Metabolomics uncovers the regulatory pathway of acyl-homoserine

Article pubs.acs.org/est

Cite This: Environ. Sci. Technol. 2018, 52, 2206−2216

Metabolomics Uncovers the Regulatory Pathway of Acyl-homoserine Lactones Based Quorum Sensing in Anammox Consortia Xi Tang,†,‡ Yongzhao Guo,†,§ Shanshan Wu,†,‡ Liming Chen,†,‡ Huchun Tao,§ and Sitong Liu*,†,‡,§ †

Key Laboratory of Water and Sediment Sciences, Ministry of Education of China, Beijing 100871, China College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China § School of Environment and Energy, Shenzhen Graduate School, Peking University, Shenzhen 518055, China ‡

S Supporting Information *

ABSTRACT: Acyl-homoserine lactones (AHLs)-mediated quorum sensing in bacterial communities have been extensively observed. However, the metabolic pathways regulated by AHLs in bacteria remain elusive. Here, we combined long-term reactor operation with microbiological and metabolomics analyses to explore the regulatory pathways for different AHLs in anammox consortia, which perform promising nitrogen removal for wastewater treatment. The results showed that no obvious shifts induced by exogenous AHLs occurred in the microbial community and, mainly, dosing AHLs induced changes in the metabolites. 3OC6-HSL, C6-HSL, and C8-HSL controlled the electron transport carriers that influence the bacterial activity. In contrast, only 3OC6-HSL regulated LysoPC(20:0) metabolism, which affected bacterial growth. AHLs mainly regulated the synthesis of the amino acids Ala, Val, and Glu and selectively regulated Asp and Leu to affect extracellular proteins. Simultaneously, all the AHLs regulated the ManNAc biosynthetic pathways, while OC6-HSL, OC8-HSL, and C6-HSL particularly enriched the UDP-GlcNAc pathway to promote exopolysaccharides, resulting in different aggregation levels of the anammox consortia. Our results not only provide the first metabolic insights into the means by which AHLs affect anammox consortia but also hint at potential strategies for overcoming the limitations of the long start-up period required for wastewater treatment by anammox processing.

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INTRODUCTION Quorum sensing (QS) communication among bacteria is one of the most important discoveries in the field of microbial research in the past 20 years. Acyl-homoserine lactones (AHLs) have been found to be used by Gram-negative bacteria as common signaling molecules and are involved in the regulation of biofilm formation and other properties.1,2 So far, more than 37 genera of Gram-negative bacteria have been identified that are regulated by AHLs signaling molecules.3 In recent years, researchers have utilized AHLs-based quorum sensing in wastewater treatment to enhance sludge aggregation and the environmental stress resistance of microorganisms.4,5 Undoubtedly, AHLs offer new approaches to solving problems related to biological wastewater treatment. Anammox has attracted much attention recently as a highefficiency, energy-saving approach for nitrogen removal from wastewater.6,7 It can achieve autotrophic ammonium conversion into nitrogen gas with nitrite as the electron acceptor via the biocatalysis of anammox bacteria.8 Although many anammox reactors have been built on a pilot scale, an additional bottleneck still remains.6 The slow growth rate and strict metabolic conditions of anammox bacteria make start-up of the reactor difficult.9 Researchers have attempted to enhance the bacterial activity and viability to achieve faster a more rapid © 2018 American Chemical Society

start-up using various methods, such as applying bacterial carriers, choosing a suitable bioreactor type and seeding sludge.10−12 These methods do not essentially regulate the metabolism of anammox bacteria, and the problem of the required long start-up time for anammox process still persists.13−16 AHLs can regulate the expression of key genes in bacteria, thereby enhancing their activity, proliferation, viability, and aggregation.17 Different AHLs have disparate regulatory pathways.18 For example, a combination of three AHLs (3OC6-, C6-, and C8-HSL) controls the maltose fermentation pathway and the glyoxylate bypass to regulate bacterial growth with specific substrates in Yersinia pestis.19 C8-HSL has been shown to regulate glucose uptake, pentose phosphate pathway, and de novo nucleotide biosynthesis via the activation of QsmR in Burkholderia glumae to enable survival.20 However, the identification of AHL-regulated metabolism is still limited and almost all the related studies focus on single bacterial strains. In complex bacterial communities, it is more difficult to uncover Received: Revised: Accepted: Published: 2206

November 7, 2017 January 25, 2018 January 29, 2018 January 29, 2018 DOI: 10.1021/acs.est.7b05699 Environ. Sci. Technol. 2018, 52, 2206−2216

Article

Environmental Science & Technology

operated, and the details are presented in the Supporting Information. Chemical Analysis and EPS Determination. The concentrations of NH4+-N, NO2−-N, and NO3−-N, as well as VSS, were measured using the standard methods of the American Public Health Association (APHA, 1998). The total nitrogen (TN) was measured using a TN analyzer (IL500, HACH, USA). The pH was obtained using a pH meter (Mettler Toledo, Columbus, OH). The biomass yield rates of the anammox consortia during the reactor operation were calculated following the methods of previous studies.28,29 Briefly, they were calculated from the biomass yield, taking the VSS increase as the main characterization parameter. The experiments were performed in triplicate to obtain average values. The SAAs were determined by a batch test according to methods described previously,30 and the experimental details are described in the Supporting Information. Floc sizes were measured in triplicate using a laser particle size analyzer (Mastersizer MS2000, Malvern Instruments, Malvern, UK) with a detection range of 0.02−2000 μm. The median diameter of floc was chosen to represent the floc size in the reactor. Additionally, the EPS extractions were performed using cation exchange resin (CER), as proposed by Hou et al.,31 such that the release of intracellular substances could be neglected.32 Extracellular polysaccharide (PS) and protein (PN) concentrations were quantified using the anthrone method with glucose as a standard and the Lowry method with bovine serum albumin as a standard, respectively.33 Both the PS and PN results were the average value of three parallel samples. To monitor the microbial community shifts during the 90 days of operation, DNA extraction and 16S rRNA gene sequencing analysis were performed. The detailed methods are presented in the Supporting Information. Extraction and Identification of AHLs. The identification of AHLs released by the anammox consortia was performed before the inoculation. AHLs were extracted from the supernatant of the anammox consortia.23 Briefly, 200 mL of the supernatants were extracted twice with an equal volume of ethyl acetate and 0.1% (v/v) formic acid. The ethyl acetate extracts were concentrated and resuspended in 200 μL of acetonitrile before analysis. All the samples were analyzed by LC-MS (Q Exactive orbitrap Thermo, CA) and the details are described in the Supporting Information. Metabolite Extraction and Profiling. The biomass samples for metabolite analysis were collected in triplicate to investigate variations in the bacterial metabolic profiles. These samples were quenched and extracted, as described previously.34 The biomass samples were quickly harvested, washed, and quenched using 60% (v/v) cold methanol at −40 °C. The biomass was separated from the quenching solution by immediate centrifugation at 9000g for 10 min at −20 °C. The pellet was resuspended in 5 mL of 80% cold methanol and sonicated at 200 W for 30 min in an ice bath. Afterward, the supernatants were harvested after centrifugation of the lysates at 10 000g for 20 min. To purify the metabolites, the samples were dried under a nitrogen flow at room temperature (25 °C) for metabolomic analysis. The mass spectrometry data were collected using a Q Exactive mass spectrometer (Thermo Fisher, Waltham, MA). The details are presented in the Supporting Information. In addition, to investigate the potential relationship between the intracellular amino acids and the extracellular PN, 15 biomass samples taken from the control reactor on different

the regulatory mechanism of AHLs. Very recently, metabolomic analysis has been applied in many phylogenetically highly diverse and complex communities to reveal their metabolic activity and changes in the metabolic pathways.20−22 Although the phenomenon of AHL-mediated QS regulation has been reported,23 the AHL-regulated metabolic pathways in anammox consortia remain unexplored. Thus, it would be significant to employ recently developed metabolomics to reveal the regulatory pathway of AHLs-based QS in anammox consortia. In this study, we used six sequencing batch reactors (SBRs), one as a control and others that were treated with five different AHLs, respectively. The AHLs-regulated metabolic pathways in the anammox consortia were explored by metabolomics and relevant phenotype monitoring. The first metabolically regulated pathways for enhancing bacterial aggregation, activity, and growth rate modulated by different AHLs, were identified in the anammox consortia. These results suggest the potential for developing strategies based on AHLs to overcome the required long start-up periods of anammox process in wastewater treatment.

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MATERIAL AND METHODS Bioreactor Operation. Six identical SBRs with working volumes of 1.0 L were operated at 37 °C for 90 days. The influent of these SBRs was synthetic wastewater, described previously.24 One reactor (R1) was incubated without AHLs as a control, while the remaining five reactors (R2−R6) were regarded as treatment groups, dosing with 2 μM 3OC6-HSL (N-(3-oxohexanoyl)-DL-homoserine lactone), 3OC8-HSL (N(3-oxooctanoyl)-DL-homoserine lactone), C6-HSL (N-hexanoyl-DL-homoserine lactone), C8-HSL (N-octanoyl-DL-homoserine lactone), and C12-HSL (N-dodecanoyl-DL-homoserine lactone), which were obtained from Sigma (St. Louis, MO, USA) (Figure S1a). The dosing concentration was determined as described previously.20 The anammox consortia, mainly composed of Candidatus jettenia,23 were seeded into the reactors with an initial biomass concentration of 0.18 g volatile suspended solids (VSS) L−1 (Figure S2). Detailed information operation are described in the Supporting Information. The concentrations of NH4+-N and NO2−-N in the influent were maintained at 240 and 200 mg L−1, respectively, with the highest concentration in the reactor was basically less than 100 mg L−1. It is a common operation strategy of SBR for anammox consortia with high concentrations of NO2−-N25−27 and was not toxic to the anammox bacteria. Hydraulic retention time (HRT) was set to 12 h on the initial day. When NO2−-N concentration in the effluent dropped below 10 mg L−1, HRT was shortened to increase the nitrogen loading rate. It was decreased to 8 h and then to 6 h. The reactor loading rate was not increased in the later period of reactor operation (days 67− 90), in order to study the potential effects of AHLs on nutrientdeficient anammox consortia (Figure S1b). Nitrite was the limiting substrate mainly responsible for nutrient deficiency in the anammox consortia. The influent and effluent were collected every 2 days for NH4+-N, NO2−-N, and NO3−-N analysis. Biomass samples were harvested from the six reactors every 15 days for analysis of the particle size, volatile suspended solids (VSS), and extracellular polymeric substances (EPS). The specific anammox activities (SAAs), biomass yield rate, and microbiome and metabolome profiles were analyzed using the biomass in the six reactors on days 30 and 90 of the reactor operation. To address the reproducibility of the experiment, six identical reactors (same as the reactors described above) were 2207

DOI: 10.1021/acs.est.7b05699 Environ. Sci. Technol. 2018, 52, 2206−2216

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Figure 1. Effects of exogenous 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL on the nitrogen removal rate (NRR). The NRR profile of the control reactor during the 90 days of operation is presented in (a). The fold-changes of the NRRs between the treatment reactor and the control are illustrated in (b−f). Three distinct phases of NRR performance are separated by dotted lines (phases I−III). One-way ANOVA with post hoc test by Dunnett’s multiple-comparison was conducted to compare the treatment reactors to the control reactor, where significant differences are indicated as follows: *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

multivariate statistical analysis and pathway analysis were performed with Mev 4.9.0 (MultiExperiment Viewer) and MetaboAnalyst 3.0,36 and hierarchical clustering was performed using Pearson distance as the distance measure. The metabolites were then filtered to identify significantly different metabolites using ANOVA with a Dunnett’s multiplecomparison test between the biomass dosed with AHLs and the control. Metabolic features with p < 0.05, and fold-changes >1.5 were considered to be significantly different.37 Thus, significant metabolites from each comparison were identified and analyzed in detail.

operation days (the sampling days are described in Table S1) were collected to measure their intracellular amino acids and PN. Separation of cells from the EPS was conducted by stripping the EPS from the biomass using CER. The extraction method and the quantification of intracellular amino acids were the same as that for the metabolites. The determination of the extracellular PN of these samples was also performed as described above. Statistical Analysis. For phenotypic data, analysis of variance (ANOVA) with a post hoc test by Dunnett’s multiple comparison was conducted using the statistical product and service solutions (SPSS) software to determine the differences between the biomass dosed with AHLs and the control without any AHL treatment. SPSS was also used to build a multiple regression model for investigating the relationship between the intracellular amino acids and extracellular PN. For the microbial community data, nonmetric multidimensional scaling (NMDS) was used to reduce the community data complexity and assess the community similarity among all the samples using the R statistical environment (version 3.2.2) based on Bray−Curtis similarities.35 The abundance of selected genera was visualized as a 100% stacked column. For metabolomic data, log-transformation followed by individual scaling (mean-centered and divided by the standard deviation of each variable) was applied initially. Next,

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RESULTS AHLs Increased Reactor Performance. First we identified the release of 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL by anammox consortia (Figure S3). To investigate whether reactor performance could be improved by these exogenous AHLs, 2 μM of 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL were added to the influent of treatment reactors R2−R6, respectively. The nitrogen removal rates (NRRs) of the six reactors over 90 days of operation are recorded in Figures 1 and S4. On the basis of the fold-changes in NRRs of all the treatment reactors compared to the control without addition of AHLs, the reactor operation was divided into three phases. 2208


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Environmental Science & Technology During phase I (the first 16 days), the NRR of the control reactor increased from 0.6 to 0.8 gN L−1 day−1. The foldchanges of the NRRs of all the treatment reactors (except for the reactor treated with C12-HSL) were close to 1, indicating that the addition of AHLs (with the exception of C12-HSL) did not significantly affect the NRRs. Phase II (days 17−66) was characterized by a dramatic increase in the fold-changes of the NRRs. The NRR of the control reactor increased from 0.8 to 1.4 gN L−1 day−1. Following the addition of 3OC6-HSL, C6HSL, and C8-HSL, the average value of the fold-changes in the NRR of reactors with additional AHLs compared to that of the control reactor during phase II was 1.14, 1.10, and 1.10, respectively. That is, 3OC6-HSL, C6-HSL, and C8-HSL significantly increased the NRRs by 14, 10, and 10%, respectively, compared to that in the control (p < 0.05). During phase III (days 67−90), the effect of AHLs on anammox consortia under the nutrient-deficient environment was studied with no further increase in the reactor loading rate. The NRR of the control reactor was maintained at approximately 1.4 gN L−1 day−1. Additionally, 3OC8-HSL, C6-HSL, and C8-HSL did not increase the NRRs during this phase. Interestingly, 3OC6-HSL still significantly increased the NRR by 8% compared to the control (p < 0.001). Moreover, this increased performance has been successfully achieved in the restarted up reactors with AHL dosing, indicative of its repeatability (Figure S5 and Text S1). When incubated with C12-HSL, the NRR decreased slightly during phase I and then significantly decreased (p < 0.05) during phases II and III compared to that of the control. This suggested that C12-HSL negatively affected the NRR. In this study, the anammox reactor was considered as successfully started up when the NRR reached approximately 1.4 gN L−1 day−1 (the maximum NRR performance of the control reactor). Thus, start-up required 64, 50, 62, 54, and 60 days in the control reactor and the reactors treated with 3OC6-HSL, 3OC8-HSL, C6-HSL, and C8-HSL, respectively. The most obvious reduction in start-up time occurred following the addition of 3OC6-HSL, which shortened the start-up time by 22%. AHLs Enhanced Bacterial Activity and Biomass Yield Rates. The increased NRRs of the reactors were influenced by either bacterial activity or biomass yield rates. Exploring these possibilities during phase II, the activity results of the anammox consortia were obtained on day 30, the time point when the most obvious differences in NRRs between the treatment reactors R2−R6 and the control reactor R1 appeared. These results showed that exogenous 3OC6-HSL, C6-HSL, and C8HSL significantly increased the SAA by 8, 11, and 7% (p < 0.05), respectively. In contrast, 3OC8-HSL had no significant effect on the SAA. Moreover, the addition of C12-HSL decreased the SAA (Figure 2a). Analysis of the biomass yield rate in the six reactors revealed that only treatment with 3OC6HSL significantly improved the biomass yield rate (38%, p < 0.05) (Figure 2b). Thus, 3OC6-HSL affected the NRR by enhancing bacterial SAA and increasing the biomass yield rate, while C6-HSL and C8-HSL only acted to enhance bacterial SAA and not the biomass yield rate (p > 0.05). The same analysis was performed on day 90 of reactor operation to investigate the influence of AHLs on the SAA and the biomass yield rates during phase III. The activities of the bacteria treated with AHLs were nearly the same as those in the control reactor, although slightly decreased (Figure S6a). Importantly, the biomass yield rate (Figure S6b) in the reactor

Figure 2. Effects of exogenous 3OC6-HSL, 3OC8-HSL, C6-HSL, C8HSL, and C12-HSL on the activity (a) and biomass yield rate (b) of anammox consortia during phase II. Significant differences were determined by one-way ANOVA with post hoc test by Dunnett’s multiple-comparison between the treatment reactors and the control (*, p < 0.05; **, p < 0.001).

improved significantly (p < 0.05) only by treatment with 3OC6-HSL. AHLs Promoted Floc Aggregation. The floc distribution was obtained using a laser particle size analyzer (Figure S7). The floc sizes inside the reactors, based on the median diameter of floc, are recorded in Figure 3a. This shows that the floc size varied among these reactors. The inoculated anammox consortia had a floc size of 300 μm. In the control reactor, the floc size during phase II increased from 470 to 560 μm, constituting a 1.5-fold increase compared to the sizes observed during phase I. Correspondingly, the floc sizes in the reactors seeded with AHLs also increased and to different extents (1.6to 1.9-fold). The fold-changes in the floc sizes in the reactors dosed with AHLs compared to the control were then calculated for all three phases (Figure 3b). No influence of exogenous AHLs on the floc size was apparent during phase I. In contrast, the differences in floc sizes between the treatment reactors and the control reactor were significant during phase II. The addition of 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL significantly increased the floc sizes by 15, 19, 8, 24, and 23%, respectively, compared to that of the control (p < 0.05). This indicates that different AHLs had variable effects on the floc aggregation. Additionally, although the floc in all reactors slightly dispersed during phase III due to the nutrientdeficiency, the consortia in the reactors fed with 3OC6-HSL, 3OC8-HSL, and C12-HSL maintained larger floc sizes compared to the control reactor (p < 0.05). Effects of Different AHLs on EPS Production. The variation profiles of the EPS in the six reactors are shown in Figure 4. Changes in trends in PN and PS content were similar to the changes in the floc sizes during the 90 days of operation (Figure 4a,c). Statistically, the PN and PS contents positively 2209


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The fold-changes in the EPS content of the consortia treated with AHLs compared to that of the control were also evaluated for the three phases (Figure 4b,d). There were no significant differences in PN and PS content during phase I. However, AHLs significantly increased the PN and PS content (PN: 21− 34%; PS: 6−17%; p < 0.05) during phase II, whereas only 3OC6-HSL, 3OC8-HSL, and C12-HSL significantly increased the PN content during phase III (p < 0.05). The increased production of PN and PS induced by AHLs is consistent with their roles in promoting aggregation. The increases in PN and PS content induced by exogenous AHLs contributes to the increase in the floc size during phase II. Meanwhile, only the increase in the PN content contributed to the increase in the floc sizes when incubated with 3OC6-HSL, 3OC8-HSL, and C12-HSL during phase III. Metabolic Output and Microbial Community Shifts Affected by Different AHLs. When the NRR, floc sizes, PN, and PS of consortia treated with AHLs were clearly different from those of the control (phase II; day 30; Figure S8), microbial community and metabolomic analyses were conducted. Analysis of shifts in the microbial community composition in the six reactors showed that the reactor operation time was the main driver of microbial community shifts (Figure S9a). Although the samples treated with C12HSL during phase II and C6-HSL during phase III fell out of the clusters due to the perturbation in the proportion of anammox bacteria (Candidatus jettenia, Figure S9b), there were no obvious shifts in the microbial community induced by exogenous AHLs in the anammox consortia. Therefore, changes in metabolites were mainly induced by the addition of AHLs rather than by the shifts in the bacterial community. To assess metabolic changes, metabolite profiles were determined and clustered (Figure 5). We obtained 310 metabolites for each sample. The obvious differences in metabolites between these reactors were indicated by the

Figure 3. Effects of exogenous 3OC6-HSL, 3OC8-HSL, C6-HSL, C8HSL, and C12-HSL on the floc sizes of anammox consortia. The profile of floc sizes in the six reactors during the 90 days of operation is shown in (a). Three distinct phases of floc sizes in six reactors are separated (phases I−III). The fold-changes in floc size between the treatment reactors and the control were calculated for all three phases, respectively (b). Error bars are defined as s.e.m. (n = 3, technical replicates). Significant differences between the treatment reactors and the control were determined by two-way ANOVA with post hoc test by Dunnett’s multiple-comparison (*, p < 0.05; ***, p < 0.001).

correlated with the floc size of the consortia (r = 0.75, p < 0.01 for PN; r = 0.91, p < 0.01 for PS).

Figure 4. Effects of exogenous 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL on EPS content. The profiles of PN and PS content in six reactors during 90 day operation in six reactors are shown in (a and c). Three distinct phases of flocs size in six reactors are separated (phases I−III). The fold-changes in extracellular PN and PS between the treatment reactors and the control were calculated for all three phases, respectively (b, d). Error bars are defined as s.e.m. (n = 3, technical replicates). Significant differences between the treatment reactors and the control were conducted by two-way ANOVA with post hoc test by Dunnett’s multiple-comparison (*, p < 0.05; **, p < 0.01; ***, p < 0.001). 2210


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biosynthesis. Moreover, 3OC6-HSL, 3OC8-HSL, and C6-HSL upregulated UDP-GlcNAc biosynthesis. In cluster 2, C12-HSL increased the levels of hydroxybutanoate and succinate semialdehyde, key components of butanoate metabolism in heterotrophic bacteria,38 indicating that C12-HSL may facilitate heterotrophic metabolism and the growth of heterotrophic bacteria in the microbial community.39 Glycerophospholipid metabolites, such as lysophosphatidylcholine (LysoPC) (20:0), were the most noteworthy metabolites in cluster 3. These were only induced by 3OC6-HSL (Figure 6c). Despite the random changes observed in cluster 4, increases in NAD following the addition of 3OC6-HSL, C6-HSL, and C8HSL were particularly interesting (Figure 6d). In addition, AHLs-induced metabolites perturbation in anammox consortia during phase III (day 90) was also confirmed. Val and Ala increased after treatment with 3OC6-HSL, 3OC8-HSL, and C12-HSL. The upregulation of glycerophospholipid metabolism induced by 3OC6-HSL during phase III consistent with the changes during phase II (Figures S13 and S14).

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DISCUSSION Existence of AHLs Quorum Sensing in Anammox Consortia. Since pure anammox bacteria have not been obtained,40 the existence of AHLs in anammox consortia was a prerequisite for AHLs regulation. Five specific kinds of AHLs were found in anammox consortia (Figure S3). The exogenous dosing concentrations were determined according to a previous study showing that the magnitude of exogenously added AHL concentration that had a positive effect on the bacterial community was in μmol/L5,41,42 and that 2 μM AHLs showed the most obvious influence in activated sludge.42 The concentration of AHLs in the reactor differed from that in the medium (2 μM), which is also supported by previous study showing that the concentration of AHLs changed after exogenous addition.23 Taking 3OC6-HSL as an example, the concentration of 3OC6-HSL in the control reactor was 0.19− 2.84 nM. The order of magnitude is similar to that of other reports.5 It was significantly lower than that of 0.33−1.3 μM in the reactor when dosing with 3OC6-HSL (p < 0.05). In addition, the hypothetic genes for AHLs production in anammox bacteria should be taken into consideration. AHL synthase could be divided into three subtypes: LuxI-type, LuxM-type, and HdtS-type.43 Although the LuxI-type of synthetic protein was extensively studied, this kind of protein could not be found in anammox bacteria by sequence alignment with the published draft genome.44−49 One of the reasons is that the gap in the draft genome may omit some potential AHLs synthase. Another reason is that LuxI-type synthase might not be responsible for AHL synthesis in anammox bacteria. The annotation of “autoinducer synthetase”(AHLs synthase) in Candidatus scalindua brodae suggested that AHL synthase of anammox bacteria was the lysophospholipid acyltransferases (LPLATs) of glycerophospholipid biosynthesis,44 which was similar to the HdtS-type.50 In other words, the AHLs synthase in anammox bacteria is likely to be HdtStype. Thus, not only from the perspective of the existence of signals but also from the genetic point of view AHLs existed in anammox consortia and may induce potential influence. Regulation Pathways Responsible for EPS Production by AHLs. The significant correlation between the increases in metabolites in cluster 1 and the increases in EPS production when dosing AHLs, combined with the previous reports that amino acids are basic components of proteins and that some

Figure 5. Hierarchical clustering analysis of metabolites in samples from the control and treatment reactors with the addition of 3OC6HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL during phase II. Clustering was performed using Pearson distance as the distance metric. The cluster tree shows how the samples and metabolites divide. Across the top are the samples, labeled by their class in different colors, and along the side are the metabolites.

hierarchical clustering tree. This is consistent with the principal component analysis (Figure S10). Addition of AHLs perturbed the metabolite levels. Interestingly, different AHLs induced disparate metabolic changes. Clustering revealed four main groups of metabolites: cluster 1, metabolites that increased in the treatment reactors; cluster 2, metabolites that only increased in samples treated with C12-HSL; cluster 3, metabolites that only increased in samples treated with 3OC6-HSL; and cluster 4, metabolites that fluctuated randomly. Significantly different metabolites were obtained from comparison of the treatment reactors and the control reactor (Figure S11). The representative metabolic pathways were obtained from the metabolite analysis and the functional metagenomic annotations of the anammox consortia (Figure S12). Increases in some amino acids contents, including valine (Val), alanine (Ala), aspartate (Asp), glutamate (Glu), and leucine (Leu), in addition to some metabolites related to amino sugar metabolic pathways including the uridine diphosphate-Nacetylgalactosamine (UDP-GlcNAc) and N-acetylmannosamine (ManNAc), were responsible for the clustering of metabolites into cluster 1. To investigate the potential relationship between these intracellular amino acids and the extracellular PN, multiple linear regression modeling was applied (Table S1). The best-fit model using SPSS (adjusted R2 = 0.850, p < 0.001; Table S2) showed that the main intracellular amino acids contributing to the variations in the extracellular PN were Ala, Val, and Glu. In addition, Ala, Val, and Glu were increased in the anammox consortia metabolites following addition of all the AHLs (Figure 6e). Distinct regulation induced by different AHLs was also observed (Figure 6a,e). 3OC6-HSL increased the amount of Asp but not of Val. 3OC8-HSL exclusively increased the amounts of Asp and Leu. C6-HSL and C12-HSL increased the amount of Leu. All the AHLs enhanced ManNAc 2211


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Figure 6. Analyzed pathways regulated by AHLs in anammox consortia during phase II. The amino acid (Ala, Val, Leu, Asp, and Glu) biosynthesis is described in (a), and amino sugar metabolism is illustrated in (b). Biosynthesis of LysoPC(20:0) in glycerophospholipid metabolism is shown in (c), and NAD involved in nitrogen metabolic pathways is presented in (d). Profile of representative metabolites in the control and the biomass with the addition of 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12-HSL is presented in (e). Metabolites that were significantly enriched by exogenous AHLs are marked in red. Metabolites that were present but not significantly enriched by AHLs are marked in black. Metabolites that could not be recognized in the metabolomic analysis are marked in gray. The dots with blue, green, purple, orange, and gray below the metabolite name represent the content of the metabolites that are elevated by 3OC6-HSL, 3OC8-SHL, C6-HSL, C8-HSL, and C12-HSL, respectively. Abbreviations for the metabolites are listed in Table S3.

amino sugars are precursors of polysaccharides,51 we propose that the enrichment of these metabolites and their metabolic pathways may mediate the increased production of PN and PS following treatment with AHLs, resulting in variable increases in floc sizes. Nearly all AHLs upregulated the levels of Ala, Val, and Glu. It could be supported by the best-fit modeling of multiple linear regression that Ala, Val, and Glu are the main amino acids that change the extracellular PN to promote extracellular PN production. Furthermore, the obviously increased Ala and Val contents following treatment with 3OC6-HSL, 3OC8-HSL, and C12-HSL correlated with the increases in extracellular PN content during phase III, which also supported this hypothesis. However, although Asp and Leu showed no correlation with increased amounts of extracellular PN in the linear regression model, the Asp and Leu contents in the extracellular PN of anammox consortia was relatively high, as previously reported.31 This indicated that Asp and Leu are important

components of the extracellular PN. Asp and Leu are also required for biofilm formation,52,53 which may influence extracellular PN production. Thus, we suggest that AHLs promote the production of extracellular PN mainly by increasing the amounts of Ala, Val, and Glu and by promoting the enrichment of Asp and Leu. Considering the diversity of extracellular PS in different microbiota,54 the regulation of PS production in these microbes varied. The metabolic synthesis of uridine diphosphate glucose (UDP-Glc), uridine diphosphate galactose (UDP-Gal), UDPGlcNAc, and ManNAc was identified here, and the syntheses of these metabolites have been implicated in PS production.55−57 This suggests that the anammox consortia are likely to regulate PS production via these metabolic pathways. Furthermore, AHLs-mediated QS influenced extracellular PS production by producing specific carbohydrate-rich polymers.58 Combined with the results of this study, it is indicated that AHLs regulated the increases in ManNAc but not the synthesis of UDP-Glc and 2212


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Regulation Characteristics of the AHL-Mediated QS System for Anammox Consortia. No differences in NRR and floc aggregation were initially observed after adding exogenous AHLs during phase I. The same phenomenon is observed in the repetitive operation of reactors. The supply of AHLs increased because of the shortened HRT during phase II. Thus, the average concentration of 3OC6-HSL in the supernatant of reactor during phase II (1.07 μM) was higher than during phase I (0.60 μM) (p < 0.05). This may be because AHLs do not affect anammox consortia at low concentrations. A previous study showed that exogenous AHLs at low concentrations did not regulate biofilm characteristics, while at high concentrations, AHLs did.66 However, the floc size of anammox consortia during phase II was larger than that during phase I. Previous study showed that the cluster of cells was importance for quorum sensing and providing benefits for the quorum sensing.67 Thus, the effects mediated by AHLs during phase II were more obvious than phase I. The discrepant effects on NRR and floc aggregation induced by AHLs between phases II and III could be explained by AHLs slowing the metabolic activity under nutrient-deficient conditions. Disintegration of bacterial communities is related to nutrient deficiency.68 The compact aggregates slightly disintegrated in phase III indicated that the anammox consortia were nutrient-deficient. Overall, the amount of most metabolites in the biomass dosing AHLs was lower than that in the control (Figure S13). Especially, some PN-related amino acids (Asp, Glu, and Leu) and PS-related amino sugars no longer increased when dosing AHLs. This suggests that the metabolic activity of anammox consortia treated with exogenous AHLs slowed down under nutrient-deficient conditions. This consumption is consistent with the results of previous studies20,69 and enables bacterial survival. Thus, the addition of exogenous AHLs to anammox consortia resulted in the loss of the advantages on NRR and aggregation during phase III because of slowed metabolic activity. Cause of Perturbations in Bacterial Activity and Growth Rate. Since accelerating the aggregation of anammox consortia is useful for increasing the bacterial activity and growth rate,70 further research is required to determine if these effects are caused by bacterial aggregation. Metabolomics analysis showed that AHLs increased the activity and growth by increasing NAD and LysoPC(20:0) biosynthesis, supporting the conclusion that AHLs regulate bacterial growth and activity in anammox consortia. Moreover, a previous study showed that AHLs increased the activity and growth of anammox consortia in batch experiments.23 In addition, the function of AHLs in short-term batch experiments is unlikely to be affected by aggregation, since this process is relatively slow. Hence, although the enhancements of bacterial activity and growth rate may be related to the simultaneous increase in floc size andAHLs addition, the main factor is the upregulated metabolism by exogenous AHLs. Our study not only provides a foundation for understanding how AHLs regulate the activity, growth rate, and EPS production of anammox consortia but also hints at potential strategies for shortening the start-up period that triggers bacterial growth rates and environmental sensitivity in anammox consortia. Because 3OC6-HSL, C6-HSL, and C8HSL had positive effects on the anammox consortia, genetically engineered bacteria carrying the signal gene for the synthesis of 3OC6-HSL, C6-HSL, and C8-HSL could be constructed for dosing the engineered bacteria in the reactor to improve the

UDP-Gal, thereby promoting PS production in anammox consortia. However, not all AHLs exhibited the same regulatory pathway during the PS production. For example, 3OC6-HSL, OC8-HSL, and C6-HSL also increase UDP-GlcNAc synthesis. This indicates that the AHLs-based regulatory mechanisms with respect to extracellular PS synthesis are diverse. Additionally, although several enzymes in the synthetic pathway of ManNAc in anammox bacteria could not be found based on the existing annotation of the anammox draft genome,49 the entire synthetic pathway of ManNAc was found in metagenomic analysis (Figure S12). This synthetic pathway may be accomplished by multiple bacteria, which is supported by a previous study showing that some metabolic process are completed through bacterial cooperation in anammox consortia.59 This suggested that the regulation of ManNAc synthesis by AHLs may not be entirely present in anammox bacteria but is beneficial to the whole consortia. Regulation of LysoPC(20:0) Biosynthesis by 3OC6HSL as an Indicator of Bacterial Growth. Bacterial growth involves multiple metabolic pathways, such as nucleotide metabolism, carbohydrate metabolism, and lipid metabolism.60 Since nucleotide metabolism and carbohydrate metabolism are involved in many metabolic processes,61 it is difficult to use variations in these metabolites as predictors of bacterial growth. When examining the differences in nucleotide metabolism and carbohydrate metabolism in the control versus the biomass dosing AHLs, nearly half of the metabolites increased, while the rest decreased. In particular, LysoPC(20:0) biosynthesis in glycerophospholipid metabolism (lipid metabolism) increased significantly, and this was coincident with the increase in the biomass yield rate of anammox consortia. Glycerophospholipid metabolism in lipid metabolism coincides with cell membrane lipid synthesis.62 In addition, bacterial growth and the accumulation of cell membrane lipids were tightly correlated.63 Thus, the increase in the growth rate mediated by 3OC6-HSL treatment was likely to be facilitated by enhancing LysoPC(20:0) biosynthesis. Although the upregulation of LysoPC(20:0) biosynthesis is not the only reason for the observed increase in growth rate, the discovery that 3OC6-HSL regulates LysoPC(20:0) biosynthesis in the anammox consortia provides a foundation for identifying key upstream metabolic pathways controlling bacterial growth. Regulation of NAD by AHLs for Increasing Bacterial Activity. According to previous studies,64 the bacterial activity is related to the expression of nitrogen-removal-related genes, particularly the typical anammox gene hzsA. However, anammox activity and its relation to metabolites remains poorly understood. In this study, we found that NAD levels were higher in anammox consortia only following treatment with 3OC6-HSL, C6-HSL, and C8-HSL during phase II. This was consistent with the results showing increased activity of anammox consortia. In metabolism, NAD is involved in redox reactions and carries electrons between reactions.61 Thus, it is considered to be an electron carrier. During the biochemical reaction that converts nitrite and ammonium into nitrogen in anammox consortia, NAD also plays an essential role in electron transfer.65 This suggests that NAD is extremely important in anammox activity. Therefore, 3OC6-HSL, C6HSL, and C8-HSL may increase the activity of anammox consortia by increasing the amount of electron transport carriers. Although many metabolites vary with increasing bacterial activity, NAD is suitable as an indicator of anammox activity for metabolite analyses. 2213


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NRR or biomass aggregation, resulting in quick start-up of the anammox reactor.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b05699. Materials and methods; schematic of experimental operation, photo of seeding sludge; LC-MS results, influent and effluent nitrogen concentrations; effects of 3OC6-HSL, 3OC8-HSL, C6-HSL, C8-HSL, and C12HSL on nitrogen removal rates (NRR), activity, and biomass yield rate; flocs distribution; changes in NRR, granular size, PN, PS, and content; microbiological community and metabolites perturbance; hierarchical clustering and heat map analysis of metabolites; linear regression results; abbreviations; additional references (PDF) Content of extracellular PN and intracellular amino acid used in multiple linear regression model from 15 different samples of anammox consortia (XLSX)

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AUTHOR INFORMATION

Corresponding Author

*Tel./Fax: 0086-10-62754290. E-mail: [email protected]. ORCID

Sitong Liu: 0000-0001-6849-8704 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundations of China (No.51478006) and Shenzhen Science and Technology Innovation Committee (No.JSGG20160429162015597) and Shenzhen Municipal Development and Reform Commission (Discipline construction of watershed ecological engineering) for financial support.

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REFERENCES

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Metabolomics uncovers the regulatory pathway of acyl-homoserine

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