Successful startup of one-stage partial nitritation and anammox system through cascade oxygen supply and potential ecological network analysis

Science of the Total Environment 696 (2019) 134065

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Successful startup of one-stage partial nitritation and anammox system through cascade oxygen supply and potential ecological network analysis Zhaolu Feng a, Yihui Wu b, Yunhong Shi c, Liwen Xiao c, Guangxue Wu a,⁎ a b c

Guangdong Province Engineering Research Center for Urban Water Recycling and Environmental Safety, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China Kunming Dianchi Water Treatment CO. LTD, Kunming 650228, Yunnan, China Department of Civil, Structural and Environmental Engineering, Trinity College Dublin, Dublin, Ireland

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• PNA was achieved through cascade oxygen supply by controlling AO durations. • The autotrophic total nitrogen removal percentage of 88.0% was obtained. • Candidatus Kuenenia and Nitrosomonas were dominant genera. • AHLs might affect the relative abundances of functional microorganisms. • Balancing functional microorganisms was regulated by the AHLs-QS system.

a r t i c l e

i n f o

Article history: Received 12 July 2019 Received in revised form 16 August 2019 Accepted 21 August 2019 Available online 22 August 2019 Editor: Yifeng Zhang Keywords: Cascade oxygen supply Partial nitritation and Anammox Functional microorganisms Quorum sensing Microbial interaction

a b s t r a c t It is challenge for the stable operation of the anaerobic ammonium oxidation (Anammox) based wastewater treatment processes. The feasibility for the startup of the partial nitritation and Anammox (PNA) process was demonstrated with cascade oxygen supply by controlling anoxic/aerobic durations. Under steady state conditions, total nitrogen removal percentages of 63.2% (pulse feeding) and 88.0% (constant feeding) were obtained with the anoxic/aerobic duration of 5 min/7 min at 26 °C, and PNA was successfully achieved. The microbial community analysis revealed that Candidatus Kuenenia and Nitrosomonas were dominant genera of Anammox bacteria and ammonia oxidizing bacteria, respectively. Microbial interactions were examined through acylhomoserine lactones-based quorum sensing (AHLs-QS) and metagenomics analyses. N-octanoyl-homoserine lactone, N-decanoyl homoserine lactone and N-dodecanoyl homoserine lactone had obvious relationships with the abundance of the Anammox bacteria. The AHLs-QS system could control microbial interactions among Anammox bacteria, nitrifiers and heterotrophs, especially for their balances in the PNA system. © 2019 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author. E-mail address: [email protected] (G. Wu).

https://doi.org/10.1016/j.scitotenv.2019.134065 0048-9697/© 2019 Elsevier B.V. All rights reserved.

Energy conservation processes are required for sustainable wastewater treatment. Regarding to nitrogen removal from wastewater, the

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Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

anaerobic ammonium oxidization (Anammox) based processes, especially the integrated partial nitritation and Anammox (PNA) process, are most promising. The PNA process can save energy for aeration, avoid organic carbon requirement for denitrification and reduce sludge production. During PNA, ammonia oxidizing bacteria (AOB) oxidize part − of ammonia (NH+ 4 -N) to nitrite (NO2 -N), and Anammox bacteria then − convert NH+ 4 -N and NO2 -N to nitrogen gas (N2). Anammox bacteria are key functional microorganisms for nitrogen removal in PNA systems, in which a continuous supply of NO− 2 -N by AOB is required. However, NO− 2 -N is also an electron donor for nitrite oxidizing bacteria (NOB) and can be easily oxidized to nitrate (NO− 3 -N). It is worth noting that the enrichment of NOB has an adverse impact on N50% of PNA installations (Lackner et al., 2014). Hence, the stable operation of the PNA system should balance Anammox bacteria and AOB, while inhibit the activity of NOB. Cooperation between Anammox bacteria and AOB affects nitrogen removal in the PNA process, while the growth conditions for these microorganisms differ significantly. On the one hand, Anammox bacteria have the optimal temperature of around 37 °C, and it is relatively difficult to enrich Anammox bacteria at room temperature (Egli et al., 2001). Meanwhile, Anammox bacteria have low growth rates with a long doubling time of 11–20 days, which are obviously much longer than that of AOB (Jetten et al., 2001). On the other hand, inhibitory NO− 2 -N concentrations might occur during the startup of the PNA process because of the faster growth of AOB than Anammox bacteria (Lackner et al., 2014). However, after the startup of the PNA system, Miao et al. (2017) found that the Anammox bacteria activity increased with the enhancement of the AOB activity through bio-augmentation. Hence, different operation patterns need to be adopted to balance activities between Anammox bacteria and AOB during acclimation. Among these, cascade oxygen supply could be an effective strategy to enrich Anammox bacteria and AOB by controlling anoxic/aerobic (A/O) durations. In addition, intermittent aeration could inhibit NOB efficiently, because NOB have a longer lag phase than AOB when transiting from anoxic to aerobic phases (Gilbert et al., 2014; Ma et al., 2015). Therefore, the cascade oxygen supply pattern with intermittent aeration would contribute to balancing activities of functional microorganisms by regulating the A/O durations, which would be an effective strategy to achieve stable operation of the PNA system. Balancing activities of functional microorganisms is also influenced by microbial interactions, such as the cooperation between Anammox bacteria and AOB and the competition between Anammox bacteria and NOB (Li et al., 2018). Microbial interactions could be achieved through quorum sensing (QS), which is the ability of microorganisms to perceive and respond to microbial density by secreting and sensing signal molecules (Waters and Bassler, 2005; Fuqua et al., 2001). Diverse microbial communities coexist in the PNA system and some microorganisms may share the same classes of signal molecules, so interactions and communications possibly occur among different microbial communities. The acyl-homoserine lactones-based QS (AHLs-QS) is one of the best-characterized QS for gram-negative bacteria and widely exists in nitrogen removal systems (Bassler, 1999; Liu et al., 2018; Tang et al., 2019). AHLs can be synthesized by functional microorganisms and regulate microbial activities. Nitrifiers could synthesize AHLs, including Ndecanoyl homoserine lactone (C10-HSL), N-octanoyl-L-homoserine lactone (C8-HSL) and N-hexanoyl-L-homoserine lactone (C6-HSL) (Mellbye et al., 2017; Tang et al., 2019). In addition, AHLs-based regulation could enhance the growth rate of Anammox bacteria (Liu et al., 2018; Zhao et al., 2018). C6-HSL and C8-HSL might increase the Anammox bacteria activities through increasing electron transport carriers (Tang et al., 2018b). On the other hand, the AHLs-QS system may also influence microbial interactions. Sun et al. (2019) observed that the AHLs-QS system might regulate interactions between Anammox bacteria and heterotrophs in PNA systems. Anammox bacteria could utilize microbial communications to regulate their activities during the startup stage and maintain their competitiveness and viability (Tang

et al., 2018b). Hence, AHLs might also play an important role in regulating activities of functional microorganisms in the PNA process. An antagonistic process of AHLs-QS is the AHLs-based quorum quenching (AHLs-QQ), in which autoinducers such as AHLs are degraded or the producers and receptors of AHLs are inhibited (Waters and Bassler, 2005). AHLs-QS genera and AHLs-QQ genera coexisted in wastewater treatment systems (Sun et al., 2019; Song et al., 2014). Therefore, microbial interactions might be affected by the niche differentiation of AHLsQS genera and AHLs-QQ genera. However, the possible regulatory mechanism of AHLs-QS system during PNA is still unclear. The purpose of the present study was to achieve the stable operation of the PNA system through cascade oxygen supply by controlling A/O durations. Relative abundances and microbial activities under different A/O durations were investigated. Both AHLs-QS and AHLs-QQ genera were identified. The possible function of the AHLs-QS system was examined and the possible regulatory mechanism was proposed. These results could provide an effective strategy for acclimating PNA, and help to clarify the regulatory mechanism of the AHLs-QS system. 2. Materials and methods 2.1. System operation Two 6 L sequencing batch biofilm reactors with 40% plastic carriers (the specific surface area of 500 m2/m3) were operated at 26 °C for the acclimation of the PNA process. Pulse feeding (PNA-P) and constant feeding (PNA-C) with the hydraulic retention time of 24 h were adopted as the feeding strategy. The PNA-P operation cycle was 360 min, compromising of 10 min filling and 330 min intermittent aeration with mixing, and 20 min decanting. The PNA-C operation cycle included 340 min mixing (10 min filling plus 330 min intermittent aeration) and 20 min decanting. The PNA reactors were operated with the cascade oxygen supply pattern through intermittent aeration. Air was intermittently supplied to create cycles of AO conditions. Under aerobic conditions, the dissolved oxygen (DO) concentration was below 1 mg/L in both PNA reactors. In addition, mechanical stirrers were used for mixing in both reactors. 382 mg/L NH4Cl was adopted in the synthetic wastewater to maintain the NH+ 4 -N concentration of 100 mg/L. The other components of the synthetic wastewater were 640 mg/L NaHCO3, 670 mg/L KHCO3, 7.2 mg/L Na2HPO4, 22 mg/L MgSO4, 60 mg/L CaCl2 and 3.2 mL/L the trace elements solution. The compositions of trace elements were according to Smolders et al. (1994). Both PNA reactors were inoculated with Anammox biofilm carriers from the lab-scale Anammox biofilm reactor. The pH inside the reactors was between 8.0 and 8.5 during the study. Three phases were applied. Specifically, during Phase I of 1–15 d, the A/O duration was 9 min/1 min and the oxygen supply rate was 40 mL/min, during Phase II of 16–48 d, the A/O duration was 9 min/3 min and the oxygen supply rate was 50 mL/min, and during Phase III of 49–90 d, the A/O duration was 5 min/7 min and the oxygen supply rate was 60 mL/min. 2.2. Microbial activity measurements Activities of AOB, NOB and Anammox bacteria in PNA reactors were examined through batch experiments. Biofilm carriers were collected from the PNA reactors in different depths, washed with the synthetic − wastewater without NH+ 4 -N and NO2 -N, and then placed in 250 mL flasks. Nitrifier activity experiments were performed at 26 °C and pH of 7.5–8 with the oxygen supply rate of 300 mL/min to ensure saturate DO. Anammox bacteria activity experiments were conducted at 30 °C and pH of 8–8.5 under anaerobic conditions by purging with N2. Biofilm was taken to test the biomass concentration in the form of volatile suspended solids (VSS), while DO and pH were measured online.

Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

The specific nitrogen consumption rates (mg/g VSS·h) of Anammox bacteria, AOB and NOB were calculated by using Eqs. (1)–(3), respectively (Strous et al., 1998).

TNAnammox ¼ ðTNinf −TNeff Þ 

1 VSS

 NitriteNOB ¼

Nitrateeff −Nitrateinf −0:26 

ð1Þ

TNAnammox 2:04

 

1 VSS

Ammonium AOB   TNAnammox 1  ¼ NitriteNOB −Nitriteeff þ Nitriteinf þ 1:32  VSS 2:04

ð2Þ

ð3Þ

where, TNAnammox is N consumption by Anammox bacteria, mg/g VSS·d; AmmoniumAOB is N consumption by AOB, mg/g VSS·d; NitriteNOB is N consumption by NOB, mg/g VSS·d; Nitriteeff, Nitriteinf, Nitrateeff, Nitrateinf, TNeff and TNinf were influent and effluent loadings − of nitrogenous compounds, mg/L·d, TN is the sum of NH+ 4 -N, NO2 -N − and NO3 -N. 2.3. High-throughput sequencing and metagenomics analysis Biofilm biomass samples were taken from different acclimation phases, and the Fast DNA Spin Kit (Illumina Inc., USA) was used for the DNA extraction. The extracted DNA in the V4-V5 regions was amplified by polymerase chain reaction (PCR) with the specific 16S rRNA gene primers of 515F and 907R (Stackebrandt and Goodfellow, 1991). The PCR products were analyzed through high-throughput sequencing by using the Illumina Hiseq2500 platform. Then, DNA sequences were parsed with the USEARCH software (Edgar, 2010) and operational taxonomic units (OTU) for tags were clustered with a similarity of 97%. The most frequently occurring sequence was extracted as the representative sequence for each OTU, and the Silva database (https://www.arbsilva.de/) was applied to obtain the taxonomic information of the representative sequence. For metagenomic analysis, sequencing of DNA was conducted by the Illumina Hiseq X-ten platform to obtain raw reads. The clean reads were obtained by using Trimmomatic for quality control, and then assembled by the MEGAHIT software (Version v1.0.6) (Li et al., 2015). Following, the open reading frames were obtained from the scaftigs (≥500 bp) by MetaGeneMark (Version 3.38) (Lukashin and Borodovsky, 1998). Then, the non-redundant gene catalogue (Unigenes) was obtained by the CD-HIT program (95% identity and 90% coverage). Comparing with the NCBI-NR database, the Unigenes results were applied for the taxonomic annotation via the DIAMOND software with an e-value cutoff of 10−5. For functional annotation, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was applied to annotate the function and metabolic pathway of genes by the DIAMOND software with an e-value cutoff of 10−5 (Kanehisa and Goto, 2000). Furthermore, the Pearson correlation analysis was conducted to clarify the potential network among detected microbial communities. 2.4. Analytical methods Standard methods were applied for the measurement of NH+ 4 -N, − NO− 2 -N, NO3 -N and VSS (APHA, 1998). The portable multi meter (HQ40d, HACH, USA) was used to test DO and pH. AHLs were extracted and detected from water, extracellular polymeric substance (EPS) and biomass phases with detailed procedures as Sun et al. (2018b). Extraction of EPS was according to Morgan et al. (1990) and the supernatant after centrifugation was recorded as the EPS phase.

3

3. Results and discussion 3.1. System performance in the PNA system Nitrogen removals in PNA-P and PNA-C are shown in Fig. 1. The A/O duration of 9 min/1 min during Phase I was applied to provide an optimal condition for Anammox biofilm carriers. The effluent NH+ 4 -N concentrations in PNA-P and PNA-C gradually decreased from 82.9 mg/L and 84.6 mg/L to 76.3 mg/L and 57.6 mg/L, respectively. The effluent NO− 2 N concentration was 0.7 mg/L in PNA-P and 2.6 mg/L in PNA-C, and the effluent NO− 3 -N concentration was about 0.7 mg/L in both PNA-P and − PNA-C. The accumulation of NO− 2 -N and NO3 -N was not observed in both PNA reactors. Besides, the TN removal percentages were 21.7% (PNA-P) and 38.3% (PNA-C) in Phase I. This implied that the activities of NOB were suppressed and the PNA reactors had been started up successfully. During Phase II, the A/O duration was changed to 9 min/3 min. − The effluent concentrations of NO− 2 -N and NO3 -N were below 2 mg/L and the TN removal percentages were 37.8% (PNA-P) and 56.6% (PNAC) at the end of Phase II. The A/O duration was 5 min/7 min in Phase III. During the initial stage of Phase III (day 49 to day 60), the TN removal percentages increased further with temporary fluctuation. After this period, steady state of PNA reactors was re-achieved and the TN removal percentages rose to 63.2% (PNA-P) and 88.0% (PNA-C), respectively. − The effluent NO− 2 -N and NO3 -N concentrations remained below 2 mg/L in both reactors. At the end of Phase III, the nitrogen removal rates were 63.4 mg N/L·d in PNA-P and 88.2 mg N/L·d in PNA-C. Nitrogen consumption rates of Anammox bacteria, AOB and NOB were further calculated. At the end of Phase I, nitrogen consumption rates of Anammox bacteria and AOB were 3.98 mg/g VSS·d and 0.91 mg/g VSS·d in PNA-P, and 8.61 mg/g VSS·d and 1.98 mg/g VSS·d in PNA-C, while those of NOB were only 0.062 mg/g VSS·d in PNA-P and 0.045 mg/g VSS·d in PNA-C. During Phase II, nitrogen consumption rates of Anammox bacteria and AOB increased continuously, but those of NOB were lower than 1 mg/g VSS·d in both reactors and the activity of NOB was inhibited. To further improve AOB activity, the PNA systems were operated with a prolonged aerobic period and a shortened anoxic period in Phase III. Nitrogen consumption rates of Anammox bacteria and AOB were 41.3 mg/g VSS·d and 5.15 mg/g VSS·d in PNA-P, and 60.8 mg/g VSS·d and 7.97 mg/g VSS·d in PNA-C. Nitrogen consumption rates of NOB maintained at low values in both reactors. The performance of PNA systems suggested that cascade oxygen supply could improve activities of Anammox bacteria and AOB effectively, while the activity of NOB was inhibited. Anammox bacteria were key functional microorganisms to achieve nitrogen removal in the PNA system (Ma et al., 2015). In this study, PNA reactors were seeded with the Anammox biofilm and the high density and suitable niche of Anammox bacteria were maintained at the startup stage. During this stage, a high A/O duration contributed to the enrichment of AOB and the formation of synergistic effect between Anammox bacteria and AOB. In Phase II, the A/O duration decreased but the concentration of DO was remained below 1 mg/L. The applied conditions could enhance the activity of AOB and inhibit the activity of NOB, thereby weakening the competitiveness of NOB for substrate. At the end of Phase II, the removal of TN increased gradually and effluent concentrations of NO− 2 -N were low, so the TN removal was possibly limited by the production rate of NO− 2 -N. The A/O duration was further reduced to enhance the NH+ 4 -N oxidation in Phase III. The PNA system showed fluctuation at the beginning of Phase III, but microorganisms exhibited strong tolerance to the fluctuation. The activity of NOB remained at low levels though the aerobic period was prolonged. Miao et al. (2016) found that intermittent aeration was a valid strategy to achieve the stable operation of the PNA system compared with constant aeration and the TN removal percentage of 77% was achieved. Qiu et al. (2019) obtained that the TN removal percentage was around 81.5% with intermittent aeration. Sun et al. (2019) and Miao et al. (2018) obtained the TN removal percentages of 42% and 77.3%,

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Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

Fig. 1. Profiles of nitrogen concentrations and total nitrogen removal percentages in PNA-P (A) and PNA-C (B), respectively.

respectively, with the relatively short A/O durations. In this study, the high TN removal percentage of 88.0% was obtained with cascade oxygen supply. Therefore, the present study provided a feasible method of cascade oxygen supply in the PNA system that could enhance the synergistic cooperation between Anammox bacteria and AOB while inhibit the activity of NOB. Meanwhile, cascade oxygen supply facilitated the stability of the PNA system and enhanced nitrogen removal. 3.2. Microbial community in the PNA system The analyzed microbial communities are shown in Fig. 2. Planctomycete and Chloroflexi were the dominant phyla followed by Proteobacteria and Bacteroidetes (Fig. 2A). Planctomycete that contained Anammox bacteria was the predominant phylum in the PNA system (Strous et al., 1999). Chloroflexi was the major heterotrophs in the autotrophic systems by metabolizing cell decay materials and soluble microbial products from autotrophs (Lawson et al., 2017; Speth et al., 2016). At the genus level, Candidatus Kuenenia and Candidatus Brocadia belonging to Anammox bacteria were detected and Ca. Kuenenia was the major genus. The relative abundances of Ca. Kuenenia increased sharply from 21.9% (PNA-P) and 23.4% (PNA-C) in Phase I to 40.3% (PNA-P) and 29.0% (PNA-C) in phase II, but decreased in Phase III (Fig. 2B). Nitrosomonas was the dominant AOB in the PNA system and the variation trend of Nitrosomonas relative abundances was similar to that of Ca. Kuenenia. Nitrospira existed in PNA with relative abundances below 0.45% in both reactors, indicating that the growth of NOB was successfully inhibited. In addition, no significant difference in microbial communities between PNA-P and PNA-C was observed, which might be

120 100 p_BRC1 p_Fibrobacteres p_Firmicutes p_Verrucomicrobia p_Armatimonadetes p_Actinobacteria p_Acidobacteria p_Patescibacteria p_Bacteroidetes p_Proteobacteria p_Chloroflexi p_Planctomycetes

80 60 40 20

120 Phase I Phase II Phase III

100 80 60 40 20 0 PN A PN -P A PN C A PN -P A PN C A PN -P A -C

0

Relative abundance (%)

B

Phase I Phase II Phase III

PN A PN -P A PN -C A PN -P A PN -C A PN - P A -C

Relative abundance (%)

A

due to the acclimation in biofilm systems. Biofilm systems could create a stable environment for functional microorganisms. The different feeding patterns in this study might have little effect on microbial community within biofilm. The change of relative abundances of Anammox bacteria and AOB might be related with the acclimation pattern of cascade oxygen supply and the niche differentiation of functional microorganisms in PNA systems. The biofilm included aerobic outer layer and anoxic inner layer due to different oxygen concentrations, so the niche differentiation existed in the PNA systems (Rittmann, 2018; Tsushima et al., 2007). Based on the niche differentiation, AOB existed in the aerobic outer layer of biofilm, which could consume DO and reduce the inhibition of DO on Anammox bacteria. This contributed to the enrichment of Anammox bacteria in the anoxic inner layer (Speth et al., 2016). During Phases I and II, the PNA system was operated with a relatively short oxygen supply duration so nitrifiers could rapidly consume DO with the increased enrichment of AOB in the aerobic outer layer. Meanwhile, more NO− 2 -N was provided for Anammox bacteria by AOB. As a result, relative abundances of Anammox bacteria and AOB increased significantly in Phase II. In Phase III, with the growth of aerobic microorganisms including nitrifiers, the relative abundance of Anammox bacteria decreased, but the Anammox bacteria activities were not inhibited due to the low concentration of DO (b1 mg/L) in both reactors. Except for functional microorganisms, Ardenticatenales_uncultured from the Chloroflexi phylum was the major heterotrophs in the PNA system with the highest relative abundance of 17%–27%. SM1A02 from the Planctomycete phylum widely existed in autotrophic systems (Wang et al., 2018; Akaboci et al., 2018). The heterotrophs accounted for 1.2%

Fig. 2. Microbial community in PNA-P and PNA-C at family (A) and genus (B) levels.

g_Pirellula g_Chryseolinea o_NB1-j;other;other o_SBR1031;other;other g_Limnobacter o_SJA-28;other;other g_SM1A02 o_Ardenticatenales;other;other g_Methanosaeta g_Methanobacterium g_Nitrolancea g_Nitrospira g_Nitrosomonas g_Candidatus_Brocadia g_Candidatus_Kuenenia

Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

to 27% and their effect included the following two aspects. On the one hand, cell decay materials and soluble microbial products produced by autotrophs could be metabolized by heterotrophs in the PNA systems without the addition of organic substrate (Lawson et al., 2017). On the other hand, nitrite produced by the denitrification process of heterotrophs could be utilized by Anammox bacteria (Wang et al., 2018). 3.3. Potential pathways for nitrogen conversions in the PNA system Relative abundances of functional genes associated with nitrogen conversion and related genera in the PNA system are shown in Fig. 3. The predominant functional genes for nitritation were ammonium monooxygenase (amo) and hydroxylamine oxidoreductase (hao). The relative abundance of amo decreased continuously while the relative abundance of hao showed an increasing trend during the acclimation, implying that the potential of NO− 2 -N production increased gradually

5

(Fig. 3A). Meanwhile, the relative abundance of nitrite oxidoreductase genes (nxr) decreased and this gene controlled the oxidization from − NO− 2 -N to NO3 -N, indicating that the ability of achieving complete nitrification weakened gradually. With the cascade oxygen supply pattern, more NO− 2 -N was available for Anammox bacteria with the improvement of potential NO− 2 -N production and partial nitritation could be maintained steadily. Unfortunately, the related functional gene of Anammox process (hydrazine dehydrogenase and hydrazine synthase) was not detected due to the limitation of KEGG annotations. Many types of microorganisms such as Anammox bacteria, nitrifiers and denitrifiers harbored genes relating to nitrogen metabolism in the PNA system. Ca. Kuenenia possessed various nitrogen removal functional genes, including hao, nxr and nir (encoding nitrite reductase) (Fig. 3B). However, Ca. Brocadia lacked the genes of nirS or nirK encoding canonical NO-forming nitrite reductases. Oshiki et al. (2016) proposed that Ca. Brocadia reduced NO− 2 -N to hydroxylamine

A 0.12 PNA-P Phase I PNA-P Phase II PNA-P Phase III PNA-C Phase I PNA-C Phase II PNA-C Phase III

Relative abundance (%)

0.10 0.08 0.06 0.04 0.02 0.01 0.00

AOB

H fA nr B sA na sZ no C rB no B pA na I H rG na

rS

rK

B

Anammox bacteria

ni

ni

rA

nx

o ha AB oC am

B

NOB

Denitrifiers

Other heterotrophs

g_Candidatus_Kuenenia g_Candidatus_Brocadia g_Nitrosomonas g_Nitrosospira g_Nitrosococcus g_Nitrospira g_Nitrobacter g_Nitrolancea g_Nitrospina g_Pseudomonas g_Sorangium g_Candidatus_Accumulibacter g_Lysobacter g_Arenimonas g_Cystobacter g_Rhodothermus g_Cupriavidus g_Micavibrio g_Burkholderia g_Roseobacter g_Comamonas g_Ruegeria g_Bradyrhizobium g_Hyphomicrobium g_Flavihumibacter g_Arenibacter g_Anaeromyxobacter g_Acidovorax g_Rubrobacter g_Candidatus_Microthrix g_Geobacter g_Caldilinea g_Bdellovibrio

PNA-P PNA-C

10-2 10-4

am ha nx nir nir na na no no na nr am ha nx nir nir na na no no na nr oC o r K S rG pA rBCsZ sABfAH oC o r K S rG pA rBCsZ sABfAH HI B HI B -6 AB AB 10

g_Candidatus_Kuenenia g_Candidatus_Brocadia g_Nitrosomonas g_Nitrosospira g_Nitrosococcus g_Nitrospira g_Nitrobacter g_Nitrolancea g_Nitrospina g_Pseudomonas g_Sorangium g_Candidatus_Accumulibacter g_Lysobacter g_Arenimonas g_Cystobacter g_Rhodothermus g_Cupriavidus g_Micavibrio g_Burkholderia g_Roseobacter g_Comamonas g_Ruegeria g_Bradyrhizobium g_Hyphomicrobium g_Flavihumibacter g_Arenibacter g_Anaeromyxobacter g_Acidovorax g_Rubrobacter g_Candidatus_Microthrix g_Geobacter g_Caldilinea g_Bdellovibrio

Phase I Phase II Phase III

Fig. 3. The relative abundances of nitrifying functional genes (A) and genera harboring nitrogen metabolism genes (B) in PNA-P and PNA-C.

Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

harbored nar while lacked of nir, which were the major producer of NO− 2 -N by denitrification. Comamonas only harbored nir that might be a competitor to Anammox bacteria for NO− 2 -N. Nevertheless, most genera did not possess complete denitrifying genes, so the transformation of nitrogen cycle intermediates and partial denitrification might play important roles in the PNA system (Speth et al., 2016). These results indicated that the complete nitrogen metabolism was achieved by the complex cooperation among functional microorganisms. Additionally, heterotrophs could also participate in various nitrogen metabolism pathways.

(NH2OH) rather than nitric oxide through yet unidentified nitrite reductase. Hence, two types of nitrogen removal pathways of Anammox bacteria existed in the PNA system. Nitrosomonas and Nitrosospira had potential to achieve nitritation by harboring the related genes of amo and hao in PNA-P. However, Nitrosospira only harbored hao and lacked of amo in PNA-C, indicating that this nitrifier might complete nitritation process by using NH2OH provided by Nitrosomonas. In addition, Nitrosomonas harbored nir and nitric oxide reductase genes, showing that Nitrosomonas might also survive in anoxic or anaerobic conditions by participating in denitrification, which was consistent with Abeliovich and Vonshak (1992). Many heterotrophs also harbored genes relating to nitrogen metabolism. Hyphomicrobium might be an important heterotroph in the nitrogen metabolism, which carried many nitrogen removal genes such as nxr, nir, nar etc., and could achieve a complete denitrification from − NO− 3 -N (or NO2 -N) to N2. Cupriavidus, Micavibrio, Burkholderia etc. all

2.5

3.0

3.5

4.0

4.5

C8-HSL in bimass phase (ng/g VSS)

5.0

F

R2=0.9821 60

70

80

90

100 110 120

4

H

3 2 1 0

R2=0.9216 60 70 80 90 100 110 120 C10-HSL in bimass phase (ng/g VSS)

EPS

Biomass

C6-HSL

200

3-oxo-C10-HSL C10-HSL

100

C12-HSL

Ph

as e Ph I as Ph e I as I eI II Ph as e Ph II as eI II

as Ph e II as eI II

C8-HSL

40 30 20 10 0 Ph

as Ph e II as eI II

Ph

D

R2=0.9186 2.0

50 40 30 20 10

Ph

as Ph e I as Ph e II as eI II

40 30 20 10 0

as Ph e II as eI II

C8-HSL

Water

300

A/O duration ratios

C12-HSL

C10-HSL in bimass phase (ng/g VSS) A/O duration ratios

G

C10-HSL

Ph

AHLs in water (ng/L), EPS and biomass phases (ng/g VSS)

E

Biomass

3-oxo-C10-HSL

100

50 40 30 20 10

EPS

400

B

C6-HSL

200

Relative abudance of Relative abudance of Anammox bacteria (%) Anammox bacteria (%)

C

Water

300

AHLs were detected in water, EPS and biomass phases in the PNA system (Fig. 4). AHLs of C6-HSL, C8-HSL, C10-HSL, N-dodecanoyl homoserine lactone (C12-HSL) and N-3-oxo-decanoyl homoserine

AHLs in water (ng/L), EPS and biomass phases (ng/g VSS)

400

A

3.4. AHLs identification and their potential functions

Relative abudance of Relative abudance of Nitrosomonas (%) Anammox bacteria (%)

6

50 40 30 20 R2=0.9972 10 30 35 40 45 50 55 60 65 70 75 4 3

C12-HSL in bimass phase (ng/g VSS)

R2=0.9654

2 1 0

15 20 25 30 35 C12-HSL in EPS phase (ng/g VSS)

4 3 2 1 0

R2=0.9153 30 40 50 60 70 80 C12-HSL in bimass phase (ng/g VSS)

Fig. 4. AHLs concentrations in the water, EPS and biomass phases in PNA-P (A) and PNA-C (B), correlations between C8-HSL (C), C12-HSL (D) and C10-HSL (E) in the biomass phase and the relative abundance of Anammox bacteria, correlations between C12-HSL in the EPS phase and the relative abundance of Nitrosomonas (F), and correlations between C10-HSL (G) and C12HSL (H) in the biomass phase and A/O duration ratios.

Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

lactone (3-oxo-C10-HSL) were detected. Microorganisms with AHLs-QS activity had been found in wastewater treatment processes to synthesize AHLs (Tang et al., 2018a; Yang et al., 2016; Cheong et al., 2013). Among them, C6-HSL and C8-HSL widely existed in Anammox systems and might affect nitrogen removal (Tang et al., 2018a; Sun et al., 2018a). Sun et al. (2019) also detected C6-HSL, C8-HSL and C10-HSL in the PNA system. In the present study, the concentration of C10-HSL in the EPS phase was 153.8 ng/g VSS (PNA-P) and 128.7 ng/g VSS (PNAC), and the concentration of C12-HSL in the biomass phase was 63.7 ng/g VSS (PNA-P) and 66.1 ng/g VSS (PNA-C) in Phase III (Fig. 4A and B). Therefore, the AHLs-QS system exited in the PNA system and C10-HSL and C12-HSL were the dominant AHLs in the PNA system. AHLs could have impact on the expression of functional genes, resulting in the enhancement of microbial activity, proliferation, viability, and aggregation (Waters and Bassler, 2005; Flickinger et al., 2011). Previous studies showed that AHLs could enhance nitrogen removal and improve the growth rate of Anammox bacteria (Tang et al., 2018a; De Clippeleir et al., 2011). Yang et al. (2016) found that C10HSL and 3-oxo-C10-HSL could promote microbial growth. C10-HSL and C12-HSL might influence the synthesis of EPS and the adhesion of biomass (Wang et al., 2018). C6-HSL and C8-HSL impacted the activity of bacteria by controlling the transport of electron (Tang et al., 2018a). To explore the role of AHLs in the PNA system, the correlations between the AHLs concentrations and the relative abundance of functional microorganisms were examined (Fig. 4C to F). Concentrations of C8-HSL, C10-HSL and C12-HSL in the biomass phase were positively correlated to the relative abundance of Anammox bacteria (R2 N 0.9), while the concentration of C12-HSL in the EPS phase was negatively correlated to the relative abundance of Nitrosomonas (R2 N 0.96). Therefore, the synthesis and secretion of C8-HSL, C10-HSL and C12-HSL might be beneficial to the enrichment of Anammox bacteria, but the high concentration of C12-HSL might have negative effect on the growth of Nitrosomonas. The concentrations of other AHLs had no significant correlation with the relative abundance of functional microorganisms. These results indicated that C8-HSL, C10-HSL and C12-HSL might regulate microbial abundance and thus affected the balance between functional microorganisms and the stability of the PNA system. In addition, the correlations between AHLs concentrations and A/O duration ratios were examined in the PNA system (Fig. 4G and H). Concentrations of C10-HSL and C12-HSL in the biomass phase were positively correlated to A/O duration ratios (R2 N 0.9), while no obvious relationships between other AHLs and A/O duration ratios were obtained. The long duration of anoxic phase (high A/O duration ratio) might benefit to the growth of Anammox bacteria. These results indicated that C10-HSL and C12-HSL might be synthesized by Anammox bacteria or other anoxic bacteria. Therefore, C10-HSL and C12-HSL were important AHLs in the PNA system and their function might have a potential impact on Anammox bacteria. The AHLs-QS related genes, i.e., synthesis protein genes, sensing protein genes and degradation genes, were obtained based on the metagenomic analysis (Fig. 5A). The hdtS was mainly responsible for AHL synthesis in the PNA system with the relative abundances approximately of 0.037% to 0.045%. The LuxI homologues (encoded by luxI) could catalyze the synthesis of AHL, and the LuxR homologues (encoded by luxR) was their sensing protein, which were both detected in the PNA system (Whiteley et al., 2017). AHLs were possibly synthesized by luxIluxR-type and HdtS-type systems. Additionally, acylases could degrade AHL by breaking irreversibly the amide linkage, which was the dominant AHLs degradation gene with relative abundances of 0.018% to 0.022% (Leadbetter and Greenberg, 2000). AHLs contents were the balance between AHLs-QS and AHLs-QQ microorganisms. Ca. Kuenenia and Ca. Brocadia, which are belonged to Anammox bacteria, both harbored hdtS and performed as the AHLs-QS genera to synthesize AHLs in the PNA system (Fig. 5B). Tang et al. (2019) also found that Candidatus Jettenia could synthesize C6-HSL and C8-HSL through the HdtS-type system. Nitrosomonas and Nitrosospira harbored hdtS and might have the ability to synthesize

7

C6-HSL, C8-HSL and C10-HSL (Burton et al., 2005). Meanwhile, Nitrosomonas could carry out intra- or inter-species communication by the AHLs-QS system (Tang et al., 2018a). In addition, hdtS was also harbored by heterotrophs, such as Hyphomicrobium, Burkholderia, and Geobacter etc. Previous studies revealed that Burkholderia could synthesize C10-HSL and Pseudomonas had the ability to secrete AHLs (Sokol et al., 2003; Davies et al., 1998). These heterotrophs could participate in the synthesis of AHLs and might interact with autotrophs through the AHLs-QS system. Bradyrhizobium, Hyphomicrobium and Methylobacterium possessed the synthesis genes of luxI, but only Bradyrhizobium harbored the sensing gene of luxR. This result indicated that Bradyrhizobium could synthesize and sense AHLs by the luxI-luxRtype system, but Hyphomicrobium and Methylobacterium only had the ability to synthesize AHLs and potentially allowed cyclic feeding (Whiteley et al., 2017). Rhodopseudomona only possessed the sensing genes of luxR and rpaR, acting as the cheater in the PNA system (Diggle et al., 2007). Rhodopseudomona could only respond to AHLs produced by other AHL-QS genera, but did not have the ability of AHLs synthesis. Besides, denitrifiers and heterotrophs had the ability to degrade AHLs due to the possession of acylase. Previous studies found that Pseudomonas had the AHLs-QQ activity and could degrade C6-HSL, C8-HSL and C12-HSL (Cheong et al., 2013). Sun et al. (2019) found that some heterotrophs harbored AHLs-QQ related genes and acted as AHLs-QQ genera in the PNA system. Hence, AHLs-QS genera and AHLs-QQ genera coexisted in the PNA system and a genus might carry both AHLs-QS and AHLs-QQ related genes. The function of these genera in the PNA system required further investigation. Additionally, the nitrogen removal functional microorganisms also harbored the AHLs related genes. Cooperation, competition and cross feeding among functional microorganisms might be regulated by the AHLs-QS system. Therefore, AHLs-QS system might play an important role in microbial communications and maintaining the balance of functional microorganisms to achieve stable PNA. 3.5. Microbial interactions in the PNA system The 41 genera were classified into five groups, including Anammox bacteria, AOB, NOB, denitrifiers and other heterotrophs. The correlations of functional microorganisms were analyzed in the PNA system (Fig. 6A). There was no significant correlation between Ca. Kuenenia and Nitrosomonas, whereas Ca. Kuenenia was negatively correlated with Nitrobacter (ρ b −0.8). Meanwhile, Ca. Kuenenia had positive correlation with some denitrifiers, such as Bradyrhizobium, Hyphomicrobium, etc., but there were negative correlations between Ca. Kuenenia and other heterotrophs. These results showed that Anammox bacteria had complex interactions with other microorganisms and were the significant functional microorganisms in the PNA system. In addition to AOB, partial denitrification was another important way to produce NO− 2 -N, so Anammox bacteria had positive correlation with some denitrifiers. Previous studies found that Rubrobacter and Bradyrhizobium harbored nitrate reductase genes, which were important sources of NO− 2 -N (Speth et al., 2016). Denitrifiers and heterotrophs had frequent microbial interactions with other bacteria (large nodes), which might be due to the complex cooperation between autotrophs and heterotrophs in the PNA system (Lawson et al., 2017). Different functional microorganisms co-existed and distributed in different layers in the biofilm system, which could achieve complex microbial interactions in the PNA system (Fig. 6B). Based on the niche differentiation, AOB and NOB mainly existed in the aerobic outer layer of biofilm, which could consume DO and reduce the inhibition of DO on − Anammox bacteria. Meanwhile, AOB could convert NH+ 4 -N to NO2 -N and provide substrate for Anammox bacteria. Anammox bacteria mainly existed in the anoxic inner layer and converted NH+ 4 -N and NO− 2 -N to N2 for achieving autotrophic nitrogen removal. Meanwhile, denitrifiers might carry out denitrification in the anoxic inner layer. These gradients of microbial communities contributed to the substrate gradient, providing more possibilities for microbial interactions.

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PNA-P Phase I PNA-C Phase I

A 0.06

PNA-P Phase II PNA-C Phase II

Relative abundance (%)

Synthesis

Sensing

PNA-P Phase III PNA-C Phase III

Degradation

0.04 0.02 0.002 0.001 0.0004 0.0002 0.0000

il

tS

I

eh on ct la s e a yl ac iA sd aR rp xR lu

rh

hd

sI

xI

tra

la

lu

yd ro la se

B

Anammox bacteria

AOB

Denitrifiers

Other heterotrophs

Phase I Phase II Phase III

se la ro yd eh on ct e la as yl ac R a rp xR lu l i rh l e as tratS ol dr hd xI hy lu ne o ct e la as yl ac R a rp xR lu l i rh l tratS hd I x lu

PNA-P PNA-C

NOB

g_Candidatus_Kuenenia g_Candidatus_Brocadia g_Nitrosomonas g_Nitrospira g_Nitrolancea g_Bradyrhizobium g_Hyphomicrobium g_Pseudomonas g_Sorangium g_Lysobacter g_Arenimonas g_Cystobacter g_Micavibrio g_Burkholderia g_Anaeromyxobacter g_Acidovorax g_Rubrobacter g_Flavihumibacter g_Geobacter g_Caldilinea g_Gemmatimonas g_Roseovarius g_Isosphaera g_Oceanithermus g_Achromobacter g_Bordetella g_Rhodopseudomonas g_Ktedonobacter g_Fischerella g_Corallococcus g_Thermus g_Anaerolinea g_Methylobacterium g_Ignavibacterium g_Pseudoxanthomonas g_Haliangium

g_Candidatus_Kuenenia g_Candidatus_Brocadia g_Nitrosomonas g_Nitrosospira g_Nitrosococcus g_Nitrospira g_Bradyrhizobium g_Hyphomicrobium g_Pseudomonas g_Sorangium g_Arenimonas g_Cystobacter g_Cupriavidus g_Micavibrio g_Burkholderia g_Anaeromyxobacter g_Acidovorax g_Flavihumibacter g_Geobacter g_Gemmatimonas g_Roseovarius g_Isosphaera g_Oceanithermus g_Achromobacter g_Bordetella g_Rhodopseudomonas g_Echinicola g_Ktedonobacter g_Fischerella g_Corallococcus g_Thermus g_Anaerolinea g_Methylobacterium g_Ignavibacterium g_Pseudoxanthomonas g_Haliangium

10-2 10-4 10-6

Synthesis Sensing Degradation

Fig. 5. The relative abundances of AHLs synthesis, sensing and degradation genes (A) and genera harboring AHLs-related genes (B) in PNA-P and PNA-C.

Most microorganisms with microbial interactions belonged to AHLsQS genera and AHLs-QQ genera (the nodes were surrounded by grey circles) (Fig. 6A). Combined with the above results and previous studies (Tang et al., 2018a; Burton et al., 2005), this study speculated that Anammox bacteria (Ca. Kuenenia) and AOB (Nitrosomonas) might perform as the AHLs-QS genus. The C6-HSL, C8-HSL and C12-HSL could be

synthesized and secreted by Anammox bacteria and the C6-HSL, C8HSL and C10-HSL could be produced by Nitrosomonas in the PNA system in this study (Fig. 6B). The synthesis and secretion of C8-HSL, C10-HSL and C12-HSL might be beneficial to the enrichment of Anammox bacteria, but the high concentration of C12-HSL might have negative effect on the growth of Nitrosomonas. These results indicated that AHLs produced

Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

B

Microbial interactions

QS

Aerobic outer layer

Nitrosomonas

C6

NO2--N NO3--N

NO2--N NO3--N

NOB

Denitrifiers

QQ

C8 C10

Bradyrhizobium Hyphomicrobium Candidatus_Kuenenia Candidatus_Brocadia Heterotrophs

C12 C6 C10-oxo

N2 Pseudomonas

AOB

C8 C10

Nitrospira

N2

Production Facilitation Inhibition Degradation

Microbial community AHLs-QS regulation

NH4+-N

Anoxic inner layer

9

QQ

C12

C6 C10-oxo

C8 C10

Anammox bacteria

C10

C12 C8

AHLs degradation

Fig. 6. Correlations between microorganisms harboring AHLs-related genes (A) and the microbial interactions and the possible regulatory mechanism by AHLs-QS systems in different biofilm layers (B). The connection representing a strong (Person's correlation coefficient r N 0.7) and significant (p-valueb0.01) correlation. The size of each node is proportional to the number of connections.

by Anammox bacteria might be conducive to the enrichment of Anammox bacteria, but might have adverse influence on the growth of Nitrosomonas. The long-term operation and the activities of functional microorganisms indicated that the cascade oxygen supply pattern benefited the acclimation of Ca. Kuenenia, acting as “ruler” in the PNA system. The nutrient requirements of Ca. Kuenenia were provided by Nitrosomonas, and Ca. Kuenenia could regulate the abundance of Nitrosomonas through the AHLs-QS system in order to protect themselves in the safe niche in the PNA system. Nitrosomonas provided substance for Ca. Kuenenia while they synthesized and secreted AHLs,

which were in favor of the enrichment of Anammox bacteria. Therefore, Nitrosomonas might become the “assistor” in the PNA system. Additionally, Pseudomonas and Acidovorax could degrade C6-HSL, C8-HSL and C12-HSL (Cheong et al., 2013). Denitrifiers and heterotrophs might act as the “protector” of AOB in the PNA system. The synthesis and degradation of AHLs benefited to the equilibrium relationships between Anammox bacteria and AOB. Therefore, AHLs-QS system might play an important role in interactions of Anammox bacteria, AOB and heterotrophs and the balance of functional microorganisms in the PNA system.

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Z. Feng et al. / Science of the Total Environment 696 (2019) 134065

4. Conclusions Cascade oxygen supply pattern was an effective strategy to balance functional microorganisms and optimize the nitrogen removal in PNA systems. Ca. Kuenenia and Nitrosomonas were dominant genera of Anammox bacteria and AOB, respectively, and NOB activity was successfully inhibited through the regulation of oxygen supply. AHLs-QS system existed in the PNA system and the concentrations of AHLs might influence the relative abundances of Anammox bacteria and AOB. The AHLs-QS system might regulate the interactions of Anammox bacteria, AOB and heterotrophs and their balances in the PNA system.

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