 40, 2972–2980. [Google Scholar] [CrossRef] [PubMed]
  38. Jia, W.; Liang, S.; Zhang, J.; Ngo, H.H.; Guo, W.; Yan, Y.; Zou, Y. Nitrous oxide emission in low-oxygen simultaneous nitrification and denitrification process: Sources and mechanisms. Bioresour. Technol. 2013, 136, 444–451. [Google Scholar] [CrossRef]
  39. Che, Y.; Liang, P.; Gong, T.; Cao, X.; Zhao, Y.; Yang, C.; Song, C. Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen cycling genes in activated sludge from WRRFs. Scientific Reports. 2017, 7, 44728. [Google Scholar] [CrossRef] [PubMed]
  40. Zhang, L.; Zeng, G.; Zhang, J.; Chen, Y.; Yu, M.; Lu, L.; Li, H.; Zhu, Y.; Yuan, Y.; Huang, A.; et al. Response of denitrifying genes coding for nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases to different physico-chemical parameters during agricultural waste composting. Appl. Microbiol. Biotechnol. 2015, 99, 4059–4070. [Google Scholar] [CrossRef]
  41. Heylen, K.; Vanparys, B.; Wittebolle, L.; Verstraete, W.; Boon, N.; Vos, P.D. Cultivation of denitrifying bacteria: Optimization of isolation conditions and cultivation of denitrifying bacteria diversity study. Appl. Environ. Microbiol. 2006, 72, 2637. [Google Scholar] [CrossRef] [Green Version]
  42. McIlroy, S.J.; Starnawska, A.; Starnawski, P.; Saunders, A.M.; Nierychlo, M.; Nielsen, P.H.; Nielsen, J.L. Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems. Environ. Microbiol. 2016, 18, 50–64. [Google Scholar] [CrossRef]
  43. Speirs, L.B.M.; Rice, D.T.F.; Petrovski, S.; Seviour, R.J. The phylogeny, biodiversity, and ecology of the Chloroflexi in activated sludge. Front. Microbiol. 2019, 10, 1–28. [Google Scholar] [CrossRef] [Green Version]
  44. Kämpfer, P.; Weltin, D.; Hoffmeister, D.; Dott, W. Growth requirements of filamentous bacteria isolated from bulking and scumming sludge. Water Res. 1995, 29, 1585–1588. [Google Scholar] [CrossRef]
  45. Wang, Q.; He, J. Complete nitrogen removal via simultaneous nitrification and denitrification by a novel phosphate accumulating Thauera sp. strain SND5. Water Res. 2020, 185, 116300. [Google Scholar] [CrossRef] [PubMed]
Figure 1. The schematic layout of the full-scale WRRFs at (A) L1, (B) H1, (C) L2, and (D) H2.
Figure 1. The schematic layout of the full-scale WRRFs at (A) L1, (B) H1, (C) L2, and (D) H2.
Water 14 00720 g001aWater 14 00720 g001b
Figure 2. Nitrogen performance with low COD:TN ratio at (A) L1 and (B) L2, and high COD:TN ratio at (C) H1 and (D) H2.
Figure 2. Nitrogen performance with low COD:TN ratio at (A) L1 and (B) L2, and high COD:TN ratio at (C) H1 and (D) H2.
Water 14 00720 g002
Figure 3. Microbial abundance of (A) amoA gene-AOA and -AOB, (B) NOB, and (C) DEN at L1, L2, H1, and H2.
Figure 3. Microbial abundance of (A) amoA gene-AOA and -AOB, (B) NOB, and (C) DEN at L1, L2, H1, and H2.
Water 14 00720 g003
Table 1. Operational parameters of the full-scale pre-anoxic zone w/- and w/o anaerobic systems by low and high COD:TN ratio.
Table 1. Operational parameters of the full-scale pre-anoxic zone w/- and w/o anaerobic systems by low and high COD:TN ratio.
Operational ParameterLow COD:TN (≤5)High COD:TN (≥8.4)
L1 (w/o)L2 (w/-)H1 (w/o)H2 (w/-)
SRT (d)30171219
HRT (total) (h)7.53.69.515.4
Anaerobic-1.0-1.3
Anoxic1.51.61.53.1
Aerobic6.01.08.011.0
DO (mg-O2/L)
Anoxic0.3 ± 0.1NegligibleNegligible0.1
Aerobic0.9 ± 0.24.0 ± 0.51.3 ± 0.42.6 ± 0.2
Table 2. Comparing the average physical and chemical characteristics of wastewater quality in full-scale pre-anoxic zone w/- and w/o anaerobic systems by low and high COD:TN ratio.
Table 2. Comparing the average physical and chemical characteristics of wastewater quality in full-scale pre-anoxic zone w/- and w/o anaerobic systems by low and high COD:TN ratio.
ParameterLow COD:TN (≤5)High COD:TN (≥8.4)
L1 (w/o)L2 (w/-)H1 (w/o)H2 (w/-)
Inlet/Outlet
pH7.2 ± 0.01/7.2 ± 0.017.2 ± 0.2/6.9 ± 0.27.0 ± 0.2/7.2 ± 0.17.2 ± 0.1/7.2 ± 0.03
Temp (°C)28.1 ± 0.5/27.7 ± 0.421.5 ± 2.5/24 ± 0.718.5 ± 2.3/18.5 ± 0.627 ± 0.1/26.9 ± 0.3
SS (mg/L)46.7 ± 5.8/8.6 ± 0.7202 ± 71.3/2 ± 0.3321.7 ± 122.5/15.3 ± 1.3178.5 ± 38.1/4.7 ± 2.5
BOD (mg/L)30.1 ± 2.7/5.0 ± 1.275 ± 26.4/3 ± 0.3283.0 ± 43.2/4.4 ± 0.7260 ± 6.52/1 ± 0.3
COD (mg/L)58 ± 25.9/19 ± 1.588 ± 25.1/10 ± 0.6452.8 ± 48.5/7.4 ± 1.8511.6 ± 36.0/40.5 ± 1.3
NH4+ (mg-N/L)11.0 ± 1.1/0.6 ± 0.210.6 ± 2.2/0.2 ± 0.128.5 ± 2.3/0.5 ± 0.255.4 ± 7.3/4.8 ± 0.2
NO3 (mg-N/L)0.2 ± 0.07/5.3 ± 1.00.1 ± 0.02/6.3 ± 2.5-/--/-
Alkalinity (mg/L)-/--/-247.8 ± 8.0/126.4 ± 7.8344.2 ± 9.3/154.3 ± 45.5
TKN (mg/L)15.4 ± 1.4/2.6 ± 0.9-/-46.2 ± 5.6/-60.8 ± 6.6/6.1 ± 0.3
TN (mg-N/L)15.6 ± 1.4/8.0 ± 0.327 ± 5.1/8 ± 0.441.3 ± 2.9/7.9 ± 0.761.2 ± 6.9/8.9 ± 0.1
TP (mg-P/L)2.3 ± 0.1/1.5 ± 0.24 ± 1.1/0.2 ± 0.02-/-7.1 ± 0.2/0.3 ± 0.1
E. Coli (MPN)-/-44,845 ± 20,782.6/22 ± 42.8-/40-/-
Removal Efficiency (%)
SS82999597
BOD83969898
COD67899892
NH4+95999891
TKN83--90
TN49708186
TP3595-96
Other information
Avg. Flow rate (m3/d)218,433220,65577,9177673
MlSS (mg/L)4509.5 ± 414.173455 ± 3803577 ± 5153315 ± 328
MLVSS (mg/L)2542 ± 4142780 ± 2802862 ± 4122610 ± 208
COD:TN ratio3.74.210.98.4
COD loading rate (kg-COD/m3·d)0.190.590.711.29
BOD loading rate (kg-BOD/m3·d)0.100.500.440.50
ALR (kg NH4 N/m3·d)0.040.070.040.14
TNLR (kg-N/L-d)0.050.180.060.15
TNRR (kg-N/L-d)0.020.250.050.13
FA (mg-N/L)0.150.170.150.28
Remark: - = Not record.
Table 3. Microorganisms’ community in four municipal WRRFs.
Table 3. Microorganisms’ community in four municipal WRRFs.
OrderSpecies%Accession No.Low COD:TN (≤5)High COD:TN (≥8.4)
L1L2H1H2
AnxAerAnaAnxAerAnxAerAnaAnaAer
Nitrifying bacteria: Ammonia oxidizing bacteria (AOB)
NitrosomonadaleNitrosomonasaestuarii90NR104818.1
Nitrosomonas eutropha93NR027566.1
Nitrosomonas communis97NR119314.1
Nitrosomonas halophila93NR104817.1
Nitrosomonas marina99NR104815.1
Nitrosomonas oligotropha96NR104820.1
Nitrosomonas stercoris98NR146824.1
Nitrosomonas ureae97NR104814.1
Nitrosospira multiformis96NR074736.1
Nitrosospira tenuis97NR114773.1
Uncultured Nitrosospira95GQ255611.1
Thiobacillus thioparus96NR117864.1
RhodocyclalesZoogloea caeni91NR043795.1
Nitrifying bacteria: Nitrite oxidizing bacteria (NOB)
NitrospiraeNitrospira lenta99NR148573.1
Heterotrophic nitrifying bacteria (HNB)
PseudomonadalesPseudomonas asturiensis98NR108461.1
Pseudomonas fragi98MT176180.1
Pseudomonas fluorescens98CP027561.1
Pseudomonasputida99MH778047.1
Denitrifying bacteria (DNB): Autotrophic denitrifying bacteria
ChloroflexiChloroflexi bacterium87KP246879.1
Uncultured Chloroflexi98GQ366686.1
RhodocyclalesAzospira restricta97NR044023.1
Thauera aromatica100NR026153.1
Thauera aminoaromatica93NR027211.1
SaprospiralesHaliscomenobacter hydrossis90NR074420.1
Denitrifying bacteria (DNB): Heterotrophic denitrifying bacteria
AcidimicrobialesIlumatobacter fluminis86NR041633.1
BurkholderialesComamonas denitrificans99NR025080.1
Comamonas phosphati96NR147778.1
Rhodoferax ferrireducens92NR074760.1
ChitinophagalesTerrimonas lutea96NR041250.1
Niabella terrae92NR132698.1
Sediminibacterium roseum82NR159130.1
RhodospirillalesTistrella mobilis91NR117256.1
MicrococcalesOryzobacter terrae98NR137270.1
Remark: ✕ is DGGE band presenting on acrylamide gel, Ana is an anaerobic system, Anx is an anoxic zone, and Aer is an aerobic zone.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Phanwilai, S.; Noophan, P.; Li, C.-W.; Choo, K.-H. Efficacies of Nitrogen Removal and Comparisons of Microbial Communities in Full-Scale (Pre-Anoxic Systems) Municipal Water Resource Recovery Facilities at Low and High COD:TN Ratios. Water 2022, 14, 720. https://doi.org/10.3390/w14050720

AMA Style

Phanwilai S, Noophan P, Li C-W, Choo K-H. Efficacies of Nitrogen Removal and Comparisons of Microbial Communities in Full-Scale (Pre-Anoxic Systems) Municipal Water Resource Recovery Facilities at Low and High COD:TN Ratios. Water. 2022; 14(5):720. https://doi.org/10.3390/w14050720

Chicago/Turabian Style

Phanwilai, Supaporn, Pongsak (Lek) Noophan, Chi-Wang Li, and Kwang-Ho Choo. 2022. "Efficacies of Nitrogen Removal and Comparisons of Microbial Communities in Full-Scale (Pre-Anoxic Systems) Municipal Water Resource Recovery Facilities at Low and High COD:TN Ratios" Water 14, no. 5: 720. https://doi.org/10.3390/w14050720

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop