Dynamics and activity measurement of nitrogen converting bacteria underlying selected bioprocesses treating animal and municipal wastewater

With a view of producing renewable energy on one side and reducing emissions of greenhouse gases and problematic nitrogenous effluents from animal wastes in Brittany on the other side, efforts were pursued in CEMAGREF-Rennes to employ animal wastes in biogas production with subsequent biological nitrogen removal (BNR). A major challenge was to develop an operationally cost-effective bioprocess that selects nitrifying consortia performing nitritationdenitritation instead of the conventional nitrification-denitrification with savings in the aeration and organic substrate requirement for the N-removal treatment. The undertaking consisted of process engineering, modelling and kinetics aspects and process microbiology. This dissertation focuses on the microbiology of the aerobic process and is divided into several parts. First, the possible sources of bias in the molecular tools used to characterize microbial communities (mainly 16S rRNA end-point polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)) in representative environmental matrices including compost, manure and soil were investigated. Measures to circumvent end-point amplification bias were then discussed. Second, the evolution of nitrifying bacteria during the start-up of stand-alone pilot-scale BNR reactor and anaerobic digestion (AD) coupled BNR (AD-BNR) reactor treating high-strength swine wastewater was investigated. Ribosomal rRNA and nosZ gene PCR-SSCP/clone library analysis were used to profile the composition of ammoniaoxidizing bacteria (AOB)/nitrite-oxidizing bacteria (NOB) and denitrifiers communities, respectively. Results suggested that these two process configurations significantly differ in the modes of biological Nremoval and the selection of distinct taxa. Third, the profiles of nitrifying bacteria during the long-term N-removal treatment of high-strength swine wastewater among the three bioprocess configurations(i) BNR, (ii) ADBNRBP with influent bypass, and (iii) ADBNRBP+R with influent bypass and inter-reactor recirculationwere investigated either by SSCP or terminal restriction fragment length polymorphism (T-RFLP) analysis. Meanwhile, the abundance of specific gene markers for nitrification (amoA, nxrB) and denitrification (napA, narG, nirS, nirK, norB, nosZ) was determined by quantitative PCR (qPCR) on a long-term basis. Analysis of reactor performance, biomass nitrification activity determined by respirometry, and qualitative and quantitative analysis of the targeted Nconverting microbial groups suggest that achievement of nitritation-denitritation in the AD BNRBP+R configuration was accompanied by the dominance of ammonia-oxidizers,suppression of nitrite-oxidizers, and fluctuating composition of heterotrophic denitrifiers. Lastly, an approach that elucidates on patterns the association between the dominant/subdominant N-converting taxa and the prevailing N-removal pathway for a given bioprocess and wastewater type was developed. For that, the specific nitrification and denitrification activities in sludge from two municipal wastewater treatment plants and two pilot-scale swine wastewater bioreactors were determined. These were correlated to the composition of AOB, NOB and denitrifiers and also the relative abundance of nitrification (amoA, nxrB) and denitrification (napA, narG, nirS, nirK, norB, nosZ) gene markers as determined by qPCR.