Microbial Molecular Analysis On The Biological Nitrogen Removal Of Landfill Leachate By Aged-refuse Bioreactor

Municipal domestic refuse generates a lot of leachate in the sanitary landfill, landfill leachate is one kind of complex mixture of wastewater, its biodegradable subtances reduced and the concentration of ammonia nitrogen (NH4+-N) increased with the landfilled time, which lead to a low C/N and scare cabon source. The traditional treatment technologies face more serve challenges when treating such mature landfill leachate.Aged-refuse is formed in the process years of filling up and stabilization after the closure of the landfill. It has loose physical structure, strong adsorption ability, rich quantity of degrading microbial. Aged-refuse bioreactor is set up with stabilized aged-refuse of more than eight to ten years as filter. It is a novel technology for treatment of landfill leachate with the use of biodegradation and adsorption. Aged-refuse technology has a character of low cast and stable high pollutant removal rate.This paper focused on the biological nitrogen removal procee of landfill leachate in aged-refuse bioreactor. Molecular technology of the quantitative real-time polymerase chain reaction (q-PCR),454high throughpurt sequencing and gene cloning library were used to analyse the microbial community structure, especially the distribution and quantity of the ammonia oxidizing bacteria (AOB), anaerobic ammonia oxidation (Anammox) bacteria. Combining with the operational condition and pollution removal rate of the bioreactor, the relationship of the pollutant remove and the microbial community construte and the probable pathway of nitrogen remove were studied.Conclusions in this paper were drawn as follows:(1) The results of PCR and q-PCR showed that there were both AOB and Anammox bacteria in the aged-refuse nitrification-denitrification (ND) reactor and Anammox reactor. Quantitive analysis results indicated that the number of AOB in the ND reactor was about107-108cells per gram filter, with a average quantity of8.58×107cells per gram filter (cells/g), which is0.14%in the total bactiera. With the increase of hydraulic loading rate, the ammonia removal rate increased and then decreased, but the number of AOB kept at a high level and gradually increased. The number of Anammox bacteria in the reactor was about107-108cells/g, with a average quantity of1.52X108cells/g,0.36%of the total bacteria. When the reactor had the highest removal rate of NH4+-N and total nitrogen (TN), the number of Anammox also reached the peak level.(2) The number of AOB in the Anammox reactor was at level of107-109cells per gram, with average quantity as8.50×107cells/g, and held a0.46%in total bacteria. The quantity of Anammox bacteria in the reactor was at107-109cells/g, and the average number was1.33×108cells/g, which held0.76%of total bacteria. The number of AOB and Anamox bacteria in the upper and middle part of the reactor were both higher than that at the bottom, which maybe result from the high concentration of pollutants from the upper influent. This indicated that Anammox was one of the probable pathways of nitrogen removal in the aged-refuse reactor.(3) Results of454high-throughput sequencing showed that there was high diversity of microbial in the ND reactor. The predominant phylum of bacteria was Proteobacteria, which had37.1%of the total bacteria. Several genus of organic degrading bacteria were deteected in bioreactor. There were other predominant bacteria, such as Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, etc. The predominant bacteria in Anammox reactor was also Proteobacteria with the share of39.4%, and the other predominant bacteria phylum were the same with ND reactor.(4) Multiple genuses of nitrogen removal bacteria were detected in both reactors, like AOB, NOB, denitrifiers and Anammox bacteria. They two shared the same genus composition of function bacteria. The AOB predominant bacteria genus was Nitrosomonas of Proteobacteria Phylum, while the NOB predominant bacteria genus was Nitrospira. The diversity of denitrifying bacteria were high in both reactors. Among the predominant denitrifying bacteria genus, there were a lot of denitrifyers with special metabolic pathway, including autotrofic denitrification, aerobic denitrification. This indicated various metabolic denitrification process in the aged-refuse reactors. There were also Planctomycetes bacteria in both ND and Anammox reactors. The predominant genus were Planctomyces and Pirellula. Two samples from Anammox reactor were proved to have Anammox bacteria sequence belong to Candidatus Kuenenia, which was normal genus of Anammox bacteria in artificial wastewater treatment plants.(5) Gene cloning library results proved that there were quite a bit of Planctomycetes bacteria distributed in Anammox reactor. Most of the Planctomycetes were uncultivated. Anammox bacteria were distributed widely but had simple genus composition. The only species of Anammox was Candidatus Kuenenia stuttgartiensis, which were regularly detected in artificial wastewater treatment plant. Therefore, it was conjected that the Anammox reactor was proceeding a comprehensive nitrogen removal procedure including nitrifying, autotrophic denitrifying and anammox reaction.The bacteria diversity and community structure in aged refuse reactor were studied by molecular technique. The results indicated that there were various genuses of nitrogen removal function bacteria existing in the aged-refuse reactors and they could perform metabolic pathway of Anammox nitrogen removal process. By adjusting of system operation condition, the quantity of functional bacteria could be increased based on the original quantity and help to improve the removal rate of pollutant, especially of TN.