Response Mechanism Of Aerobic Granular Sludge Under Ribavirin Stress

The outbreak of Corona Virus Disease 2019(COVID-19)has led to a large number of pharmaceutical pollutants entering wastewater treatment plants(WWTPs).Meanwhile,since the traditional wastewater treatment process cannot completely remove the pharmaceuticals,it is necessary to select a new treatment process to simultaneously remove conventional pollutants and pharmaceutical pollutants.The aerobic granular sludge(AGS)is widely used in the removal of emerging contaminants due to its 3D structure,high biomass and strong toxicity tolerance.However,AGS technology also possesses some peoblems such as long-term stable operation,which restricts its large-scale promotion and application.Thus,it is necessary to further explore whether AGS can show excellent removal efficiency and stable operation under pharmaceutical stress.In this study,typical pharmaceuticals in WWTPs with different technological process were identified to summarize the occurrence of different pharmaceutical pollutants and evalute the ecological risk assessment,and the antiviral pharmaceutical Ribavirin(RBV)was finally selected to act on the AGS system.On this basis,compared with the conventional AGS,the physicochemical properties,extracellular polymeric substances(EPS),information transfer,energy conversion,microbial characteristics and protein expression of the AGS system under stress were studied for correlation analysis.In addition,the role of AGS signaling molecules,the regulation of genes,and the micro response mechanisms of energy and electron transfer in response to external stimuli were further elucidated.Besides,the regulatory factors affecting the growth and stability of AGS were explored,and the stability maintenance mechanism of AGS was further analyzed.This study aimed to explore the application field of AGS and clarify the micro factors affecting AGS life activities,so as to provide theoretical basis and data reference for ensuring the application of AGS in practical engineering field and maintaining its stable operation.The results obtained in this paper were as follows:(1)The investigation results of 6 typical pharmaceutical pollutants in two WWTPs with different technological processes showed that the detection rate of pharmaceuticals was 100%,and the concentration range was 612.04-2322.71 ng/L.Compared with previous studies,the detection frequency and concentration of RBV(the COVID-19 treatment pharmaceutical)were higher in this study and with the highest concentration reaching 314.30 ng/L.The pharmaceutical occurrence in different processes exhibited that the Moving-Bed Biofilm Reactor(MBBR)process had higher removal capacity for most pharmaceuticals than the Anoxic-Anaerobic-Anoxic-Oxic(AAAO)process,but the removal rates of RBV and ranitidine hydrochloride in both processes were all lower than 42.96%.The biodegradation mechanism showed that the biodegradation process of RBV in the activated sludge system was as follows:firstly,it was decomposed into 1H-1,2,4-triazole-3-carboxamide and an oxygen-containing five-membered heterocycle under the reaction of nucleosidase,and then 1H-1,2,4-triazole-3-carboxamide was formed by amide hydrolysis to form 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylic acid.The ecological risk assessment showed that the pharmaceuticals in the influent of the WWTPs presented a medium-high risk,and the RQ values decreased after MBBR and AAAO treatment.Meanwhile,clozapine and ranitidine hydrochloride could still show high and medium risk in effluent,respectively,and had potential risks to aquatic ecosystems.(2)The change trend of AGS removal rate of conventional pollutants in the conventional group and the stress group was consistent,and both showed good removal rates.RBV stress accelerated the granulation process of AGS and maintained the particle size of AGS relatively small.The removal rate of RBV by AGS gradually increased from the initial 61.9% to more than 99% and remained stable.RBV was mainly removed by the biodegradation of AGS,while the biodsorption removal ratio was relatively low.There are three possible transformation pathways of RBV in AGS.Compared with ordinary flocculent sludge,the degradation pathways of RBV in AGS were diverse,which may be attributed to the more complex microbial community structure in AGS.In AGS,tightly bound protein is the main component of EPS,accounting for more than 90%.The content of EPS,hydrophobic protein and hydrophobic polysaccharide increased in the early stage of RBV stress system,which may be beneficial to form a network structure and protect microorganisms.Meanwhile,it may be beneficial to improve the efficiency of electron transfer between functional microorganisms and enhance the biodegradation efficiency of RBV.In addition,the two systems exhibited different coping mechanisms to the filamentous bulking,which were mainly manifested in the increase of the contents and proportions of different amino acids.(3)The main components of EPS are tyrosine-type protein,tryptophan-type protein,humic acid-type organic matter and soluble microbial metabolites.RBV stress caused AGS to exhibit a stress protection mechanism of increased tryptophan-like proteins,and at the same time increased the proportion of structures unfavorable for aggregation in AGS.Consequently,AGS maintained a small particle size range under this condition.As a medium for electron transfer,the concentration of cytochrome under RBV stress was correspondingly higher than that of AGS in normal culture during the same period.It may be attributed that the stress condition prompts AGS to secrete more cytochrome during the granulation process to resist external stress and degrade pollutants.C4-HSL,C6-HSL,3OC6-HSL and C8-HSL were the most important Acyl-homoserine lactones in the formation of AGS.Under RBV stress,C8-HSL was more responsible for instructing the synthesis of polysaccharides,interfering or inhibiting the pathway of tightly bound protein synthesis.C4-HSL was negatively correlated with soluble metabolic products polysaccharide,loosely bound polysaccharide and loosely bound protein,indicating that RBV stress led to changes in the metabolic pathways of sludge microorganisms.Microbial community analysis showed that RBV stress promoted the growth of Acidovorax(signal molecule-synthesizing bacteria),Acinetobacter(pharmaceutical-resistant bacteria)and Delftia(biotoxic bacteria).Meanwhile,RBV stress changed the marker species in AGS,increasing the complexity of the system.RBV stress led to an increase in the abundance of sphingolipid metabolism,sphingolipid signaling and polysaccharide degradation pathways in the stress group.At the same time,it increased the modular center point Pseudoxanthomonas in the molecular ecological network of the stress group,which was conducive to pharmaceutical degradation.Therefore,RBV stress did not change the organization of major metabolic pathways and molecular ecological networks in AGS,but only changed the abundance of some non-major metabolic pathways and strengthened the role of microorganisms with pharmaceutical degradation as key species in the network.Proteomics further showed that RBV stress affected the proteins in various pathways of microorganisms and changed the microbial activity pathways of microorganisms.In this context,the stress enhanced the expression of related proteins in the carbon metabolism pathway and enhanced the degradation of RBV,thereby maintaining the stability of the system.