Quantification of Nitrosomonas oligotropha -Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Abstract

Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotrophalike ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ؎ 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ؎ 0.28% of the biosludge population in the municipal WWTP and 0.37% ؎ 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.Nitrification, the bio-oxidation of ammonia (NH 3 ) to nitrate (NO 3 Ϫ ), is a key process in the removal of ammonia from wastewater, which alleviates problems of aquatic toxicity, high oxygen demand in receiving waters, and nutrient contributions to eutrophication (3, 13). The number and physiological activity of nitrifying bacteria in wastewater treatment reactors are considered the rate-limiting parameters for the bioconversion of nitrogen in sewage (48). The slow growth rate of nitrifying bacteria and the sensitivity of these organisms to several environmental factors (e.g., pH, O 2 , temperature, substrate concentration, and the presence of toxic substances) influence the minimum sludge age during wastewater treatment required to establish stable nitrification (34). Consequently, early detection of a decline in the nitrifying population by rapid and reliable molecular methods may improve process control and prevent washout of these organisms from a wastewater treatment system.Chemolithotrophic nitrification is a two-step biochemical process involving sequential transformation of NH 3 to NO 3 Ϫ via NO 2 Ϫ (6). In wastewater treatment plants (WWTPs), the reactions are catalyzed by two phylogenetically distinct groups of bacteria, the ammonia-oxidizing bacteria (AOB) belonging to the ␤ subclass of the class Proteobacteria and nitrite-oxidizing bacteria (NOB). In AOB, oxidatio...

A robust nitrifying community in a bioreactor at 50 °C opens up the path for thermophilic nitrogen removal

“…The SYBR Green assay (Power SyBr Green, Applied Biosystems, Carlsbad, CA, USA) was used to quantify the 16S rRNA of Nitrospira spp. (Dionisi et al, 2002) and the functional archaeal amoA gene (Tourna et al, 2008).…”

Comparison of Partial and Full Nitrification Processes Applied for Treating High-Strength Nitrogen Wastewaters: Microbial Ecology through Nitrous Oxide Production

“…to preferentially grow at lower nitrite concentrations. [32][33][34] However, the sustained lack of detection of Nitrospira spp. under low nitrite concentrations during full-nitrification (0.75 ( 0.69 mg-N/L, n = 15) relative to partial nitrification (360 ( 42 mg-N/L, n = 71) was unexpected and again possibly because of competition with a well-established culture of Nitrobacter spp.…”

Nitrogen removal and ammonia-oxidising bacteria in a vertical flow constructed wetland treating inorganic wastewater

“…Nitrosomonas nitrosa like sequences have often been isolated from systems with high ammonia concentration such as industrial wastewater treatment plants (Dionisi et al, 2002) and at the same time from low ammonia environments such as freshwater estuaries (Cebron et al, 2003) and a submerged biofilm for drinking water pretreatment in China (Qin et al, 2007). Nitrosomonas nitrosa-like sequences were abundant in the wetland cell 1 which receives highly variable NH 3 -N concentrations, corroborating the claims of ecological versatility for this group.…”