Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

Abstract

The phylogenetic affiliation and physiological characteristics (e.g., K_s and maximum specific growth rate [μ_max]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

TEXT

Anaerobic ammonium oxidation (anammox) is a microbial process in which ammonium is oxidized to nitrogen gas with nitrite as the electron acceptor under anoxic conditions (1–3). At least five candidate genera have been tentatively proposed in this taxon (4). The “Candidatus Scalindua” group is primarily found in marine environments (5–10). Previous studies indicate that “Candidatus Scalindua” contains taxonomically diverse members, while only a few members of the “Candidatus Scalindua” group have been successfully grown in enrichment cultures so far (11–15). Physiological characteristics of anammox bacteria affiliated to “Candidatus Scalindua” have been demonstrated only partially (11, 12, 16, 17) compared with the freshwater anammox bacteria (18–23). In this study, the phylogenetic affiliation and physiological characteristics of an anammox bacterium previously enriched from marine sediments of Hiroshima Bay, Japan, were determined. Anaerobic batch experiments were performed to determine the following physiological parameters: (i) growth temperature, pH, and salinity ranges, (ii) inhibition by ammonium and nitrite, (iii) half-saturation constants (K_s) for nitrite and ammonium, (iv) accumulation and consumption of hydrazine after the addition of hydroxylamine, and (v) biomass yield. The maximum specific growth rate (μ_max) and the ultrastructure of the anammox bacterium were also determined.

Biomass samples were obtained from a 7-liter membrane bioreactor (MBR) inoculated with anammox biofilms (13, 14) to obtain free-living anammox bacterial cells related to “Candidatus Scalindua.” Cells in the MBR were collected for fluorescence in situ hybridization (FISH) and phylogenetic analyses, transmission electron microscopy (TEM), and anaerobic batch experiments. The detailed procedures are described in the supplemental material.

A single species of “Candidatus Scalindua sp.” was successfully enriched in free-living cells (see Fig. S1 in the supplemental material) using the MBR in this study, where the anammox bacteria that hybridized with the Sca1129b probe accounted for 88.8% ± 3.1% of all the bacteria. The partial sequences of the 16S rRNA gene from 83 clones were grouped into a single operational taxonomic unit (OTU) based on 97% sequence identity. Four nearly full-length 16S rRNA gene sequences from the OTU (99.7% identity among the four sequences) shared 96.9% to 97.0% identity with the sequence of “Candidatus Scalindua wagneri” (Fig. 1; see also Table S2 in the supplemental material). Such low sequence similarity suggests that the members of “Candidatus Scalindua sp.” are affiliated with a different anammox species in the “Candidatus Scalindua” lineage. The cellular structure of “Candidatus Scalindua sp.” has shown three separate compartments that include electron-dense particles and no pilus-like appendages (Fig. 2), as reported by van Niftrik et al. (24, 25).

Fig 1.

Maximum-likelihood tree based on 16S rRNA gene sequences. The GenBank/EMBL/DDBJ accession numbers are indicated. The scale bar represents the number of nucleotide changes per sequence position. Pie charts at the nodes represent the confidence of the branch topology results, and bootstrap values (1,000 resamplings) greater than 85% are filled in black (the neighbor-joining method, NJ, for the upper-left sector, the maximum-likelihood method, ML, for the upper-right sector, or the maximum-parsimony method, MP, for the bottom sector). The designation of “Ca. Scalindua” is indicated on the right. The Planctomyces brasiliensis sequence (CP002546) served as an outgroup to root the tree. ID, identification; IMG/M, Integrated Microbial Genomes with Microbiome Samples.

Fig 2.

Transmission electron micrographs of “Candidatus Scalindua sp.” enriched in the MBR, showing the cells which were about to be divided. All cells in panels A and B were divided into three separate compartments by individual membranes: the paryphoplasm, the riboplasm, and the anammoxosome compartments. In panel A, all cells were occupied by the voluminous anammoxosome. In panel B, the white arrows indicate condensed and electron-dense particles. Scale bars, 500 nm.

Table 1 summarizes the physiological characteristics of “Candidatus Scalindua sp.” and other anammox bacteria (10, 12, 18–23, 26). The optimal temperature and pH ranges of “Candidatus Scalindua sp.” (10 to 30°C and pH 6.0 to 8.5, respectively) (Fig. 3A and B) were lower than those of other anammox species (i.e., 15 to 45°C and pH 6.5 to 9.0, respectively) (10, 12, 19, 23, 26). Anammox activities of “Candidatus Scalindua sp.” were observed under conditions of 0.8% to 4.0% salinity, whereas no activity was detected in the absence of salinity. This outcome indicates that “Candidatus Scalindua sp.” is a halophilic bacterium. “Candidatus Scalindua sp.” accumulated hydrazine after the spike addition of hydroxylamine (see Fig. S2 in the supplemental material), which is a phenomenon commonly observed in the known anammox bacteria (12, 18, 21, 23).

Table 1.

Physiological characteristics of “Candidatus Scalindua sp.” and four anammox bacteria identified elsewhere: “Candidatus Brocadia sinica,” “Candidatus Brocadia anammoxidans,” “Candidatus Kuenenia stuttgartiensis,” and “Candidatus Scalindua profunda”

Parameter	Value(s) for:
	“Ca. Scalindua sp.”	“Ca. Brocadia sinica”	“Ca. Brocadia anammoxidans”	“Ca. Kuenenia stuttgartiensis”	“Ca. Scalindua profunda”
Growth temp (°C)	10–30	25–45	20–43	25–37	15–45
Growth pH	6.0–8.5	7.0–8.8	6.7–8.3	6.5–9.0	7.4
Growth salinity (%) or level of salinity (mmol)	1.5–4.0	<3	Not determined	200 mmol (chloride)	3.3
Biomass yield (mmol C [mmol N]−1)	0.030	0.063	0.07	Not determined	Not determined
Ks for ammonium (μM)	3	28 ± 4	<5	Not determined	Not determined
Ks for nitrite (μM)	0.45	86 ± 4	<5	0.2–3	Not determined
Activation energy (kJ mol−1)	81.4 ± 3	56 ± 3	70	Not determined	Not determined
Protein content of biomass (g protein [g VSS]−1)	0.64	0.61	0.6	Not determined	Not determined
μmax (h−1)	0.0020	0.0041	0.0027	0.0026–0.0035	Not determined
Tolerance
Nitrite (mM)	7.5	<16	7	13, 25	Not determined
Ammonium (mM)	>16	Not determined	Not determined	Not determined	Not determined

Fig 3.

The results of batch experiments. (A to C) Influence of temperature, pH, and salinity on anammox activity determined by the ²⁹N₂ gas production rate. (D) Incorporation of [¹⁴C]bicarbonate and ammonium consumption by anammox bacteria. Incorporation of [¹⁴C]bicarbonate (vertical axis) increased with increases in the ammonium consumption rate (horizontal axis). Filled circles represent the value of “Candidatus Scalindua sp.” at 28°C, whereas an open circle represents the value at 37°C. Filled and open squares represent the values of “Candidatus Brocadia sinica” at 28 and 37°C, respectively. Biomass yields, 0.030 and 0.063 mol C (mol NH₄⁺)⁻¹, were obtained by dividing the amount of incorporated [¹⁴C]bicarbonate by the amounts of consumed ammonium obtained under different temperature conditions. All values are means ± standard deviations of the results of independent triplicate experiments.

The K_s values for nitrite and ammonium of “Candidatus Scalindua sp.” (Table 1; see also Fig. S3 in the supplemental material) were lower than those of other anammox bacteria, suggesting that the high affinity for nitrite is necessary for the bacteria survive in marine environments with extremely low levels of nitrite concentrations. Indeed, the ammonium, nitrite, and nitrate concentrations at the sediment sampling point (the sediment was used as the inoculum in this study) were 17.8, 2.1, and 5.7 μM, respectively (15), which suggests no occurrence of the substrate limitation for the growth of “Candidatus Scalindua sp.” in such marine environments.

Interestingly, the biomass yield of “Candidatus Scalindua sp.” was half the reported value (19, 23). The biomass yield of “Candidatus Scalindua sp.” was determined to be 0.030 mol C (mol NH₄⁺)⁻¹ at 28°C and 37°C (Fig. 3D). The biomass yield of “Candidatus Brocadia sinica” was also not dependent on temperature (0.063 mol C [mol NH₄⁺]⁻¹ at 28 and 37°C) (Fig. 3D). The reason for the low biomass yield is not clear at present. The maximum volumetric ammonium oxidation rate (q_max; 4.02 g N liter⁻¹ day⁻¹) was obtained in an up-flow column reactor after 50 days of operation (see Fig. S4 in the supplemental material). The biomass concentration at that point was 4.8 g volatile suspended solids (VSS) liter⁻¹ (corresponding to 3.07 g protein liter⁻¹). Based on the biomass yield, q_max, and biomass concentration, the μ_max was calculated to be 0.0020 h⁻¹ (doubling time = 14.4 days). The μ_max of “Candidatus Scalindua sp.” (0.0020 h⁻¹) was significantly lower than those of other anammox bacteria (0.0027 to 0.0041 h⁻¹) (18, 22, 23). The low μ_max of “Candidatus Scalindua sp.” was derived from the low biomass yield, as the q_max of “Candidatus Scalindua sp.” (65 μmol NH₄⁺ [g protein]⁻¹ min⁻¹) was comparable to those of other anammox bacteria (3, 23, 26).

In conclusion, the fundamental physiological characteristics of “Candidatus Scalindua sp.” enriched from the sediment of Hiroshima Bay, Japan, were investigated. This microorganism is a halophilic bacterium and exhibits higher affinity for nitrite and a lower growth rate than the known anammox species. “Candidatus Scalindua sp.” could maintain its anammox activity under low-temperature conditions. These physiological characteristics support the idea of the predominance of “Candidatus Scalindua sp.” in marine sediments. The findings contribute to our understanding of the niche adaptation of “Candidatus Scalindua sp.”

Nucleotide sequence accession numbers.

The 16S-23S rRNA gene sequence data were deposited in the GenBank/EMBL/DDBJ databases under accession numbers AB811945 to AB811948.