Bacterial communication intelligently regulates their interactions in anammox consortia under decreasing temperatures.

Bacterial communication could affect their interactions, but whether this regulation has "intelligence" is still unknown. Here, we operated an anammox reactor under temperature gradient from 35 °C to 15 °C. As results, expression abundance of bacterial communication genes increased by 12 % significantly after temperature declined. Division of labor among distinct signal molecules was evidenced by complementary roles of acyl-homoserine lactones (AHLs) and diffusible signal factor (DSF) in affecting bacterial interactions and niche differentiation respectively. DSF based inter-and intra-communication helped bacteria match their investments and rewards during cross-feedings. When temperature was below 25 °C, transcription regulator Clp governed by DSF inclined to promote folate and molybdenum cofactor biosynthesis, which coincidentally benefited one anammox species more than another. Meanwhile, for the anammox species with lower benefits, Clp also inclined to decrease biosynthesis of costly tryptophan and vitamin B1 rewarding others. Interestingly, bacterial communication inclined to influence the bacteria with many cooperators in the community or with high capacity to export cofactors for cross-feedings when temperature decreased. As results, these bacteria were enriched which could lead to closer interactions in whole community to adapt to low temperatures. The discovered intelligence of bacterial communication opened another window for understanding bacterial sociobiology.

Response of amino acid metabolism to decreased temperatures in anammox consortia: Strong, efficient and flexible.

Although amino acid (AA) metabolism is basis of bacterial activities, unique characteristics of its response to decreased temperatures are not fully understood. Achieving nitrogen removal rate of 130-150 mg N/ (L∙d), metabolic differences of anammox consortia between 35 °C and four decreased temperatures (15-30 °C) were revealed respectively. 0-11.4-fold abundance variation of marker metabolites evidenced change of key metabolism (metabolism of AA, lipid and energy production) at decreased temperatures. However, AA metabolism varied more obviously than others, implying stronger response and higher functional potential. Efficiently, network topology confirmed more cellular processes represented by growth metabolism and biofilm formation were influenced by AA metabolism. Flexibly, down-regulated biosynthesis of unfavorable AAs for psychrophilic enzyme differed from enhanced biosynthesis of costly AAs, which only matched partial decreased temperatures to save energy. This work elucidates advantages of AA metabolism over others, exogenous amino acids could significantly promote activity of anammox bacteria at decreased temperatures.

Metabolic patterns reveal enhanced anammox activity at low nitrogen conditions in the integrated I-ABR.

Substrate concentrations greatly influence bacterial growth and metabolism. However, optimal nitrogen concentrations for anammox bacteria in nitrogen-limited environments remain unclear. Here, we observed enhanced nitrogen metabolism and anabolism of anammox bacteria at low nitrogen conditions. Efficient nitrogen removal was achieved at ammonium and nitrite influent concentration of 30 mg/L under HRT of 1 hr, with an average nitrogen removal rate (NRR) of 0.73 kg N/(m3 ·day) in I-ABR composed of four compartments. The highest anammox activity of 6.25 mmol N/ (gVSS·hr) was observed in the fourth compartment (C4) with the lowest substrate levels (ammonium and nitrite of 11.6 mg/L and 7 mg/L). This could be resulted from the highest expression level of genes involved in nitrogen metabolism in C4, which was 1.49-1.67 times higher than that in other compartments. Besides, the second compartment (C2) exhibited the most active anabolism at ammonium and nitrite of 17 mg/L and 13 mg/L, respectively, which contributed to the most active amino acid synthesis and thus the highest EPS (1.35 times higher) in C2. This enhanced amino acid auxotrophy between anammox bacteria with heterotrophs, and consequently, heterotrophs thrived and competed for nitrite. These results hint at the potential application of anammox process in micro-polluted water. PRACTITIONER POINTS: High nitrogen removal and efficient biomass retention at low nitrogen concentrations under short HRT was achieved in I-ABR. Optimal concentrations for anammox nitrogen removal and anabolism were discussed under low nitrogen concentrations. More active anabolism contributed to enhanced amino acid synthesis and thus higher EPS contents. Low substrate levels led to enhanced expression of genes involved in nitrogen metabolism and thus high anammox activity.

Metabolic acclimation of anammox consortia to decreased temperature.

Widespread application of anammox process has been primarily limited to the high sensitivity of anammox consortia to fluctuations of temperature. However, the metabolic acclimation of anammox consortia to decreased temperature remains unclear, which is the core of developing potential strategies for improving their low-temperature resistance. Here, we operated anammox reactors at 25 °C and 35 °C to explore the acclimation mechanism of anammox consortia in terms of metabolic responses and cross-feedings. Accordingly, we found that the adaptation of anammox consortia to ambient temperature (25 °C) was significantly linked to energy conservation strategy, resulting in decreased extracellular polymeric substance secretion, accumulation of ATP and amino acids. The expression patterns of cold shock proteins and core enzymes caused the apparent metabolic advantage of Candidatus Brocadia fulgida for acclimation to ambient temperature compared to other anammox species. Importantly, strengthened cross-feedings of amino acids, nitrite and glycine betaine benefited adaptation of anammox consortia to ambient temperature. Our work not only uncovers the temperature-adaptive mechanisms of anammox consortia, but also emphasizes the important role of metabolic cross-feeding in the temperature adaptation of microbial community.

Enhance the treatment of low strength wastewater at low temperature with the coexistence system of AnAOB and heterotrophic bacteria: Performance and bacterial community.

The application of anammox process in mainstream wastewater treatment process is still facing challenges especially at the low temperature. To resolve this problem, the coexistence system of anaerobic ammonia-oxidizing bacteria (AnAOB) and heterotrophic bacteria (HB) was built in this study. The nitrogen removal efficiency mainly maintained at above 90% during the process of temperature reducing from 35 °C to 10 °C. The nitrogen removal rate were 0.30 g N·L-1·d-1 at both 25 and 15 °C and 0.10 g N·L-1·d-1 at 10 °C, respectively. Analysis of 16S rRNA genus sequencing revealed that as the temperature reduced to 10 °C, the Denutrotisoma genera presented a downward trend but Comamonadaceae genera showed an upward trend. At 10 °C, the contrast of anammox activities between granular and flocculent sludge in the system revealed that although the abundance of anammox genera was much lower in flocculent sludge than that in granular sludge, the anammox activities showed no significant discrepancy. And the abundance of Comamonadaceae and Chloroflexales genera were much higher in flocculent sludge than those in granular sludge, presenting their key roles to anammox activity at low temperature. The Circos diagram and Cluster of orthologous Group of protein functional predication showed that the functional abundance related to interaction among microbial communities were higher in flocculent sludge but those related to self-growth was higher in granular sludge. This result indicated the significance of the interactions based on the microbial diversity in the application of annamox process at low temperature.

Enhanced microbial metabolism in one stage partial nitritation-anammox system treating low strength wastewater by novel composite carrier.

One stage partial nitritation-anammox (PN-A) process has attracted more and more attention due to the low investment cost but the instability in treating low strength wastewater. In this study, for producing a novel composite carrier that could provide high ammonia microenvironment in low strength wastewater, the zeolites and floating materials were combined in the spherical shell and distributed evenly by the spherical polyhedron. And a moving bed biofilm reactor (MBBR) with the composite carriers and ordinary carriers without zeolites as control group was operated for nearly 120 days. The PN-A process were realized in 53 days, and the total nitrogen removal efficiency reached around 85% at influent ammonium concentration of 50 mg/L finally. Analysis of 16S rRNA gene sequencing revealed that the composite carriers showed significant promotion on the proliferation of ammonium oxidizing bacteria (AOB) and enrichment of anaerobic ammonium oxidizing bacteria (AnAOB), accounting for 19.14% and 41.65% on the surface, respectively. Moreover, the existence of relative higher abundance of ammonia on the composite carrier surface was validated by the metabolite biomarker of glutamate and especially spermidine. The metabolomics analysis and 16S rRNA function prediction showed that the protein synthesis pathway was obviously upregulated on the composite carriers surface compared with that on the ordinary carriers surface. The higher abundance of glutamate and putrescine indicated that the composite carrier could stimulate the metabolism and growth of bacteria. The present study provided a functional carrier to realize the transformation of activated sludge system into PN-A system treating low strength wastewater, which is significant to the application of the process in mainstream.