Microbial nitrogen transformation and metabolic versatilities of ammonia oxidizers in full-scale wastewater treatment systems

Abstract

Microbial community composition and functions are critically important to the operational efficiency of wastewater treatment plants (WWTPs). The overall microbial communities of four full-scale WWTPs treating landfill leachate were analyzed using 16S rRNA gene high-throughput sequencing technology and the active microbial groups were identified by reverse-transcribed 16S rRNA sequencing. Transcriptionally active organisms tended to be more abundant compared to the inactive organisms in activated sludge. Microbial migration from influent wastewater likely has considerable effect on the microbial communities and abundances in activated sludge, in which some heterotrophic microbes predominated the microbial community despite their low activities. Biomass immobilized onto carrier materials could significantly promote the accumulation of the slow-growing anaerobic ammonium oxidation (anammox) bacteria in WWTPs. Using the initially enriched anammox bacterial sludge as an inoculum for the nitrogen removal in WWTPs is a practical means for the application of the slow-growing anammox bacteria. The complex microbial driven nitrogen cycling network in three full-scale anammox-inoculated conventional WWTPs were analyzed by the combined metagenomic and metatranscriptomic approaches, showing the partial-nitrification anammox (PNA), simultaneous nitrification, anammox and denitrification (SNAD), and nitrification-denitrification for nitrogen removal processes from the WWTPs. In the PNA system, ammonia-oxidizing bacterium (AOB) Nitrosomonas and anammox bacteria Ca. Brocadia and Ca. Kuenenia were the active contributors for nitrogen removal. In the SNAD system, ammonia-oxidizing archaea (AOA) Thaumarchaeota unexpectedly predominated the nitrifying community. Unexpectedly, the complete ammonia oxidizer (comammox) Nitrospira species was the dominant one even in the presence of AOB Nitrosomonas in the non-aeration and organic carbon-rich nitrification-denitrification tanks of the WWTP. Four comammox metagenome-assembled genomes were retrieved from two of the above three metagenomic datasets of the WWTPs. Active genes for the utilization of urea, amines, and cyanate suggest that comammox bacteria can use diverse organic nitrogen compounds in addition to free ammonia as substrates for complete nitrification. They also possessed multiple alternative energy metabolisms including respiration with hydrogen, formate, and sulfite as electron donors. One of the comammox metagenomes encoded and transcribed a pathway for homoacetate fermentation linked to the release of H2 by a putative membrane-bound type 4f [NiFe] hydrogenase, which was highly unexpected in a nitrifying bacterium. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely assist comammox bacteria survive unfavorable conditions and facilitate switches between different lifestyles in fluctuating environments. Meanwhile, three Group 1.1b AOA metagenome-assembled genomes were retrieved from one WWTP and one of the three AOA metagenomes predominated the nitrifying community. Genes for the utilization of urea and amines indicate that AOA use diverse organic compounds in addition to free ammonia as substrates, but these genes were not found in the dominant novel metagenomes. Instead, it appears to acquire substrate competitive advantage by highly expressing ammonium transporter and probably oxidizing ionized ammonium for chemolithoautotrophic growth. Two of the AOA metagenomes encoded a type 3b [NiFe] hydrogenase and a putative membrane-bound type 4f [NiFe] hydrogenase, reflecting the unexpected flexible hydrogen metabolism in wastewater AOA. Pathways for the biosynthesis of polyphosphate and polyhydroxyalkanoates as intracellular storage compounds likely contribute to AOA survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. The encoded amino acids, peptides, and dicarboxylates transporters and metabolic pathways provide them the capability to use exogenous organic carbon for mixotrophic growth.