Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies

Abstract

Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member’s ecophysiology in a variety of habitats.

Enrichment and Physiological Characterization of a Cold-Adapted Nitrite-Oxidizing Nitrotoga sp. from an Eelgrass Sediment

“…The cell size and tendency to aggregation vary between species. Although other AOB and NOB isolated from activated sludge by a cell sorting system form single-species microcolonies (32)(33)(34), such microcolonies of Nitrotoga were not found in this study. The enriched Nitrotoga cells were planktonic or aggregated with heterotrophs (Fig.…”

Functional Characterization and Diversity of Ammonia-Oxidizing Microorganisms in Streams South of the Dabie Mountains, China

“…Out of the AOB genes (Supplementary Figure S1), 61 sequences were collected, and these sequences were clustered into three OTUs. With a 99% similarity for each OTU, as previously predicted [42], Nitrosomonas cluster is usually found in river ecosystems [43]. Our results also confirmed this phenomenon; 99.7% of the total AOB sequences were obtained from S7 and S9 downstream.…”

Ecophysiology and Comparative Genomics of Nitrosomonas mobilis Ms1 Isolated from Autotrophic Nitrifying Granules of Wastewater Treatment Bioreactor

“…The genome included 3075 protein-coding DNA sequences (CDSs), 41 tRNA genes, and a single copy of the 16S-23S-5S rRNA operon. Based on phylogenetic analyses of N. mobilis Ms1 with other AOB (Fujitani et al, 2015), Ms1 showed the greatest similarity to Nitrosomonas sp. strains Nm107 and Nm104 (99.7–100%) at the 16S rRNA and amoA genes levels and 99.6% similarity to Nitrosococcus mobilis Nc2 at the 16S rRNA gene level, therefore Ms1 was attributed to the N. mobilis lineage.…”

“…Nitrosomonas mobilis Ms1 isolated from nitrifying granules (Fujitani et al, 2015) was kept in liquid culture of mineral medium containing 30 mg-N L -1 of ammonium substrate (NH 4 Cl) with the following components per liter of distilled water: NaCl (0.116 g), MgSO 4 ⋅7H 2 O (0.4 g), CaCl 2 ⋅2H 2 O (0.073 g), KCl (0.038 g), KH 2 PO 4 (0.034 g), FeCl 2 (0.002 g), EDTA (0.0043 g), MnCl 2 ⋅4H 2 O (0.1 mg), CoCl 2 ⋅6H 2 O (0.024 mg), NiCl 2 ⋅6H 2 O (0.024 mg), CuCl 2 ⋅2H 2 O (0.017 mg), ZnCl 2 (0.068 mg), Na 2 WO 4 ⋅2H 2 O (0.033 mg), Na 2 MoO 4 (0.024 mg), and H 3 BO 3 (0.062 mg). The pH of the culture media was adjusted to 8.0 by NaHCO 3 (0.84 g L -1 ), and phenol red (0.4 w/v %) was used as the pH indicator.…”

“…Recently, the pure culture of Nitrosomonas mobilis Ms1 has been successfully isolated from the autotrophic nitrifying granules by selective isolation using a cell-sorting system (Fujitani et al, 2015). These nitrifying granules were produced in the aerobic upflow fluidized bed reactor to treat inorganic wastewater and exhibited an effective ammonia removal rate (Tsuneda et al, 2003).…”