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Functional and compositional characteristics of nitrifiers reveal the failure of achieving mainstream nitritation under limited oxygen or ammonia conditions
Bioresource Technology 275 (2019) 272–279
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Functional and compositional characteristics of nitrifiers reveal the failure of achieving mainstream nitritation under limited oxygen or ammonia conditions
T
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Wenru Liua, , Wenjing Chenb, Dianhai Yangc, Yaoliang Shena a
National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology, Suzhou 215009, China b School of Environmental Engineering and Science, Yangzhou University, Yangzhou, Jiangsu 225127, China c School of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrification Nitritation Mainstream Dissolved oxygen Nitrosomonas Nitrospira
For understanding the potential of achieving nitritation under different oxygen and ammonia levels, two activated sludge reactors with high (RH) and low (RL) dissolved oxygen (DO) were parallelly operated. During over two months continuous operation, rare nitrite accumulation was observed in both reactors. K-strategists Nitrosomonas oligotropha and r-strategists Nitrosomonas europaea were enriched in the RH and RL, respectively, yet their response to DO variations was almost identical. Although K-strategists Nitrospira defluvii dominated both reactors, species cultured with low-DO exhibited higher oxygen affinity. Instead of DO, ammonia and nitrite availability should be the key factor for the selective enrichment of these nitrifiers. Taken together, the limiting ammonia for ammonia oxidizing bacteria and the better oxygen-uptake capacity of nitrite oxidizing bacteria was respectively responsible for the failure of nitrite accumulation in the RH and RL. This study supported that high DO coupled with excess ammonia would favor the achievement of mainstream nitritation.
1. Introduction Sewage treatment including high-rate and stable mainstream shortcut or nitrite pathway nitrogen removal processes, such as nitritation/
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denitritation and partial nitritation/anammox, has the potential to become energy-neutral or even energy-producing (Kartal et al., 2010). The successful implementation of the nitrite pathway relies on the reliable nitritation, which achieved through effective selection of the
Corresponding author. E-mail address: [email protected] (W. Liu).
https://doi.org/10.1016/j.biortech.2018.12.065 Received 27 November 2018; Received in revised form 16 December 2018; Accepted 20 December 2018 Available online 21 December 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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wastewater containing 190 mg·L−1 NH4Cl, 50 mg·L−1 K2HPO4, 20 mg·L−1 CaCl2·2H2O, 25 mg·L−1 MgSO4·7H2O and 1 mL·L−1 of trace element solution as described by Bellucci et al (2011). For all working periods, the influent loading rate (150 g N·m−3·d−1), the hydraulic retention time (8 h) and the SRT (20 days) were almost constant. The pH in each reactor was maintained between 7.0 and 7.5 by dosing a buffer containing NaHCO3. Both reactors were operated at 22 °C constant temperature. Compressed air was introduced by a fine bubble diffuser placed at the bottom of each reactor. The RH was run at DO concentrations above 2.0 mg O2·L−1, while the RL was operated with DO near to 0.3 mg O2·L−1. After process operating for more than three SRTs, sludge samples were taken from RH and RL on day 70. These samples were immediately centrifuged at 10000 rpm for 3 min to separate water and solids. The sludge pellet was persevered under −20 °C for DNA extraction within one month.
ammonium oxidizing bacteria (AOB) but stable washout of the nitrite oxidizing bacteria (NOB) (Agrawal et al., 2018). However, considering the difficulty of suppression of NOB in continuous mainstream activated sludge processes under the dilute and cold conditions, till now only limited and/or nascent approaches for achieving nitritation have been reported reported (Ge et al., 2014; Isanta et al., 2015; Ma et al., 2009; Regmi et al., 2014; Wu et al., 2016). In addition to the unfavourable mainstream conditions, the challenge of achieving mainstream nitritation also relates to the complicated nitrifiers composition and their quite unpredictable life-history strategies (i.e., growth and reproductive rates). As new discoveries confirmed through combining different molecular techniques that the genus Nitrospira, generally being the main culprit for failure of mainstream nitrite pathway, had an unexpected high biodiversity and a surprisingly physiological versatility (Gruber-Dorninger et al., 2015; Koch et al., 2015). The metabolic versatility, as basis of ecological niche partitioning within the genus Nitrospira, indicated that members of Nitrospira may survive and coexist with other microorganisms in diverse microniches and even in the nitrite-limited and/or anoxic micro-environment (Annavajhala et al., 2018; Daims et al., 2016; Lücker et al., 2010). Consequently, the commonly used low-DO control for inhibiting Nitrospira like-NOB to achieve long-term mainstream nitritation was often proved to be ineffective. One of the preconditions for the establishment of nitritation is the difference between ammonium and nitrite oxidization rates. Note that in the mainstream the AOB-NOB growth rate (or metabolic activity) differential are dependent on optimal oxygen and nitrogen substrate levels, according to their individual Mond kinetics (Regmi et al., 2014). Moreover, from the ecological perspective, the selection of specific nitrifiers (e.g., fast growth AOB) is generally linked with the environmental conditions such as the availability of substrate (oxygen and/or ammonia) (Dytczak et al., 2008). Therefore, one of the key problems for achieving mainstream nitritation is how to correctly control the DO and ammonia levels in the bulk liquid. Previous studies have demonstrated that the concurrently low DO and low ammonia condition was unsuitable for nitrite accumulation (Bellucci et al., 2011; Fitzgerald et al., 2015; Liu & Wang, 2013; Park & Noguera, 2004). However, a relatively high DO coupled with adequate residual ammonia in the bulk liquid would be in favor of achieving nitritation (Liu et al., 2017; Regmi et al., 2014; Wu et al., 2016). The potential and mechanisms for nitrite accumulation under high DO but with limited residual ammonia or high residual ammonia but limited DO conditions have not been well described or clarified till now, since these operating conditions were frequently present at the conventional wastewater treatment plants and the emerging autotrophic nitrogen removal processes. In this study, two nitrifying processes treating low-strength wastewater were parallelly operated under two different oxygen and ammonium levels. The purpose of this study was to understand the nitrite accumulation potential under the given DO and ammonium levels. Functional and compositional analysis of microbial communities was conducted to provide insights into the microbial community structure of the two nitrifying processes, and reveal the link between microbial community structure and process performance.
2.2. In-situ and ex-situ batch experiments In-situ batch tests were conducted inside the RH and RL, respectively, to evaluate the short-time performance of the reactors under the two conditions, (1) high DO and excess ammonium condition, and (2) low DO and excess ammonium condition. When the batch tests were conducted, continuous operation was stopped. The bulk liquid was exchanged with new substrate of the desired initial ammonium concentration (25–30 mg N·L−1). After the bulk liquid had been exchanged the air supply was switched on again. During the batch tests, the operational conditions, except DO, was maintained the same as the normally operated RH and RL. Ex-situ batch experiments were conducted in some 300 mL reactors to evaluate the activities of the nitrifying biomass under the different levels of DO. Specific ammonium uptake/utilization rate (SAUR) and specific nitrite uptake/utilization rate (SNUR) were used to represent the activities of communities. For testing the SAUR, ammonium (25 mg N·L−1) was supplied in excess; to evaluate the SNUR, nitrite (20 mg N·L−1) was used instead of ammonium. pH was maintained between 7 and 7.5 by adding sodium bicarbonate. Triplicate experiments were conducted at every DO level. All batch experiments were carried out in three replicates at 22 °C. 2.3. Analytical methods Concentrations of NH4+-N, NO2−-N, NO3−-N, total suspended solids (TSS) and volatile suspended solids (VSS) were analyzed in accordance with standard methods (APHA, 2005). Temperature, DO and pH were monitored by pH/oxi1970i meter with DO and pH probes (WTW Multi1970i, Germany). The morphology of granules was measured by microscope (OLYMPUS CX41, Japan) with an attached digital camera. 2.4. DNA extraction and amplicon sequencing Identification of the microbial population was performed using next-generation sequencing. DNA was extracted from sludge samples collected in the RH and RL by using FastDNA SPIN Kits for Soil (MP Biomedicals, LLC, Solon, OH) following the manufacturer’s instructions. The quality and quantity of extracted DNA were checked by Nanodrop spectral photometer (Thermo Fisher Scientific, Beverly, MA, USA). A minimum concentration of 20 ng·L−1 of extracted DNA should be reached. Paired-end sequencing of the extracted DNA was performed on an Illumina MiSeq platform according to standard protocols. The primers set F515 (5′-GTGCCAGCMGCCGCGG-3′) and R907 (5′-CCGTCAATTCMTTTRAGTTT-3′) were used for PCR amplification. To minimize the effects of random sequencing error, low-quality sequences were removed by eliminating those without an exact match to the forward primer. After demultiplexing, quality trimming and alignment, chimeric
2. Materials and methods 2.1. Reactors description and sludge sampling Two identical laboratory-scale complete-mixing reactors (working volume of 2.4 L each) as previously described (Liu & Yang, 2017), namely RH and RL, were set up. Both reactors were inoculated with the same suspended biomass from Quyang WWTP (Shanghai, China), which operates an anaerobic/anoxic/aerobic (AAO) process with approximately a 10-day sludge retention time (SRT). After inoculation, the obtained initial MLVSS concentration in each reactor was about 2.2 ± 0.2 g VSS L−1. The reactors were fed continuously with synthetic 273
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sequences were removed after finally being checked with a Chimer Slayer algorithm. The reads flagged as chimeras were extracted out, and the non-chimera reads then formed the database of ‘effective reads’ for each sample. Eventually, the two samples resulted in 36,966 high quality sequences (18,483 for each sample on average) with an average length of 396 bp.
ammonium excess and oxygen limited condition. Interestingly, similar ammonium removal performance (full nitrification) was maintained under both operating conditions. 3.1.2. Batch experiments Fig. 2 gives further information about the nitrification characteristics of each reactor through in-suit batch experiments. For the batch experiment with a DO of 2.0 ± 0.1 mg O2·L−1 in the RH (Fig. 2A), ammonium was oxidized gradually with the production of nitrate. Concurrently, and unexpectedly, transient nitrite accumulation was also observed and reached the peak value when the ammonium was nearly exhausted. These observations partially contradicted the longterm nitrification performance of the RH. Since nitrite accumulation should be resulted from the different rates between ammonium oxidation and nitrite consumption, the observed transient nitrite accumulation suggested the higher activity of AOB than that of NOB under the specific conditions (high DO and excess ammonium). Interestingly, during the batch experiments with the oxygen-limited condition (Fig. 2B), nitrite accumulation was also observed. Similar nitrification performance was observed in the RL during batch experiments. As shown in Fig. 2C and D, the concentration of ammonium gradually decreased with time; meanwhile, a corresponding increase of nitrate was observed. However, the nitrite accumulation only occurred during batch experiments with the high DO concentration (Fig. 2D). In brief, independent of the concentration of ammonium in the reactor bulk liquid, the produced nitrite by AOB could be rapidly oxidized to nitrate by NOB under the oxygen-limited condition, resulting in the little nitrite accumulated in the RL.
2.5. Biodiversity analysis and phylogenetic classification The sequences were clustered into operational taxonomic units (OTUs) at a cutoff of 97% sequence identity using the MOTHUR program. Raw sequencing data that obtained from this study were deposited to the NCBI Sequence Read Archive with the project accession number of PRJNA509048. Differences in order-based Hill numbers (based on the number of OTUs, Shannon index and Simpson index) were determined via MOTHUR program. Phylogenetic trees were constructed using MEGA7 based on neighborjoining method. The relative abundance of a given phylogenetic group was set as the number of sequences affiliated with that group divided by the total number of sequences per sample. 3. Results 3.1. Nitrification performance
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3.1.1. Long-term operation The two reactors were continuously operated for more than three SRTs with the constant influent volumetric nitrogen loading rate of 0.15 kg N·m−3·d−1. Almost all the oxidized ammonium was converted to nitrate without nitrite accumulation during the entire experimental run (Fig. 1), that is, full nitrification was maintained in both reactors with different operating DO. Note that starting from the first day, nearly 100% ammonium removal efficiency had been obtained in the RH with the stabilized effluent ammonium concentration below 0.1 mg N·L−1 (Fig. 1A). However, likely due to the limited oxygen supply, the ammonium removal rate of the RL only reached 0.09 kg N m−3·d−1 on day 70 and thereby the RL was operated with the excess ammonium concentrations detected in the bulk liquid throughout the investigation (Fig. 1B). In other words, the RH was run with ammonium limitation but oxygen excess, while the biomass for the RL was cultivated under
3.2. Short-term effect of DO on response nitrification activity Batch tests were also conducted to investigate the short-term effect of DO on nitrifying activity (specific ammonium and nitrite oxidization rates) of the sludge cultivated in RH and RL, since operating DO was the primary stress driven the different performance between the two reactors. The obtained SAUR and SNUR values were normalized at high DO applied in each case, as shown in Fig. 3. For the sludge cultivated in the RH (Fig. 3A), a low DO (0.2–0.3 mg O2·L−1) greatly inhibited both the SAUR and SNUR, with an average inhibition efficiency of 73% and 70% respectively. For the sludge cultivated in the RL (Fig. 3B), a low DO also greatly inhibited the SAUR (71.5% reduction), but only lead to an average decrease of 26% in SNUR. The different response of SNUR to DO concentrations indicated the less oxygen sensitivity of NOB cultivated in the low DO reactor (RL) as compared to the high DO reactor (RH). Interestingly, similar observations were also reported by Liu and Wang (2013), although the operating conditions in their study were different from the present research. They demonstrated that the similar response of the ammonium oxidizing rate (AOR) to a low DO concentration resulted from the same dominant AOB community (Nitrosomonas europaea/eutropha) in both sludges (cultivated with a high DO and a low DO), whereas the nitrite oxidizing rate (NOR) for the sludge cultivated with low DO became less sensitive to low DO concentrations due to the enrichment of Nitrospira-like NOB (with high oxygen affinity) instead of Nitrobacter like-NOB. To further reveal the failure of achieving nitritation in the present study, the bacterial communities were also investigated using Illumina MiSeq high-throughput sequencing technology.
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3.3.1. Microbial community diversity The bacterial community diversity in both reactors was evaluated on day 70 by means of the Hill diversity order numbers, as shown in Fig. 4. Each of the three order numbers of the bacterial community in the RH was significantly lower than that in the RL, suggesting the significantly higher bacterial diversity of sludge cultivated under a low DO
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3.3.2. Nitrifying community composition The structure and abundance of nitrifying bacteria in both reactors was analyzed based on the Illumina sequencing results, as shown in Fig. 5. Almost all the identified sequences belonging to the known AOB in both reactors were affiliated to Nitrosomonas genus. However, the preponderant Nitrosomonas-AOB species was different between the reactors (Fig. 5A). More specifically, the OTU0002, closely related to Nitrosomonas europaea, was the most abundant AOB in the RL, accounting for 14.73% of the total bacterial community. Instead OTU0001, most similar to the Nitrosomonas oligotropha, represented 57.17% of the total bacterial community, with the highest levels in the RH. Additionally, the relative abundance of total AOB was significantly higher in RH (61.52%) than in RL (17.62%). However, the known-AOB community in both reactors had the similar species diversity. For nitrite oxidation, three different OUT clusters within the Nitrospira genus were
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Fig. 4. Microbial community diversity of the samples for both reactors on day 70. The three Hill order diversity numbers H0 (number of OTUs), H1 (exponential value of the Shannon index), and H2 (inverse Simpson index) were calculated for bacteria.
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Fig. 2. Profile of ammonium, nitrite, and nitrate concentrations during a representative in-suit batch experiment performed on day 70 in the RH with a DO of 2.0 ± 0.1 mg O2·L−1 (A) and 0.2–0.3 mg O2·L−1 (B), and in the RL with a DO of 0.2–0.3 mg O2·L−1 (C) and 2.0 ± 0.1 mg O2·L−1 (D).
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Fig. 3. SAUR and SNUR under a high DO (4.0 ± 0.1 mg O2 L−1) and a low DO (0.2–0.3 mg O2·L−1) for the sludge obtained from RH (A) and RL (B) on day 70.
in comparison with the biomass cultivated under a high DO. Moreover, the significantly higher microbial community diversity for biomass in the RL also suggested that the RL had greater microbial community redundancy and more metabolic potential compared with the RH. 275
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Fig. 5. Relative abundance and phylogenetic affiliation of (A) AOB and (B) NOB based on 16S rRNA gene sequence. Sequences with 0.1obtained in this study are shown with “OUT” in the names. Other sequences were obtained from GenBank. Numbers at the branch nodes are bootstrap values. The scale bar represents 1% estimated sequence divergence. Relative abundance is defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample (%). OUTs making up less than 0.1% of the total composition in both libraries are not shown. 276
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commonly used start-up strategy of nitritation at low ammonium loading rates or low DO should, therefore, be reconsidered. Additionally, results from our short-term batch experiments indicated that obviously nitrite accumulation, though transient, was observed with the both high ammonium concentrations and oxygen levels (Fig. 2). The results are consistent with previous proposed strategies of operating nitritation process with high DO and enough residual ammonium in the bulk liquid (Park et al., 2010; Regmi et al., 2014; Wu et al., 2016). Therefore, our results also supported that operating nitrification process at a relatively high level of DO and ammonium concentrations will be a better alternative to achieving nitrite accumulation under mainstream conditions. Note that the results obtained in the present study and the abovementioned discussions are based on the flocculent sludge processes, and in fact it is not enough for flocculent sludge process to just run with the high level of bulk DO and ammonium concentration for achieving stable nitritation. Strategies for outcompeting NOB and maintaining nitritation in continuous floccular sludge process particularly treating low-strength wastewater have necessitated a combination of several factors such as combination with aerobic SRT control, imposing transient anoxia and optimizing nitrite competitor (Ge et al., 2014; Regmi et al., 2014; Wu et al., 2016). Interestingly, for biofilm and aerobic granular systems mainstream nitritation could be readily achieved just by the suitable control of bulk DO and ammonium concentrations, more specifically, maintain an appropriate ratio between the DO and the total ammonia nitrogen (TAN) concentrations in the bulk (Bian et al., 2017; Isanta et al., 2015; Perez et al., 2014).
identified. Interestingly, as exhibited in Fig. 5B, the OTU0004 belonging to the Nitrosomonas oligotropha lineage dominated both reactors, contributing to 0.96% (RL) and 5.42% (RH) of the community genetic information, respectively. However, the abundance of total NOB was also markedly higher in RH (5.7%) than in RL (1.17%). One should be pointed out that the AOB/NOB abundance ratio in both reactors was very high and reached up to 10–15, suggesting that AOB vastly outnumbered NOB under the both operating conditions. 4. Discussion 4.1. Different mechanisms for failure of nitrite accumulation Nitrite accumulation is indeed a result of the ammonia oxidation rate higher than the nitrite oxidation rate. Therefore, achieving nitritation should not only focus on the suppression of the NOB, but also try to promote the AOB activity. Recent success of mainstream nitritation in activated sludge process generally involved high dissolved oxygen (DO, > 1.0 mg O2·L−1) and adequate residual ammonium in the bulk liquid (Liu et al., 2017; Regmi et al., 2014; Wu et al., 2016). As they explained, selecting fast growth AOB or promoting AOB activity could be achieved under the high DO and residual ammonium conditions, and consequently maximized the difference between AOB and NOB growth rates. Then nitrite accumulation would be readily obtained based on the low solids retention time (SRT) coupled with other control strategies, although the fast growth NOB Nitrobacter were also present. Building on these past observations, it can be speculated that the selection of fast growth AOB against NOB is one of the key points for achieving high-rate mainstream nitritation. In the present study, although the two nitrifying reactors were operated with relatively high ammonium or oxygen levels, full nitrification still was observed in both reactors during the over two months continuous operation (Fig. 1). In other words, the conversion of ammonium to nitrite was the rate-limiting step of nitrification under both operating conditions. Interestingly, our results suggested that the mechanisms of achieving full nitrification between the two reactors were different, as shown in Fig. 6. More specifically, for the RH run with a high DO (> 2.0 mg O2·L−1), the observed little nitrite accumulation likely resulted from the low bulk ammonium concentration (< 0.1 mg N·L−1) or the limited ammonium supply. The proposed mechanism was supported by the evidence obtained from batch experiments that obviously nitrite accumulation occurred when the bulk ammonium in excess (Fig. 2A). Regarding to the RL, which was operated under a low DO concentration (0.3 mg N·L−1), the obtained full nitrification should be ascribed to the better competition for limited oxygen of NOB than AOB (Fig. 3B), though the RL also run with excess ammonium in the bulk. NOB became a better oxygen competitor than AOB and, as a result, no nitrite accumulated during reactor operation under long-term low DO conditions were also confirmed by previous study (Liu & Wang, 2013; Liu & Yang, 2017; Park & Noguera, 2004). Information obtained in the present work allowed the suggestion that for achieving nitritation, nitrifying process should not be operated under oxygen-limited (low DO) or ammonium-limited conditions. The
4.2. Linking system function with community structure Microbiomes are the engines that power system-level bioprocesses. To further understanding of the failure of nitrite accumulation in the two reactors, relating the reactors performance to their community structure will be necessary. As exhibited in Fig. 5, the dominant nitrifying populations in the RH were closely associated with Nitrosomonas oligotropha-like AOB and Nitrospira defluvii-like NOB, which are both well known as K-strategists (lower specific growth rates and higher substrate affinity) (Bollmann et al., 2002; Park et al., 2017). This observation agrees well with the extremely low bulk ammonia and nitrite concentrations in the RH during the long-term operation. Moreover, these Nitrosomonas oligotropha and Nitrospira defluvii populations cultured under the high oxygen concentration (> 2.0 mg O2·L−1) seemingly had similar sensitivity to oxygen, as indicated by Fig. 3A, and yet further validation is required. It should be noted that the observed nitrite accumulation during the short-term batch experiments with a DO of 0.2–0.3 mg O2·L−1 (Fig. 2B) might be caused by the mass transfer effects within the large flocs (data not shown) (Manser et al., 2005; Picioreanu et al., 2016). For the RL run with a DO of 0.2–0.3 mg O2·L−1, the dominant AOB and NOB species were Nitrosomonas europaea and Nitrospira defluvii. As opposed to Nitrospira-like NOB (suggested to be Kstrategists), Nitrosomonas europaea were generally thought to be rstrategists with higher specific growth rates and low substrate affinity (Dytczak et al., 2008). However, in concordance with the present study Nitrosomonas europaea were also enriched under low DO conditions by previous studies (Liu & Wang, 2013; Park & Noguera, 2004). Even so, Nitrospira-like NOB seemingly still be a better oxygen competitor than Nitrosomonas europaea and, as a result, no nitrite accumulated under long-term low DO conditions (Liu & Wang, 2013). This statement can also be supported by the experimental results that the response of SAUR to DO change was much stronger than that of SNUR (Fig. 3B). Moreover, AOB outnumbered NOB more than ten times in both full nitrification reactors (Fig. 5), which further indicated that the activity of AOB were limited by the lacking adequate ammonium supply (for the RH) or the absence of competitive advantage for insufficient oxygen (for the RL). Given the above, the linking community structure with functions lead to a better understanding of the failure of nitrite
Fig. 6. Schematic representation of the potential for nitrite accumulation under different bulk DO and ammonium levels conditions based on experimental evidence obtained from previous reports and the present study. 277
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Environmental adaptations and alternative metabolisms might be reasons for the enrichment of Nitrospira defluvii under the both low and high DO conditions. As suggested by Fig. 3, these Nitrospira defluvii cultivated under the different DO levels did have a difference in metabolic characteristic. Furthermore, another possible interpretation was that activated sludge offered microhabitats due to the spatial heterogeneity of flocs that lead to strong gradient of electron acceptor (O2) for the Nitrospira defluvii, since the flocs in the RH was much more compact and denser than that in the RL (data not shown). Additionally, the microbial communities in the both reactors comprised hundreds of species (Fig. 4), the tight microbe-microbe interactions might also contribute to the observed nitrifiers activity and composition (Daims et al., 2016; Freilich et al., 2011), and yet the microbial community assembly and most bacterial behavior is quite unpredictable. In summary, the results obtained in the present study suggested that the selective pressure or ecological niche imposed by the specific operational condition likely governed the nitrifiers composition and their metabolic characteristic in each reactor.
accumulation in the two activated sludge reactors. 4.3. Relationships between operating conditions and nitrifiers composition Note that the predominant AOB species between the two reactors was different. This result implied the ecological niche differentiation between Nitrosomonas oligotropha and Nitrosomonas europaea with the difference in their kinetic and physiological characteristics (e.g., affinity to a limiting substrate (ammonium or oxygen), mixotrophic metabolism, or susceptibility to various compounds). Since the parallel two reactors just run with different DO and ammonium concentrations in the bulk, and thus the bulk DO and/or ammonium concentrations will be the key driver of the niche differentiation in the present study. The relationship between AOB community composition and operational DO concentrations has been previously investigated. However, those previous reports were controversial. For example, Bellucci et al. (2011) indicated that members of the Nitrosomonas oligotropha were the prevalent AOB in both high and low DO reactors, while none of sequences retrieved from low-DO reactors was phylogenetically close to Nitrosomonas europaea/eutropha. Nevertheless, many studies reported that Nitrosomonas europaea was the dominant AOB lineage in reactors, independent of the DO (Arnaldos et al., 2013; Liu & Wang, 2013). Additionally, Park & Noguera (Park & Noguera, 2004) found that the AOB community in a high-DO reactor shifted from the Nitrosomonas oligotropha lineage to the Nitrosomonas europaea lineage. The inconsistency of these previous reports indicated that the AOB community composition was determined not only by the operational DO concentrations. Note that the abovementioned nitrification studies were characterized by the limited or undetected residual ammonium in effluent. Data from the present study suggested that the effect of bulk ammonium concentrations would be more important. As Bollmann et al. suggested that bacteria belonging to the Nitrosomonas oligotropha cluster were better adapted to growth at low ammonium concentrations than Nitrosomonas europaea (Bollmann et al., 2002; Bollmann & Laanbroek, 2001). Park & Noguera (Park & Noguera, 2007) also demonstrated that unlike the Nitrosomonas europaea, the adaptation of Nitrosomonas oligotropha-like AOB to the low-DO condition was due to their high affinity for ammonia rather than a high affinity for oxygen per se. Regarding the findings, it could be concluded that the observed different predominant AOB species in our study resulted from the ammonium-based niche differentiation between the two reactors, that is, Nitrosomonas oligotropha with a high affinity for ammonia were dominant in the RH with lower ammonium levels, whereas Nitrosomonas europaea with a low affinity to ammonia were the prevalent AOB in the RH with higher ammonium levels. As most literature reported, Candidatus Nitrospira defluvii (Nitrospira lineage I) were generally enriched from activated sludge by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations (Lücker et al., 2010; Nowka et al., 2015; Park et al., 2017; Spieck et al., 2006). Consistent with the predicted advantages of their high affinity for nitrite, the results presented in this manuscript revealed that the consistently nondetectable nitrite most likely is the key factor underlying the successful enrichment of Nitrospira defluvii-like NOB in both nitrifying reactors. Intriguingly, our results indicated that species closely related to Nitrospira defluvii can also be selected under a relatively high DO condition. The result seemed to contradict the finding that Nitrospira defluvii lacks any catalase, superoxide dismutase (SOD), or superoxide reductase, which are widely distributed key enzymes for the defense against ROS (Lücker et al., 2010). Although the discrepancy is hard to explain by our experimental evidence, the effective growth of Nitrospira defluvii under high DO conditions could be highly supported by other previous reports. As reported by Park & Noguera (2008), the Nitrospira defluvii dominated in an extremely high DO (8.5 mg O2·L−1) chemostat for up to six months. Moreover, increasing discoveries indicated that members of Nitrospira are surprisingly versatile with diverse ecological functions (Daims et al., 2016).
5. Conclusions
• Although different AOB species were respectively enriched under • • • •
the high and low DO conditions, their response to DO variations was almost identical. Nitrospira defluvii-like NOB dominated both reactors, but species cultured with low-DO exhibited higher oxygen affinity. Instead of DO, ammonia or nitrite availability should be the key factor for the selective enrichment of the nitrifier community. Limited ammonia supply for AOB and better oxygen-uptake capacity of NOB were respectively responsible for failure of nitrite accumulation under the high and low DO conditions. High DO couple with excess ammonia would be better for achieving nitritation.
Conflict of interest All authors declare that they have no conflict of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 51808367, No. 51578353), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 18KJD610003) and the National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology (No. 2018KF05). Authors also acknowledge support from the Jiangsu Key Laboratory of Environmental Science and Engineering, and Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment. References Agrawal, S., Seuntjens, D., Cocker, P., Lackner, S., Vlaeminck, S.E., 2018. Success of mainstream partial nitritation/anammox demands integration of engineering, microbiome and modeling insights. Curr. Opin. Biotechnol. 50, 214–221. Annavajhala, M.K., Kapoor, V., Santo-Domingo, J., Chandran, K., 2018. Comammox functionality identified in diverse engineered biological wastewater treatment systems. Environ. Sci. Tech. Lett. 5 (2), 110–116. APHA, 2005. Standard methods for the examination for water and wastewater american public health association, 21th ed. American Public Health Association, Washington, DC. Arnaldos, M., Kunkel, S.A., Stark, B.C., Pagilla, K.R., 2013. Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions. Appl. Microbiol. Biotechnol. 97 (23), 10211–10221. Bellucci, M., Ofiteru, I.D., Graham, D.W., Head, I.M., Curtis, T.P., 2011. Low-dissolvedoxygen nitrifying systems exploit ammonia-oxidizing bacteria with unusually high yields. Appl. Environ. Microbiol. 77 (21), 7787–7796. Bian, W., Zhang, S., Zhang, Y., Li, W., Kan, R., Wang, W., Zheng, Z., Li, J., 2017. Achieving nitritation in a continuous moving bed biofilm reactor at different temperatures through ratio control. Bioresour. Technol. 226, 73–79.
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Damsté, J.S.S., Spieck, E., Le Paslier, D., 2010. A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proc. Natl. Acad. Sci. U.S.A. 107 (30), 13479–13484. Ma, Y., Peng, Y., Wang, S., Yuan, Z., Wang, X., 2009. Achieving nitrogen removal via nitrite in a pilot-scale continuous pre-denitrification plant. Water Res. 43 (3), 563–572. Manser, R., Gujer, W., Siegrist, H., 2005. Consequences of mass transfer effects on the kinetics of nitrifiers. Water Res. 39 (19), 4633–4642. Nowka, B., Daims, H., Spieck, E., 2015. Comparison of oxidation kinetics of nitrite-oxidizing bacteria: nitrite availability as a key factor in niche differentiation. Appl. Environ. Microbiol. 81 (2), 745–753. Park, H.-D., Noguera, D.R., 2008. Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels. J. Microbiol. Biotechnol. 18 (8), 1470–1474. Park, H.D., Noguera, D.R., 2007. Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations. J. Appl. Microbiol. 102 (5), 1401–1417. Park, H.D., Noguera, D.R., 2004. Evaluating the effect of dissolved oxygen on ammoniaoxidizing bacterial communities in activated sludge. Water Res. 38 (14–15), 3275–3286. Park, M.-R., Park, H., Chandran, K., 2017. Molecular and kinetic characterization of planktonic nitrospira spp. selectively enriched from activated sludge. Environ. Sci. Technol. 51 (5), 2720–2728. Park, S., Bae, W., Rittmann, B.E., Kim, S., Chung, J., 2010. Operation of suspendedgrowth shortcut biological nitrogen removal (SSBNR) based on the minimum/maximum substrate concentration. Water Res. 44 (5), 1419–1428. Perez, J., Lotti, T., Kleerebezem, R., Picioreanu, C., van Loosdrecht, M.C.M., 2014. Outcompeting nitrite-oxidizing bacteria in single-stage nitrogen removal in sewage treatment plants: a model-based study. Water Res. 66, 208–218. Picioreanu, C., Perez, J., van Loosdrecht, M.C., 2016. Impact of cell cluster size on apparent half-saturation coefficients for oxygen in nitrifying sludge and biofilms. Water Res. 106, 371–382. Regmi, P., Miller, M.W., Holgate, B., Bunce, R., Park, H., Chandran, K., Wett, B., Murthy, S., Bott, C.B., 2014. Control of aeration, aerobic SRT and COD input for mainstream nitritation/denitritation. Water Res. 57, 162–171. Spieck, E., Hartwig, C., McCormack, I., Maixner, F., Wagner, M., Lipski, A., Daims, H., 2006. Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge. Environ. Microbiol. 8 (3), 405–415. Wu, J., He, C., van Loosdrecht, M.C.M., Pérez, J., 2016. Selection of ammonium oxidizing bacteria (AOB) over nitrite oxidizing bacteria (NOB) based on conversion rates. Chem. Eng. J. 304, 953–961.
Bollmann, A., Bär-Gilissen, M.-J., Laanbroek, H.J., 2002. Growth at low ammonium concentrations and starvation response as potential factors involved in niche differentiation among ammonia-oxidizing bacteria. Appl. Environ. Microbiol. 68 (10), 4751–4757. Bollmann, A., Laanbroek, H.J., 2001. Continuous culture enrichments of ammonia-oxidizing bacteria at low ammonium concentrations. FEMS Microbiol. Ecol. 37 (3), 211–221. Daims, H., Lucker, S., Wagner, M., 2016. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends Microbiol. 24 (9), 699–712. Dytczak, M.A., Londry, K.L., Oleszkiewicz, J.A., 2008. Activated sludge operational regime has significant impact on the type of nitrifying community and its nitrification rates. Water Res. 42 (8), 2320–2328. Fitzgerald, C.M., Camejo, P., Oshlag, J.Z., Noguera, D.R., 2015. Ammonia-oxidizing microbial communities in reactors with efficient nitrification at low-dissolved oxygen. Water Res. 70, 38–51. Freilich, S., Zarecki, R., Eilam, O., Segal, E.S., Henry, C.S., Kupiec, M., Gophna, U., Sharan, R., Ruppin, E., 2011. Competitive and cooperative metabolic interactions in bacterial communities. Nat. Commun. 2, 589. Ge, S., Peng, Y., Qiu, S., Zhu, A., Ren, N., 2014. Complete nitrogen removal from municipal wastewater via partial nitrification by appropriately alternating anoxic/ aerobic conditions in a continuous plug-flow step feed process. Water Res. 55, 95–105. Gruber-Dorninger, C., Pester, M., Kitzinger, K., Savio, D.F., Loy, A., Rattei, T., Wagner, M., Daims, H., 2015. Functionally relevant diversity of closely related Nitrospira in activated sludge. ISME J. 9 (3), 643–655. Isanta, E., Reino, C., Carrera, J., Perez, J., 2015. Stable partial nitritation for low-strength wastewater at low temperature in an aerobic granular reactor. Water Res. 80, 149–158. Kartal, B., Kuenen, J., Van Loosdrecht, M., 2010. Sewage treatment with anammox. Science 328 (5979), 702–703. Koch, H., Luecker, S., Albertsen, M., Kitzinger, K., Herbold, C., Spieck, E., Nielsen, P.H., Wagner, M., Daims, H., 2015. Expanded metabolic versatility of ubiquitous nitriteoxidizing bacteria from the genus Nitrospira. Proc. Natl. Acad. Sci. U.S.A. 112 (36), 11371–11376. Liu, G., Wang, J., 2013. Long-term low do enriches and shifts nitrifier community in activated sludge. Environ. Sci. Technol. 47 (10), 5109–5117. Liu, W., Yang, D., 2017. Evaluating the feasibility of ratio control strategy for achieving partial nitritation in a continuous floccular sludge reactor: experimental demonstration. Bioresour. Technol. 224, 94–100. Liu, W., Yang, Q., Ma, B., Li, J., Ma, L., Wang, S., Peng, Y., 2017. Rapid achievement of nitritation using aerobic starvation. Environ. Sci. Technol. 51 (7), 4001–4008. Lücker, S., Wagner, M., Maixner, F., Pelletier, E., Koch, H., Vacherie, B., Rattei, T.,
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Functional and compositional characteristics of nitrifiers reveal the failure of achieving mainstream nitritation under limited oxygen or ammonia conditions
T
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Wenru Liua, , Wenjing Chenb, Dianhai Yangc, Yaoliang Shena a
National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology, Suzhou 215009, China b School of Environmental Engineering and Science, Yangzhou University, Yangzhou, Jiangsu 225127, China c School of Environmental Science and Engineering, Tongji University, Shanghai 200092, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Nitrification Nitritation Mainstream Dissolved oxygen Nitrosomonas Nitrospira
For understanding the potential of achieving nitritation under different oxygen and ammonia levels, two activated sludge reactors with high (RH) and low (RL) dissolved oxygen (DO) were parallelly operated. During over two months continuous operation, rare nitrite accumulation was observed in both reactors. K-strategists Nitrosomonas oligotropha and r-strategists Nitrosomonas europaea were enriched in the RH and RL, respectively, yet their response to DO variations was almost identical. Although K-strategists Nitrospira defluvii dominated both reactors, species cultured with low-DO exhibited higher oxygen affinity. Instead of DO, ammonia and nitrite availability should be the key factor for the selective enrichment of these nitrifiers. Taken together, the limiting ammonia for ammonia oxidizing bacteria and the better oxygen-uptake capacity of nitrite oxidizing bacteria was respectively responsible for the failure of nitrite accumulation in the RH and RL. This study supported that high DO coupled with excess ammonia would favor the achievement of mainstream nitritation.
1. Introduction Sewage treatment including high-rate and stable mainstream shortcut or nitrite pathway nitrogen removal processes, such as nitritation/
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denitritation and partial nitritation/anammox, has the potential to become energy-neutral or even energy-producing (Kartal et al., 2010). The successful implementation of the nitrite pathway relies on the reliable nitritation, which achieved through effective selection of the
Corresponding author. E-mail address: [email protected] (W. Liu).
https://doi.org/10.1016/j.biortech.2018.12.065 Received 27 November 2018; Received in revised form 16 December 2018; Accepted 20 December 2018 Available online 21 December 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
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wastewater containing 190 mg·L−1 NH4Cl, 50 mg·L−1 K2HPO4, 20 mg·L−1 CaCl2·2H2O, 25 mg·L−1 MgSO4·7H2O and 1 mL·L−1 of trace element solution as described by Bellucci et al (2011). For all working periods, the influent loading rate (150 g N·m−3·d−1), the hydraulic retention time (8 h) and the SRT (20 days) were almost constant. The pH in each reactor was maintained between 7.0 and 7.5 by dosing a buffer containing NaHCO3. Both reactors were operated at 22 °C constant temperature. Compressed air was introduced by a fine bubble diffuser placed at the bottom of each reactor. The RH was run at DO concentrations above 2.0 mg O2·L−1, while the RL was operated with DO near to 0.3 mg O2·L−1. After process operating for more than three SRTs, sludge samples were taken from RH and RL on day 70. These samples were immediately centrifuged at 10000 rpm for 3 min to separate water and solids. The sludge pellet was persevered under −20 °C for DNA extraction within one month.
ammonium oxidizing bacteria (AOB) but stable washout of the nitrite oxidizing bacteria (NOB) (Agrawal et al., 2018). However, considering the difficulty of suppression of NOB in continuous mainstream activated sludge processes under the dilute and cold conditions, till now only limited and/or nascent approaches for achieving nitritation have been reported reported (Ge et al., 2014; Isanta et al., 2015; Ma et al., 2009; Regmi et al., 2014; Wu et al., 2016). In addition to the unfavourable mainstream conditions, the challenge of achieving mainstream nitritation also relates to the complicated nitrifiers composition and their quite unpredictable life-history strategies (i.e., growth and reproductive rates). As new discoveries confirmed through combining different molecular techniques that the genus Nitrospira, generally being the main culprit for failure of mainstream nitrite pathway, had an unexpected high biodiversity and a surprisingly physiological versatility (Gruber-Dorninger et al., 2015; Koch et al., 2015). The metabolic versatility, as basis of ecological niche partitioning within the genus Nitrospira, indicated that members of Nitrospira may survive and coexist with other microorganisms in diverse microniches and even in the nitrite-limited and/or anoxic micro-environment (Annavajhala et al., 2018; Daims et al., 2016; Lücker et al., 2010). Consequently, the commonly used low-DO control for inhibiting Nitrospira like-NOB to achieve long-term mainstream nitritation was often proved to be ineffective. One of the preconditions for the establishment of nitritation is the difference between ammonium and nitrite oxidization rates. Note that in the mainstream the AOB-NOB growth rate (or metabolic activity) differential are dependent on optimal oxygen and nitrogen substrate levels, according to their individual Mond kinetics (Regmi et al., 2014). Moreover, from the ecological perspective, the selection of specific nitrifiers (e.g., fast growth AOB) is generally linked with the environmental conditions such as the availability of substrate (oxygen and/or ammonia) (Dytczak et al., 2008). Therefore, one of the key problems for achieving mainstream nitritation is how to correctly control the DO and ammonia levels in the bulk liquid. Previous studies have demonstrated that the concurrently low DO and low ammonia condition was unsuitable for nitrite accumulation (Bellucci et al., 2011; Fitzgerald et al., 2015; Liu & Wang, 2013; Park & Noguera, 2004). However, a relatively high DO coupled with adequate residual ammonia in the bulk liquid would be in favor of achieving nitritation (Liu et al., 2017; Regmi et al., 2014; Wu et al., 2016). The potential and mechanisms for nitrite accumulation under high DO but with limited residual ammonia or high residual ammonia but limited DO conditions have not been well described or clarified till now, since these operating conditions were frequently present at the conventional wastewater treatment plants and the emerging autotrophic nitrogen removal processes. In this study, two nitrifying processes treating low-strength wastewater were parallelly operated under two different oxygen and ammonium levels. The purpose of this study was to understand the nitrite accumulation potential under the given DO and ammonium levels. Functional and compositional analysis of microbial communities was conducted to provide insights into the microbial community structure of the two nitrifying processes, and reveal the link between microbial community structure and process performance.
2.2. In-situ and ex-situ batch experiments In-situ batch tests were conducted inside the RH and RL, respectively, to evaluate the short-time performance of the reactors under the two conditions, (1) high DO and excess ammonium condition, and (2) low DO and excess ammonium condition. When the batch tests were conducted, continuous operation was stopped. The bulk liquid was exchanged with new substrate of the desired initial ammonium concentration (25–30 mg N·L−1). After the bulk liquid had been exchanged the air supply was switched on again. During the batch tests, the operational conditions, except DO, was maintained the same as the normally operated RH and RL. Ex-situ batch experiments were conducted in some 300 mL reactors to evaluate the activities of the nitrifying biomass under the different levels of DO. Specific ammonium uptake/utilization rate (SAUR) and specific nitrite uptake/utilization rate (SNUR) were used to represent the activities of communities. For testing the SAUR, ammonium (25 mg N·L−1) was supplied in excess; to evaluate the SNUR, nitrite (20 mg N·L−1) was used instead of ammonium. pH was maintained between 7 and 7.5 by adding sodium bicarbonate. Triplicate experiments were conducted at every DO level. All batch experiments were carried out in three replicates at 22 °C. 2.3. Analytical methods Concentrations of NH4+-N, NO2−-N, NO3−-N, total suspended solids (TSS) and volatile suspended solids (VSS) were analyzed in accordance with standard methods (APHA, 2005). Temperature, DO and pH were monitored by pH/oxi1970i meter with DO and pH probes (WTW Multi1970i, Germany). The morphology of granules was measured by microscope (OLYMPUS CX41, Japan) with an attached digital camera. 2.4. DNA extraction and amplicon sequencing Identification of the microbial population was performed using next-generation sequencing. DNA was extracted from sludge samples collected in the RH and RL by using FastDNA SPIN Kits for Soil (MP Biomedicals, LLC, Solon, OH) following the manufacturer’s instructions. The quality and quantity of extracted DNA were checked by Nanodrop spectral photometer (Thermo Fisher Scientific, Beverly, MA, USA). A minimum concentration of 20 ng·L−1 of extracted DNA should be reached. Paired-end sequencing of the extracted DNA was performed on an Illumina MiSeq platform according to standard protocols. The primers set F515 (5′-GTGCCAGCMGCCGCGG-3′) and R907 (5′-CCGTCAATTCMTTTRAGTTT-3′) were used for PCR amplification. To minimize the effects of random sequencing error, low-quality sequences were removed by eliminating those without an exact match to the forward primer. After demultiplexing, quality trimming and alignment, chimeric
2. Materials and methods 2.1. Reactors description and sludge sampling Two identical laboratory-scale complete-mixing reactors (working volume of 2.4 L each) as previously described (Liu & Yang, 2017), namely RH and RL, were set up. Both reactors were inoculated with the same suspended biomass from Quyang WWTP (Shanghai, China), which operates an anaerobic/anoxic/aerobic (AAO) process with approximately a 10-day sludge retention time (SRT). After inoculation, the obtained initial MLVSS concentration in each reactor was about 2.2 ± 0.2 g VSS L−1. The reactors were fed continuously with synthetic 273
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sequences were removed after finally being checked with a Chimer Slayer algorithm. The reads flagged as chimeras were extracted out, and the non-chimera reads then formed the database of ‘effective reads’ for each sample. Eventually, the two samples resulted in 36,966 high quality sequences (18,483 for each sample on average) with an average length of 396 bp.
ammonium excess and oxygen limited condition. Interestingly, similar ammonium removal performance (full nitrification) was maintained under both operating conditions. 3.1.2. Batch experiments Fig. 2 gives further information about the nitrification characteristics of each reactor through in-suit batch experiments. For the batch experiment with a DO of 2.0 ± 0.1 mg O2·L−1 in the RH (Fig. 2A), ammonium was oxidized gradually with the production of nitrate. Concurrently, and unexpectedly, transient nitrite accumulation was also observed and reached the peak value when the ammonium was nearly exhausted. These observations partially contradicted the longterm nitrification performance of the RH. Since nitrite accumulation should be resulted from the different rates between ammonium oxidation and nitrite consumption, the observed transient nitrite accumulation suggested the higher activity of AOB than that of NOB under the specific conditions (high DO and excess ammonium). Interestingly, during the batch experiments with the oxygen-limited condition (Fig. 2B), nitrite accumulation was also observed. Similar nitrification performance was observed in the RL during batch experiments. As shown in Fig. 2C and D, the concentration of ammonium gradually decreased with time; meanwhile, a corresponding increase of nitrate was observed. However, the nitrite accumulation only occurred during batch experiments with the high DO concentration (Fig. 2D). In brief, independent of the concentration of ammonium in the reactor bulk liquid, the produced nitrite by AOB could be rapidly oxidized to nitrate by NOB under the oxygen-limited condition, resulting in the little nitrite accumulated in the RL.
2.5. Biodiversity analysis and phylogenetic classification The sequences were clustered into operational taxonomic units (OTUs) at a cutoff of 97% sequence identity using the MOTHUR program. Raw sequencing data that obtained from this study were deposited to the NCBI Sequence Read Archive with the project accession number of PRJNA509048. Differences in order-based Hill numbers (based on the number of OTUs, Shannon index and Simpson index) were determined via MOTHUR program. Phylogenetic trees were constructed using MEGA7 based on neighborjoining method. The relative abundance of a given phylogenetic group was set as the number of sequences affiliated with that group divided by the total number of sequences per sample. 3. Results 3.1. Nitrification performance
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3.1.1. Long-term operation The two reactors were continuously operated for more than three SRTs with the constant influent volumetric nitrogen loading rate of 0.15 kg N·m−3·d−1. Almost all the oxidized ammonium was converted to nitrate without nitrite accumulation during the entire experimental run (Fig. 1), that is, full nitrification was maintained in both reactors with different operating DO. Note that starting from the first day, nearly 100% ammonium removal efficiency had been obtained in the RH with the stabilized effluent ammonium concentration below 0.1 mg N·L−1 (Fig. 1A). However, likely due to the limited oxygen supply, the ammonium removal rate of the RL only reached 0.09 kg N m−3·d−1 on day 70 and thereby the RL was operated with the excess ammonium concentrations detected in the bulk liquid throughout the investigation (Fig. 1B). In other words, the RH was run with ammonium limitation but oxygen excess, while the biomass for the RL was cultivated under
3.2. Short-term effect of DO on response nitrification activity Batch tests were also conducted to investigate the short-term effect of DO on nitrifying activity (specific ammonium and nitrite oxidization rates) of the sludge cultivated in RH and RL, since operating DO was the primary stress driven the different performance between the two reactors. The obtained SAUR and SNUR values were normalized at high DO applied in each case, as shown in Fig. 3. For the sludge cultivated in the RH (Fig. 3A), a low DO (0.2–0.3 mg O2·L−1) greatly inhibited both the SAUR and SNUR, with an average inhibition efficiency of 73% and 70% respectively. For the sludge cultivated in the RL (Fig. 3B), a low DO also greatly inhibited the SAUR (71.5% reduction), but only lead to an average decrease of 26% in SNUR. The different response of SNUR to DO concentrations indicated the less oxygen sensitivity of NOB cultivated in the low DO reactor (RL) as compared to the high DO reactor (RH). Interestingly, similar observations were also reported by Liu and Wang (2013), although the operating conditions in their study were different from the present research. They demonstrated that the similar response of the ammonium oxidizing rate (AOR) to a low DO concentration resulted from the same dominant AOB community (Nitrosomonas europaea/eutropha) in both sludges (cultivated with a high DO and a low DO), whereas the nitrite oxidizing rate (NOR) for the sludge cultivated with low DO became less sensitive to low DO concentrations due to the enrichment of Nitrospira-like NOB (with high oxygen affinity) instead of Nitrobacter like-NOB. To further reveal the failure of achieving nitritation in the present study, the bacterial communities were also investigated using Illumina MiSeq high-throughput sequencing technology.
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3.3.1. Microbial community diversity The bacterial community diversity in both reactors was evaluated on day 70 by means of the Hill diversity order numbers, as shown in Fig. 4. Each of the three order numbers of the bacterial community in the RH was significantly lower than that in the RL, suggesting the significantly higher bacterial diversity of sludge cultivated under a low DO
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3.3.2. Nitrifying community composition The structure and abundance of nitrifying bacteria in both reactors was analyzed based on the Illumina sequencing results, as shown in Fig. 5. Almost all the identified sequences belonging to the known AOB in both reactors were affiliated to Nitrosomonas genus. However, the preponderant Nitrosomonas-AOB species was different between the reactors (Fig. 5A). More specifically, the OTU0002, closely related to Nitrosomonas europaea, was the most abundant AOB in the RL, accounting for 14.73% of the total bacterial community. Instead OTU0001, most similar to the Nitrosomonas oligotropha, represented 57.17% of the total bacterial community, with the highest levels in the RH. Additionally, the relative abundance of total AOB was significantly higher in RH (61.52%) than in RL (17.62%). However, the known-AOB community in both reactors had the similar species diversity. For nitrite oxidation, three different OUT clusters within the Nitrospira genus were
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Fig. 3. SAUR and SNUR under a high DO (4.0 ± 0.1 mg O2 L−1) and a low DO (0.2–0.3 mg O2·L−1) for the sludge obtained from RH (A) and RL (B) on day 70.
in comparison with the biomass cultivated under a high DO. Moreover, the significantly higher microbial community diversity for biomass in the RL also suggested that the RL had greater microbial community redundancy and more metabolic potential compared with the RH. 275
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Fig. 5. Relative abundance and phylogenetic affiliation of (A) AOB and (B) NOB based on 16S rRNA gene sequence. Sequences with 0.1obtained in this study are shown with “OUT” in the names. Other sequences were obtained from GenBank. Numbers at the branch nodes are bootstrap values. The scale bar represents 1% estimated sequence divergence. Relative abundance is defined as the number of sequences affiliated with that taxon divided by the total number of sequences per sample (%). OUTs making up less than 0.1% of the total composition in both libraries are not shown. 276
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commonly used start-up strategy of nitritation at low ammonium loading rates or low DO should, therefore, be reconsidered. Additionally, results from our short-term batch experiments indicated that obviously nitrite accumulation, though transient, was observed with the both high ammonium concentrations and oxygen levels (Fig. 2). The results are consistent with previous proposed strategies of operating nitritation process with high DO and enough residual ammonium in the bulk liquid (Park et al., 2010; Regmi et al., 2014; Wu et al., 2016). Therefore, our results also supported that operating nitrification process at a relatively high level of DO and ammonium concentrations will be a better alternative to achieving nitrite accumulation under mainstream conditions. Note that the results obtained in the present study and the abovementioned discussions are based on the flocculent sludge processes, and in fact it is not enough for flocculent sludge process to just run with the high level of bulk DO and ammonium concentration for achieving stable nitritation. Strategies for outcompeting NOB and maintaining nitritation in continuous floccular sludge process particularly treating low-strength wastewater have necessitated a combination of several factors such as combination with aerobic SRT control, imposing transient anoxia and optimizing nitrite competitor (Ge et al., 2014; Regmi et al., 2014; Wu et al., 2016). Interestingly, for biofilm and aerobic granular systems mainstream nitritation could be readily achieved just by the suitable control of bulk DO and ammonium concentrations, more specifically, maintain an appropriate ratio between the DO and the total ammonia nitrogen (TAN) concentrations in the bulk (Bian et al., 2017; Isanta et al., 2015; Perez et al., 2014).
identified. Interestingly, as exhibited in Fig. 5B, the OTU0004 belonging to the Nitrosomonas oligotropha lineage dominated both reactors, contributing to 0.96% (RL) and 5.42% (RH) of the community genetic information, respectively. However, the abundance of total NOB was also markedly higher in RH (5.7%) than in RL (1.17%). One should be pointed out that the AOB/NOB abundance ratio in both reactors was very high and reached up to 10–15, suggesting that AOB vastly outnumbered NOB under the both operating conditions. 4. Discussion 4.1. Different mechanisms for failure of nitrite accumulation Nitrite accumulation is indeed a result of the ammonia oxidation rate higher than the nitrite oxidation rate. Therefore, achieving nitritation should not only focus on the suppression of the NOB, but also try to promote the AOB activity. Recent success of mainstream nitritation in activated sludge process generally involved high dissolved oxygen (DO, > 1.0 mg O2·L−1) and adequate residual ammonium in the bulk liquid (Liu et al., 2017; Regmi et al., 2014; Wu et al., 2016). As they explained, selecting fast growth AOB or promoting AOB activity could be achieved under the high DO and residual ammonium conditions, and consequently maximized the difference between AOB and NOB growth rates. Then nitrite accumulation would be readily obtained based on the low solids retention time (SRT) coupled with other control strategies, although the fast growth NOB Nitrobacter were also present. Building on these past observations, it can be speculated that the selection of fast growth AOB against NOB is one of the key points for achieving high-rate mainstream nitritation. In the present study, although the two nitrifying reactors were operated with relatively high ammonium or oxygen levels, full nitrification still was observed in both reactors during the over two months continuous operation (Fig. 1). In other words, the conversion of ammonium to nitrite was the rate-limiting step of nitrification under both operating conditions. Interestingly, our results suggested that the mechanisms of achieving full nitrification between the two reactors were different, as shown in Fig. 6. More specifically, for the RH run with a high DO (> 2.0 mg O2·L−1), the observed little nitrite accumulation likely resulted from the low bulk ammonium concentration (< 0.1 mg N·L−1) or the limited ammonium supply. The proposed mechanism was supported by the evidence obtained from batch experiments that obviously nitrite accumulation occurred when the bulk ammonium in excess (Fig. 2A). Regarding to the RL, which was operated under a low DO concentration (0.3 mg N·L−1), the obtained full nitrification should be ascribed to the better competition for limited oxygen of NOB than AOB (Fig. 3B), though the RL also run with excess ammonium in the bulk. NOB became a better oxygen competitor than AOB and, as a result, no nitrite accumulated during reactor operation under long-term low DO conditions were also confirmed by previous study (Liu & Wang, 2013; Liu & Yang, 2017; Park & Noguera, 2004). Information obtained in the present work allowed the suggestion that for achieving nitritation, nitrifying process should not be operated under oxygen-limited (low DO) or ammonium-limited conditions. The
4.2. Linking system function with community structure Microbiomes are the engines that power system-level bioprocesses. To further understanding of the failure of nitrite accumulation in the two reactors, relating the reactors performance to their community structure will be necessary. As exhibited in Fig. 5, the dominant nitrifying populations in the RH were closely associated with Nitrosomonas oligotropha-like AOB and Nitrospira defluvii-like NOB, which are both well known as K-strategists (lower specific growth rates and higher substrate affinity) (Bollmann et al., 2002; Park et al., 2017). This observation agrees well with the extremely low bulk ammonia and nitrite concentrations in the RH during the long-term operation. Moreover, these Nitrosomonas oligotropha and Nitrospira defluvii populations cultured under the high oxygen concentration (> 2.0 mg O2·L−1) seemingly had similar sensitivity to oxygen, as indicated by Fig. 3A, and yet further validation is required. It should be noted that the observed nitrite accumulation during the short-term batch experiments with a DO of 0.2–0.3 mg O2·L−1 (Fig. 2B) might be caused by the mass transfer effects within the large flocs (data not shown) (Manser et al., 2005; Picioreanu et al., 2016). For the RL run with a DO of 0.2–0.3 mg O2·L−1, the dominant AOB and NOB species were Nitrosomonas europaea and Nitrospira defluvii. As opposed to Nitrospira-like NOB (suggested to be Kstrategists), Nitrosomonas europaea were generally thought to be rstrategists with higher specific growth rates and low substrate affinity (Dytczak et al., 2008). However, in concordance with the present study Nitrosomonas europaea were also enriched under low DO conditions by previous studies (Liu & Wang, 2013; Park & Noguera, 2004). Even so, Nitrospira-like NOB seemingly still be a better oxygen competitor than Nitrosomonas europaea and, as a result, no nitrite accumulated under long-term low DO conditions (Liu & Wang, 2013). This statement can also be supported by the experimental results that the response of SAUR to DO change was much stronger than that of SNUR (Fig. 3B). Moreover, AOB outnumbered NOB more than ten times in both full nitrification reactors (Fig. 5), which further indicated that the activity of AOB were limited by the lacking adequate ammonium supply (for the RH) or the absence of competitive advantage for insufficient oxygen (for the RL). Given the above, the linking community structure with functions lead to a better understanding of the failure of nitrite
Fig. 6. Schematic representation of the potential for nitrite accumulation under different bulk DO and ammonium levels conditions based on experimental evidence obtained from previous reports and the present study. 277
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Environmental adaptations and alternative metabolisms might be reasons for the enrichment of Nitrospira defluvii under the both low and high DO conditions. As suggested by Fig. 3, these Nitrospira defluvii cultivated under the different DO levels did have a difference in metabolic characteristic. Furthermore, another possible interpretation was that activated sludge offered microhabitats due to the spatial heterogeneity of flocs that lead to strong gradient of electron acceptor (O2) for the Nitrospira defluvii, since the flocs in the RH was much more compact and denser than that in the RL (data not shown). Additionally, the microbial communities in the both reactors comprised hundreds of species (Fig. 4), the tight microbe-microbe interactions might also contribute to the observed nitrifiers activity and composition (Daims et al., 2016; Freilich et al., 2011), and yet the microbial community assembly and most bacterial behavior is quite unpredictable. In summary, the results obtained in the present study suggested that the selective pressure or ecological niche imposed by the specific operational condition likely governed the nitrifiers composition and their metabolic characteristic in each reactor.
accumulation in the two activated sludge reactors. 4.3. Relationships between operating conditions and nitrifiers composition Note that the predominant AOB species between the two reactors was different. This result implied the ecological niche differentiation between Nitrosomonas oligotropha and Nitrosomonas europaea with the difference in their kinetic and physiological characteristics (e.g., affinity to a limiting substrate (ammonium or oxygen), mixotrophic metabolism, or susceptibility to various compounds). Since the parallel two reactors just run with different DO and ammonium concentrations in the bulk, and thus the bulk DO and/or ammonium concentrations will be the key driver of the niche differentiation in the present study. The relationship between AOB community composition and operational DO concentrations has been previously investigated. However, those previous reports were controversial. For example, Bellucci et al. (2011) indicated that members of the Nitrosomonas oligotropha were the prevalent AOB in both high and low DO reactors, while none of sequences retrieved from low-DO reactors was phylogenetically close to Nitrosomonas europaea/eutropha. Nevertheless, many studies reported that Nitrosomonas europaea was the dominant AOB lineage in reactors, independent of the DO (Arnaldos et al., 2013; Liu & Wang, 2013). Additionally, Park & Noguera (Park & Noguera, 2004) found that the AOB community in a high-DO reactor shifted from the Nitrosomonas oligotropha lineage to the Nitrosomonas europaea lineage. The inconsistency of these previous reports indicated that the AOB community composition was determined not only by the operational DO concentrations. Note that the abovementioned nitrification studies were characterized by the limited or undetected residual ammonium in effluent. Data from the present study suggested that the effect of bulk ammonium concentrations would be more important. As Bollmann et al. suggested that bacteria belonging to the Nitrosomonas oligotropha cluster were better adapted to growth at low ammonium concentrations than Nitrosomonas europaea (Bollmann et al., 2002; Bollmann & Laanbroek, 2001). Park & Noguera (Park & Noguera, 2007) also demonstrated that unlike the Nitrosomonas europaea, the adaptation of Nitrosomonas oligotropha-like AOB to the low-DO condition was due to their high affinity for ammonia rather than a high affinity for oxygen per se. Regarding the findings, it could be concluded that the observed different predominant AOB species in our study resulted from the ammonium-based niche differentiation between the two reactors, that is, Nitrosomonas oligotropha with a high affinity for ammonia were dominant in the RH with lower ammonium levels, whereas Nitrosomonas europaea with a low affinity to ammonia were the prevalent AOB in the RH with higher ammonium levels. As most literature reported, Candidatus Nitrospira defluvii (Nitrospira lineage I) were generally enriched from activated sludge by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations (Lücker et al., 2010; Nowka et al., 2015; Park et al., 2017; Spieck et al., 2006). Consistent with the predicted advantages of their high affinity for nitrite, the results presented in this manuscript revealed that the consistently nondetectable nitrite most likely is the key factor underlying the successful enrichment of Nitrospira defluvii-like NOB in both nitrifying reactors. Intriguingly, our results indicated that species closely related to Nitrospira defluvii can also be selected under a relatively high DO condition. The result seemed to contradict the finding that Nitrospira defluvii lacks any catalase, superoxide dismutase (SOD), or superoxide reductase, which are widely distributed key enzymes for the defense against ROS (Lücker et al., 2010). Although the discrepancy is hard to explain by our experimental evidence, the effective growth of Nitrospira defluvii under high DO conditions could be highly supported by other previous reports. As reported by Park & Noguera (2008), the Nitrospira defluvii dominated in an extremely high DO (8.5 mg O2·L−1) chemostat for up to six months. Moreover, increasing discoveries indicated that members of Nitrospira are surprisingly versatile with diverse ecological functions (Daims et al., 2016).
5. Conclusions
• Although different AOB species were respectively enriched under • • • •
the high and low DO conditions, their response to DO variations was almost identical. Nitrospira defluvii-like NOB dominated both reactors, but species cultured with low-DO exhibited higher oxygen affinity. Instead of DO, ammonia or nitrite availability should be the key factor for the selective enrichment of the nitrifier community. Limited ammonia supply for AOB and better oxygen-uptake capacity of NOB were respectively responsible for failure of nitrite accumulation under the high and low DO conditions. High DO couple with excess ammonia would be better for achieving nitritation.
Conflict of interest All authors declare that they have no conflict of interest. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 51808367, No. 51578353), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 18KJD610003) and the National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology (No. 2018KF05). Authors also acknowledge support from the Jiangsu Key Laboratory of Environmental Science and Engineering, and Jiangsu Collaborative Innovation Center of Technology and Material of Water Treatment. References Agrawal, S., Seuntjens, D., Cocker, P., Lackner, S., Vlaeminck, S.E., 2018. Success of mainstream partial nitritation/anammox demands integration of engineering, microbiome and modeling insights. Curr. Opin. Biotechnol. 50, 214–221. Annavajhala, M.K., Kapoor, V., Santo-Domingo, J., Chandran, K., 2018. Comammox functionality identified in diverse engineered biological wastewater treatment systems. Environ. Sci. Tech. Lett. 5 (2), 110–116. APHA, 2005. Standard methods for the examination for water and wastewater american public health association, 21th ed. American Public Health Association, Washington, DC. Arnaldos, M., Kunkel, S.A., Stark, B.C., Pagilla, K.R., 2013. Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions. Appl. Microbiol. Biotechnol. 97 (23), 10211–10221. Bellucci, M., Ofiteru, I.D., Graham, D.W., Head, I.M., Curtis, T.P., 2011. Low-dissolvedoxygen nitrifying systems exploit ammonia-oxidizing bacteria with unusually high yields. Appl. Environ. Microbiol. 77 (21), 7787–7796. Bian, W., Zhang, S., Zhang, Y., Li, W., Kan, R., Wang, W., Zheng, Z., Li, J., 2017. Achieving nitritation in a continuous moving bed biofilm reactor at different temperatures through ratio control. Bioresour. Technol. 226, 73–79.
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