Rapid enrichment of Ca. Nitrospira inopinata using anammox and kanamycin: a path toward sustainable nitrification

Abstract

The identification of complete ammonia oxidizers (comammox) within the nitrite-oxidizing bacteria (NOB) genus Nitrospira, capable of oxidizing ammonia directly to nitrite and nitrate, represents a pivotal advancement in elucidating the microbial and metabolic pathways underlying nitrification in the global nitrogen cycle. Although comammox Nitrospira have been consistently identified across diverse environmental habitats, their rapid enrichment from complex activated sludge systems remains challenging owing to their intrinsically low growth rates and the restricted availability of cultured isolates. In this study, Candidatus Nitrospira inopinata (Ca. N. inopinata) was successfully enriched from anammox-inoculated sludge within 70 d under low ammonia concentrations (~4.88 mg/L), ambient temperatures (21.6–28.4 °C), and minimal aeration (0–0.01 mg/L), facilitated by the application of kanamycin (KAN). By employing 16S rRNA gene amplicon sequencing and quantitative polymerase chain reaction (qPCR) targeting the functional marker gene amoA, Ca. N. inopinata was identified as the dominant ammonia oxidizer, achieving a relative abundance of 95.22% in the nitrifying community. Kanamycin was shown to exert significant selective pressure, further enhancing the enrichment of Ca. N. inopinata. These findings demonstrate the feasibility of establishing comammox Nitrospira through direct inoculation of anammox pellets coupled with kanamycin, offering a robust and efficient strategy for rapid enrichment of comammox Nitrospira for biotechnological applications, while underscoring the energy-efficient advantages of comammox-driven ammonia oxidation processes.

Graphical abstract

Keywords

Comammox Nitrospira / Kanamycin / anammox / Enrichment

Highlight

	● Ca. N. inopinata was rapidly enriched from anammox granules within 70 d.
	● Antibiotic kanamycin addition further enhanced Ca. N. inopinata enrichment.
	● Ca. N. inopinata dominated ammonia oxidation at 95.22% of the nitrifying community.
	● This study presents a practical strategy for enriching comammox Nitrospira .

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