6 Feng et al.Wuter Research 165 (2019)114974 none was 00s up-regul P).whi with ac addition(fold change 2.02p<0.01byt but i ase (ALD lated in the nce of a 179.p<0.01 i,CFX 2.CFX3.andCYA1.bu nd exp ld nJ.ca was depen dent on acetate kinase(AckA. 412.9 an (xrp. ion levels of o ted in ackA further transforme Genes exp narZ, in CFX2 p<01 by t-te 2 test)were nCtenmedanmoadosyfetingnamnammoxonsorta ata th transformed mainly through ind n distinct route: communit type of im AMP- (AMP-AC)(EC 62.1.1)and ADP-forming effect of acetat mino acid e e in anam 13).wh vel 4(a with acetate ange 8,p TPM ma mi synthe athways ddi ialty for pro1 a Route cetate Acetyl-CoA amino pheny DP-ac put their degra 170 AMP-ac of Iv signif n the pr Acetate ALDH (Me)m (n (er)were ac genome.and the expres of Tyr and degradation ALDH s enc ute Route 3 e Ack Acetyl-P 4cd).The rotal PRO1 and transpo were also ted ()and e gene? CYAI and PRO1 (P<005) athways in anammox con tia.Heatmap ex contents of the inte d,the reader is re to th corresponded to the gene expression profile in the consortia.1234 up-regulated genes were expressed. Abundant, increasing COG functions in B. sinica included [C] energy production and conversion (105 significantly up-regulated genes expressed), [E] amino acid transport and metabolism (81 significantly up￾regulated genes expressed) and [J] translation, ribosomal struc￾ture and biogenesis (76 significantly up-regulated genes expressed). N-cycle-related functional genes (hdh, hzs, hao, nar, nir, and nrf) were found to be highly expressed in anammox bacteria. By comparing TPM values in J. caeni and B. sinica, between the auto￾trophic and mixotrophic groups, it was observed that transcription level of nrfA for DNRA function in B. sinica was significantly up￾regulated in the presence of acetate (fold change ¼ 1.79, p < 0.01), but no significant difference of nrfA expression was found in J. caeni (fold change ¼ 0.95, p > 0.05). TPM values of nrfA in B. sinica was 6363e14565, which was higher than that in J. caeni (TPM value ¼ 208e231) by nearly 1 to 2 orders of magnitude. No signif￾icant difference was found between the expression levels of other functional genes mentioned above, in the anammox bacteria of the two groups. Denitrification genes, including narG, narZ, nxrA, nirS, nosZ, and napA, were detected in bacteria of the anammox con￾sortia. Genes expression of narG, narZ, nxrA in CFX2 (fold change ¼ 2.11, p < 0.05 by t-test), nirS in CFX3 (fold change ¼ 5.06, p < 0.01 by t-test), nosZ in CYA1 (fold change ¼ 3.72, p < 0.01 by t￾test) and napA in PRO1 (fold change ¼ 2.11, p < 0.05 by t-test) were stimulated by acetate. Importantly, genes involved in acetate transformation were selected to analyze acetate metabolism by anammox consortia. Acetate was transformed mainly through three distinct routes (Fig. 3). The first route involved two types of acetyl-CoA synthetases (Acs), AMP-forming Acs (AMP-Acs) (EC 6.2.1.1) and ADP-forming Acs (ADP-Acs) (EC 6.2.1.13), which could catalyze acetate to form acetyl-CoA. AMP-acs expression level was up-regulated signifi- cantly with acetate addition in B. sinica (fold change ¼ 1.78, p < 0.01 by t-test) and PRO1 (fold change ¼ 5.93, p < 0.05 by t-test). Comparative genome analysis indicated that a response regulator, atoC was located up-stream of AMP-acs in B. sinica, but none was found in the proximity of AMP-acs in J. caeni (Fig. S4). Expression of atoC in B. sinica was significantly up-regulated with acetate addi￾tion (fold change ¼ 1.92, p < 0.05 by t-test), but it did not change significantly in J. caeni. cAMP receptor protein (CRP), which encodes acs transcription factor, was found in B. sinica and was up-regulated with acetate addition (fold change ¼ 2.02, p < 0.01 by t-test), but it was missing in J. caeni. The second route is composed of aldehyde dehydrogenase (ALDH, EC 1.2.1.3) catalyzing the reversible reaction of acetate to acetaldehyde. This gene was detected in B. sinica, J. caeni, CFX2, CFX3, and CYA1, but was found expressing more only in CFX3 with acetate addition (fold change ¼ 2.15, p < 0.05 by t￾test). The third route was dependent on acetate kinase (AckA, EC 2.7.2.1) and D-xylulose-5-phosphate phosphoketolase (Xfp, EC 4.1.2.9), and termed AckA-Xfp pathway. The first step was the transformation of acetate to acetyl phosphate. Gene ackA only existed in B. sinica and PRO1. Acetyl phosphate in B. sinica could be further transformed to D-Xylulose 5-phosphate, catalyzed by Xfp. Thus, acetate metabolism pathways in B. sinica were more versatile than those in J. caeni. 3.4. Strengthened amino acid cross-feeding in anammox consortia with acetate Previous studies indicate that amino acid exchange occurs in microbial communities, as a type of important inter-species inter￾action (Embree et al., 2015; Lawson et al., 2017). Therefore, the effect of acetate on amino acid exchange in anammox community was investigated. Expression levels of 20 amino acid synthetic and degradation pathways in six abundant bacteria are shown (Fig. 4 (a) and (b)). Among six abundant bacteria species, only PRO1 had intact synthetic pathways of all 20 amino acids. Interestingly, TPM values of many amino acids synthetic pathways were significantly increased with acetate addition (p < 0.05), especially for PRO1 and B. sinica. For CFX2, amino acids tryptophan (Trp), phenylalanine (Phe), histidine (His), lysine (Lys) and arginine (Arg) could not be synthesized independently, but their degradation pathways were found in the genome, and the expression of Lys and Arg degrada￾tion pathways were significantly up-regulated in CFX2 with acetate addition (fold change (Lys) ¼ 1.78, fold change (Arg) ¼ 1.55). Meanwhile, the synthetic pathways of Lys and Arg were signifi- cantly up-regulated in PRO1 and B. sinica in the presence of acetate (Fig. 4(a)). For CYA1, synthetic pathways of amino acids Trp, Tyr, His, methionine (Met), proline (Pro), and serine (Ser) were lacking, but degradation pathways of these amino acids were found in the genome, and the expression of Trp, Tyr and Met degradation pathways were up-regulated significantly, with fold changes of 2.48, 1.82 and 1.96, respectively. Genes encoding extracellular, outer membrane and periplasmic peptidase involved in extracellular protein degradation, except the periplasmic peptidase of AMX1 and extracellular peptidase of PRO1, were detected in all six abundant bacteria in the community (Fig. 4(d)). The total expression of these peptidase genes were significantly up-regulated in CFX2, PRO1 and B. sinica (p < 0.05). Genes involved in amino acid and oligopeptide transport system were also detected (Fig. 4(e)), and the genes expression levels were significantly promoted by acetate in CFX2, CYA1 and PRO1 (p < 0.05). Metabolomic analysis was conducted to evaluate metabolites contents of the whole consortia. A total of 18 amino acids were detected (Fig. S5), and 10 amino acids including Met and isoleucine (Ile), were up-regulated significantly with acetate addition, which corresponded to the gene expression profile in the consortia. Fig. 3. Three main acetate metabolic pathways in anammox consortia. Heatmap ex￾hibits gene expression profiles (Z score) in autotrophic and mixotrophic. Red indicates significant difference (p < 0.05 by two-tail t-test). *: p < 0.05; **: p < 0.01. (For inter￾pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6 Y. Feng et al. / Water Research 165 (2019) 114974