The Mechanism Of Enhancing The Performance Of Nitrogen Removal By Anaerobic Ammonium Oxidation At Ambient And Low Temperature Based On Quorum Sensing

The anaerobic ammonium oxidation(anammox)process is an advanced wastetwater treatment technology with advantages in high nitrogen removal efficiency,low carbon demand and low energy consumption advantages.However,the activity of anammox bacteria is usually inhibited under condition of ambient and low temperatures(less than 15 ℃),resulting in the low efficiency of treatment which limits application of anammox-based wastewater treatment technology.Based on the quorum sensing mechanism,the anammox biofilm reactor was regulated at ambient and low temperature.This study aimed to strengthen the anammox bacteria activity and treatment performance at ambient and low temperatures through exogenous dosing of quorum sensing signal molecules(i.e.,acyl homoserine lactone,AHLs)and redox media(i.e.,fulvic acid).Moreover,the detailed mechanism underlying enhanced anammox performance was explored by biotechnology of high-throughput amplicon sequencing,metagenomics sequencing as well as metabolomics analysis.Subsequently,objectives of this study were to solve the bottleneck problems of low nitrogen removal rate and efficiency in the anammox process under conditions of ambient and low temperature.A lab-scale anammox biofilm reactor was operated to explore variations of nitrogen removal performance,concentrations of quorum sensing signal molecules and the microbial structure under conditions of different temperatures.Results showed that the removal rate of total inorganic nitrogen(TIN)dropped rapidly from 80% to 66%when the temperature dropped from 36 ℃ to 15 ℃.Candidatus Kuenenia and Candidatus Brocadia were the dominant genera of anammox bacteria in the system,nonetheless,the relative abundance of Candidatus Kuenenia continued to increase from1.43% to 22.89% with the decrease of temperature.Concentrations of AHLs in the water and biomass phases showed a decrease,suggesting that the quorum sensing effect in the system was weakened.These results indicated that decreasing temperature reduced the activity of bacteria and its communications with other bacteria,which lead to the deterioration of anammox performance.To improve the performance of the anammox system at 15 ℃,quorum sensing signal molecules were amended to the anammox system.It was found that separate introduction of C6-HSL and C8-HSL promoted the activity of anammox bacteria by 43.5% and 89.7%,respectively,which subsequently improved the removal efficiency of TIN.Moreover,the secretion of EPS and the expression of anammox-related functional genes(i.e.,hzo,hzs B and ccs B)were enhanced after amendment C6-HSL or C8-HSL.Metagenomic analysis demonstrated that amendment of quorum sensing molecules effectively increased the relative abundance of genes involved in the tricarboxylic acid cycle,and biosynthesis of amino sugar and nucleotide sugar,and amino acid,particularly genes related to the biosynthesis of leucine and methionine as well as the synthesis of UDP-Gal and UDP-GLc NAc.C6-HSL and C8-HSL were simultaneously amended to the anammox system to investigate its efficacy in enhancing anammox process under conditions of 15 ℃~5 ℃.The result showed that simultaneous amendment of C6-HSL and C8-HSL improved the TIN removal efficiency,achieving to 80% at 15 ℃ and 60% at 5 ℃.The growth rate and doubling time of anammox bacteria were measured and results showed that the addition of composite quorum sensing signal molecules promoted the growth of anammox bacteria,and its maximum specific growth rate was 58.3% higher than that of the control group,and correspondingly the doubling time of anammox bacteria was shortened to 5.6 days.Additionally,metagenomic analysis found that the addition of composite quorum sensing signal molecules increased the relative abundance of microorganisms containing Lux R family transcription regulator genes,which could potentially strengthen communications among microorganisms and regulate activities of microorganisms under conditions of low temperatures.Non-targeted metabolome analysis showed that the higher concentration of tyrosine and glutamic acid may help anammox bacteria adapt to low temperature conditions,and promote their growth at ambient and low temperatures,consequently improving the nitrogen removal performance at anbient and low temperatures.A redox media,i.e.,fulvic acid,was also used to regulate the anammox system at ambient and low temperatures.Exogenous fulvic acid could stimulate the secretion and release of microbial quorum sensing signaling molecules in the anammox system.Besides,the addition of 0.1 m M fulvic acid improved the nitrogen removal performance,microbial activity and the expression level of anammox-related functional genes(hzo and hzs B)in the anammox system at 15 ℃.The removal efficiency of TIN reached 58.6%,which was higher than that of the control group of 42.1%.And the dominant genus of anammox showed a shift from Candidatus Brocadia to Candidatus Kuenenia.Co-occurrence network analysis showed that the addition of fulvic acid strengthened potential interations of microorganisms in the anammox system.Metagenomics analysis further revealed that amendment of fulvic acid promoted relative abundances of genes involved in the tricarboxylic acid cycle metabolism pathway.In addition,metabolomic analysis showed that the presence of fulvic acid promoted concentrations of several metabolites such as amino sugars(glucose-1-phosphate,UDP-D-glucuronic acid and UDP),and also concentrations of metabolites related to redox reactions(NAD+ and NADH),indicating that fulvic acid stimulated the metabolic activity of microorganisms in the anammox system,which could be benefical to improve the nitrogen removal performance under conditions of ambient and low temperatures.