Differential responses of nitrifying archaea and bacteria to methylene blue toxicity

Abstract

Methylene blue, a heterocyclic aromatic chemical compound used to treat fish diseases in the ornamental fish aquaculture industry, is believed to impair nitrification as a side effect. However, very little is known about the toxicity of methylene blue to nitrifying micro-organisms. Here, we report the susceptibility of six bacterial and one archaeal ammonia-oxidizing micro-organisms to methylene blue within the range of 10 ppb to 10 ppm. Remarkably high susceptibility was observed in the archaeal species Nitrosopumilus maritimus compared to the bacterial species. Ammonia oxidation by Nitrosopumilus maritimus was inhibited 65% by 10 ppb of methylene blue. Of the bacterial species examined, Nitrosococcus oceani was the most resistant to methylene blue toxicity. For similar inhibition of Nitrosococcus oceani (75% inhibition), one thousand times more methylene blue (10 ppm) was needed. The examination of single cell viability on Nitrosomonas marina demonstrated that methylene blue is lethal to the cells rather than reducing their single cell ammonia oxidation activity. The level of susceptibility to methylene blue was related to the cell volume, intracytoplasmic membrane arrangement and the evolutionary lineage of nitrifying micro-organisms. Our findings are relevant for effectively using methylene blue in various aquaculture settings by helping minimize its impact on nitrifiers during the treatment of fish diseases. In the future, resistant nitrifiers such as Nitrosococcus oceani may be purposely added to aquaculture systems to maintain nitrification activity during treatments with methylene blue.

Significance and impact of the study: The susceptibility of six bacterial and one archaeal nitrifying micro-organisms to methylene blue was tested. Remarkably high susceptibility was observed in the archaeal species compared to the bacterial species. The level of resistance to methylene blue was related to the cell volume, cytomembrane system and the taxonomic position of the nitrifying micro-organisms. This may be significant in the design and management of engineered nitrification systems and the stability of the nitrification process in various ecosystems if these systems are exposed to harmful chemicals or toxins.

Keywords: ammonia-oxidizing archaea; ammonia-oxidizing bacteria; aquaculture; aquarium; methylene blue; nitrification; ornamental fish.