Sealing solid agar in serum bottles for rapid isolation and long-term preservation of chemoautotrophic ammonia-oxidizing bacteria

• Published in: Water research (Oxford) Vol. 260; p. 121916
• Main Authors: Wu, Jiajie, Zhan, Manjun, Yuan, Lingling, Zhu, Yueyue, Lin, Weitie, Luo, Jianfei
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15.08.2024
• Subjects: Ammonia-oxidizing bacteria; Isolation; Oxygen concentration; Preservation; Sealed solid agar plate; Isolation; Oxygen concentration; Preservation; Sealed solid agar plate; Ammonia-oxidizing bacteria
• ISSN: 0043-1354; 1879-2448; 1879-2448
• DOI: 10.1016/j.watres.2024.121916

•A solid agar plate inside of a sealed bottle was developed to isolate and preserve AOB.•21 % O2 delays or suppresses AOB colony formation on solid agar plates.•5 % O2 is beneficial for AOB growth either on A solid plate or in liquid culture.•Two novel AOB species were isolated using the sealed solid agar plates.•Preservation of AOB on a solid plate could be maintained for more than ten months. Ammonia-oxidizing bacteria (AOB) are ubiquitous on the earth and have broad applications in bioremediation. However, the number of their species with standing in nomenclature and deposited in Microbial Culture Collections still remains low. Moreover, only a few novel species have been reported over the last decades. In this study, we sealed agar in serum bottles to develop a kind of solid agar plate with the oxygen concentration in the headspace maintained at low levels. By using these plates, eight AOB isolates including two novel species were obtained. When AOB cells were grown on the sealed solid agar plates, the time to form visible colonies was largely reduced and the maximum diameter of colonies reached 2 mm, which makes the process of AOB isolation rapid and efficient. Based on five AOB isolates, the headspace oxygen concentration had a significant influence on AOB growth either on solid plate or in liquid culture. Especially, when grown under 21 % O2, the number of colonies formed on solid agar plates was very low and sometimes no visible colony formed. Besides the application on AOB isolation, the sealed solid agar plate was also effective for the enumeration and preservation of AOB cells. When preserved under room temperature for more than ten months, the AOB colonies on the plate could still be recovered. This method provides a feasible way to isolate more novel AOB species from the environment and deposit more species in Microbial Culture Collections. [Display omitted]