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Feng eta/Water Research 16(201)14974 n of diffe 2 Materials and methods mple it was reported wth under 2.1.Sample collection stud 3 mmox consortia tch ratio tion o da species still remain (Shu et al. 2015 and Noi N in the d we tigate the m metabolism of nammox ba 1at6.8 en was ren n order to faster and thusshorten thereactorstt-up period 250ml um bottle with the effecti 0f200m Nutrient s robiom ractions may help to maintair N 0)to e three serum bo les of the contro gro (autotro (20)and a 0.3 as withina constant ran of72-7sduri raction(St With the rapid d inte lopment c being sprayed with a gas mixt ure of N -c02(95 in order to teztionandurme into at 3 and agitated at 150 rpmin e dark.1 mL su tan .2008.and ntrations of NHA-N.NOZ-N.NOT- d COD.which itrate tration between controland ter d nd n g betw a by he oph have been fou mox red into R tubes.After bot the produc and the mic,metatransc and l und tial and cro sition andassem 017. com vever,the proces 22.Metagenome and metatranscriptome sequencing inte and heterotrophs in the consortia still remain Ve investigated ween two typical ion with the FastDNA autotrophic and evels of a actedfrom triplicate ophic and mix pant resp potentia Midi Kit (Omegao r055. their metabolicinteractionsin the Nano chip (total RNA)in an Agilent 2100 B nalyzer and wa cate autotr ented to ar average size of -300 bp with Covaris M220(Gene Companystructure (Leal et al., 2016). Responses of different anammox species to organics seem to be distinct. For example, it was reported that Candidatus Jettenia asiatica (J. aiatica) showed no superiority in growth under mixotrophic conditions compared to autotrophs (Huang et al., 2014), while other study found that the biomass of Candidatus Brocadia fulgida (B. fulgida) showed an increase under certain C/N ratios (Jenni et al., 2014). However, the process by which, organics affect the gene functional potential of different anammox species still remains unclear (Shu et al., 2015). As anammox bacteria grow extremely slowly, it may be meaningful to investigate the mixotrophic metabolism of anammox bacteria further, as they may be acquiring extra energy from organic matter in order to grow faster and thus shorten the reactor start-up period (Kartal et al., 2012). Nutrient sources also affect metabolic interactions of the microbiome. Metabolic interactions are ubiquitous in microbial communities, especially in microscale cell aggregates, which play an important role in the functioning of microbial communities (Cordero and Datta, 2016). From an ecological aspect, metabolic interactions may help to maintain a stable coexistence between bacteria as a strategy to decrease the energy consumption of the community (Guo et al., 2018; Pande et al., 2014). The emergence and maintenance of metabolic interactions depends on many factors, such as nutrient sources (Benomar et al., 2015) and spatial organization (Jiang et al., 2018). Variation in nutrient conditions may influence gene transcription and thereby impact metabolic interaction (Steffen et al., 2014). With the rapid development of meta-omics technology, the subject of metabolic interactions in microbial communities has drawn wide attention and turned into an important topic (Ponomarova and Patil, 2015). Pure anammox culture is extremely hard to obtain (Kuenen, 2008), and many heterotrophs, such as Chloroflexi and Chlorobi, are abundant in these communities (Speth et al., 2016). Recently, metabolic interactions which could perform energy-efficient nitrogen removal from wastewater, such as degradation of extracellular peptide substrates of anammox bacteria by heterotrophs and nitrogen and metabolite cross-feeding between anammox bacteria and heterotrophs, have been found in anammox consortia (Lawson et al., 2017). Cross-feeding is a kind of microbial interaction, in which metabolites could be shared by both the producer and the receiver, thus they can benefit from this process (Zhao et al., 2018). An investigative study of the mechanism underlying metabolic interactions in microbial communities may broaden our insight in regard to the composition and assembly of these communities (Zengler and Zaramela, 2018). However, the process by which organics influence interactions between anammox bacteria and heterotrophs in the consortia still remain unresolved. We investigated competition between two typical anammox species, J. caeni and B. sinica, under autotrophic condition and mixotrophic condition with acetate addition based on batch tests. This phenotype was mapped to the underlying microbiome and further determined by sampling and analyzing autotrophic and mixotrophic anammox consortia. We characterized the gene functional potential, as well as the metabolic network, using levels of anammox species in the anammox community, in order to explore the hypothesis that different anammox species have discrepant responses to acetate addition. As well, potential mechanisms associated with individual anammox species and their metabolic interactions in the consortia were analyzed. Our study provides a novel, detailed insight into mixotrophic metabolism of anammox bacteria, and suggests the possibility of predicting an increase in anammox performance under low C/N ratio. 2. Materials and methods 2.1. Sample collection The anammox consortia used in this study was collected from a 3 L lab-scale sequencing batch reactor (SBR), which had been in operation at 37 C for 280 days (Tang et al., 2018a). It was fed with a synthetic medium solution (Van de Graaf et al., 1995), and the concentration of NH4 þ-N and NO2 -N in the influent were 300 mg L1 . The hydraulic retention time (HRT) was 0.75 d. The pH was maintained at 6.8e7.5, and dissolved oxygen was removed by sparging with N2-CO2 (95/5%) gas. The batch tests were performed in six 250 mL serum bottles with the effective volume of 200 mL, each containing 0.315 g volatile suspended solids (VSS)/L anammox consortia inoculum mentioned above. By referring to the previous study about heterotrophic metabolism of anammox bacteria (Güven et al., 2005), the initial concentrations of NH4 þ-N and NO2 -N in synthetic medium solution were set at a ratio of 1:1 with the concentrations of 50 mg L1 . No sodium acetate was added (COD/ TN ¼ 0) to the three serum bottles of the control group (autotrophic group), while acetate was added to the three serum bottles of the experiment group (mixotrophic group), to maintain a final COD/TN ratio of 0.3 as per a previous study (Feng et al., 2018). The pH of the medium was adjusted to 7.2 by adding 0.1 M NaOH solution (Carvajal-Arroyo et al., 2014). Although the pH was not controlled, it was within a constant range of 7.2e7.5 during the experiment. After being sprayed with a gas mixture of N2-CO2 (95/5%) in order to maintain strict anaerobic conditions, all serum bottles were incubated at 37 C and agitated at 150 rpm in the dark. 1 mL supernatant sample from each bottle were collected using syringes to determine the concentrations of NH4 þ-N, NO2 -N, NO3 -N, and COD, which was done for five times during the experiment. At the point where the nitrate concentration between control and experiment group had statistical difference (p < 0.05 by t-test) and the difference of average nitrate concentration of control and experiment group was more than 10% (Kartal et al., 2007a), both autotrophic and mixotrophic anammox consortia were collected from each serum bottle and transferred into RNase-free tubes. After being rapidly frozen in liquid nitrogen, consortia samples were stored at 80 C for subsequent metagenomic, metatranscriptomic, and metabolomic analyses, which could be applied to explore microbial gene functional potential and cross-feedings (Bahram et al., 2018; Lawson et al., 2017). 2.2. Metagenome and metatranscriptome sequencing Autotrophic anammox consortia samples and mixotrophic anammox consortia samples collected in triplicate from each bottle in batch tests were used for total DNA extraction with the FastDNA Spin Kit for Soil (MP Biotechnology, CA, U.S.). DNA concentration and purity was determined using TBS-380 and NanoDrop 2000, respectively. 1% agarose gels electrophoresis system was used to examine DNA quality. Respective triplicate autotrophic and mixotrophic anammox consortia DNA samples were mixed thoroughly for metagenome sequencing (Jia et al., 2018). Total RNA was extracted from triplicate autotrophic and mixotrophic anammox consortia samples from batch tests using the E.Z.N.A® Soil RNA Midi Kit (Omega BioTek, Norcross, GA, U.S.) according to manufacturer's protocols. RNA quality was assessed with a RNA6000 Nano chip (total RNA) in an Agilent 2100 Bioanalyzer and was determined by the RNA integrity number (RIN). Respective triplicate autotrophic and mixotrophic anammox consortia RNA samples were used for metatranscriptome sequencing. To construct paired-end library, DNA was fragmented to an average size of ~300 bp with Covaris M220 (Gene Company 2 Y. Feng et al. / Water Research 165 (2019) 114974