Effects Of Glutaraldehyde And Other Antimicrobial Substances In Swine Wastewater On The Autotrophic Nitrogen Removal Process Efficiency And Microbial Community Structure

The intensive and large-scale development of pig farming has led to difficulties in returning wastewater from large or extra-large pig farms,serious separation of planting and breeding,and wastewater treatment in compliance with standards.The digested piggery wastewater is a typical low C/N ratio wastewater,and it is difficult to remove nitrogen.The traditional nitrification-denitrification process requires a large amount of carbon sources and cannot meet its demand for nitrogen removal.Therefore,the development of a new biological nitrogen removal process is imminent.The Autotrophic Nitrogen Removal（ANR）process,which with aerobic ammonia oxidizing microorganisms（AOM）and anammox bacteria as the main functional flora,requires no additional carbon source,have low sludge output,low power consumption,and high efficiency,It is one of the most promising new biological nitrogen removal processes.Affected by pig feeding methods and the African swine fever epidemic,there are a large number of potential heavy metals,antibiotics and disinfectants in the digested piggery wastewater,which may inhibit the functional flora in the ANR process.It hinders the application of this process in piggy wastewater.However,it is still unknown whether the piggery disinfectant represented by glutaraldehyde remains in the digested piggery wastewater and whether it will inhibit the nitrogen removal flora.For these many reasons,in this research,we analyzed the types and contents of potential antimicrobial factors in piggery wastewater;explored the effects of main antimicrobial substances on the main nitrogen removal functional flora in autotrophic nitrogen removal granular sludge;glutaraldehyde is a typical piggery disinfectant component,analyzed its long-term impact on Anammox bacteria,the main functional group of ANR process,and analyzed its effects from the aspects of sludge characteristics,microbial community structure and the expression level of nitrogen removal functional genes.The main research contents are as follows:（1）With simulated wastewater as a control,the efficiency of the autotrophic nitrogen removal process in treating digested piggery wastewater was tested.The results show that the digested piggery wastewater contains certain antimicrobial substances,which makes the ANR sludge smaller and the particles disintegrate;when the total nitrogen load is equivalent,the total nitrogen removal rate and total nitrogen removal efficiency of treating digested piggery wastewater are only 1.90±0.98 kg-N/m³/day and 35.24%,which is much lower than treating the simulated wastewater（4.29±0.49 kg-N/m³/day and 78.22%）.it is difficult to improve through the adjustment of process parameters such as dissolved oxygen.（2）In order to explore the existence of potential antimicrobial substances in piggery wastewater,this study collected 20 piggery wastewater in different regions or at different treatment stages,and tested its biochemical characteristics,heavy metal content,typical disinfectant glutaraldehyde and their by-products.It is found that the organic matter,Cu²⁺,and glutaraldehyde residues in the digested piggery wastewater may be the main antimicrobial factors that make the efficiency of the ANR process difficult to improve.（3）The effect of the main antimicrobial substances on the activity of the nitrogen removal microorganisms of the autotrophic nitrogen removal granular sludge was further explored through the activity experiment.The results show that organic matter mainly inhibits the activity of AOM in the ANR granular sludge.When 2160 mg/L sodium acetate is added,the AOM activity decreases by about 36%;Cu²⁺mainly affects the ANR by inhibiting the activity of Anammox bacteria.When the Cu²⁺concentration is 2.5 mg/L,the activity of Anammox is reduced by 30%;and glutaraldehyde has strong inhibitory effect on the activity of AOM,Anammox bacteria,denitrifying bacteria and other nitrogen removal microorganisms in the ANR granular sludge.When the concentration of glutaraldehyde is 50 mg/L,the activities of AOM,Anammox bacteria,and denitrifying bacteria decrease by 87%,71%,and 80%,respectively.In the experiment,it is found that glutaraldehyde can cause sludge depolymerization.The extracellular polysaccharides in the sludge continue to dissolve into the environment,leading to the disintegration of granular sludge.（4）Through 160 days of reactor operation,the long-term influence of glutaraldehyde on the Anammox flora,which is the main functional flora of the ANR process,is explored.When the concentration of glutaraldehyde is lower than 20 mg/L,the Anammox flora has a good tolerance to it,the effect of glutaraldehyde on the nitrogen removal efficiency is limited,and it had no significant effect on the particle size and morphology of the granular sludge;When its concentration increases to 40 mg/L,its inhibitory effect is significant,the sludge particle size becomes smaller,the flocculent sludge increases,the nitrogen removal efficiency deteriorates,and the anammox cell autolyzes,which turns from high enrichment to low enrichment state;the abundance of microbial diversity in the sludge has decreased overall.（5）The Anammox flora under the long-term influence of glutaraldehyde was analyzed by metagenomic sequencing and q PCR analysis,and explored the changes of microbial community structure on the anammox system causing by glutaraldehyde.The results show that after adding glutaraldehyde,the basic metabolism and material conversion of all microorganisms in the system are inhibited,and the number of microorganisms is significantly reduced;Candidatus brocadia is the main Anammox bacteria in the system,and glutaraldehyde makes it gradually falls off granular sludge.Aggregate into floc sludge,the abundance in granular sludge is significantly reduced.Further,the abundance of filamentous bacteria such as Candidatus promineofilum is reduced,which eventually leads to the inhibition of the agglomeration of anammox bacteria and the gradual depolymerization,caused performance deteriorates.In addition,although denitrifying bacteria have a certain tolerance to glutaraldehyde,their abundance in granular sludge has increased,becoming the main substitute for anammox bacteria under the stress of glutaraldehyde,however,its activity is low and its substitution effect is limited because of the autotrophic system.