Chemoautotrophic growth of ammonia-oxidizing Thaumarchaeota enriched from a pelagic redox gradient in the Baltic Sea

Abstract

Ammonia-oxidizing archaea (AOA) are an important component of the planktonic community in aquatic habitats, linking nitrogen and carbon cycles through nitrification and carbon fixation. Therefore, measurements of these processes in culture-based experiments can provide insights into their contributions to energy conservation and biomass production by specific AOA. In this study, by enriching AOA from a brackish, oxygen-depleted water-column in the Landsort Deep, central Baltic Sea, we were able to investigate ammonium oxidation, chemoautotrophy, and growth in seawater batch experiments. The highly enriched culture consisted of up to 97% archaea, with maximal archaeal numbers of 2.9 × 10(7) cells mL(-1). Phylogenetic analysis of the 16S rRNA and ammonia monooxygenase subunit A (amoA) gene sequences revealed an affiliation with assemblages from low-salinity and freshwater habitats, with Candidatus Nitrosoarchaeum limnia as the closest relative. Growth correlated significantly with nitrite production, ammonium consumption, and CO2 fixation, which occurred at a ratio of 10 atoms N oxidized per 1 atom C fixed. According to the carbon balance, AOA biomass production can be entirely explained by chemoautotrophy. The cellular carbon content was estimated to be 9 fg C per cell. Single-cell-based (13)C and (15)N labeling experiments and analysis by nano-scale secondary ion mass spectrometry provided further evidence that cellular carbon was derived from bicarbonate and that ammonium was taken up by the cells. Our study therefore revealed that growth by an AOA belonging to the genus Nitrosoarchaeum can be sustained largely by chemoautotrophy.

Keywords: Baltic Sea; CO2-fixation; Thaumarchaeota; ammonia-oxidizing archaea; chemoautotrophy; enrichment.

Figure 1

Epifluorescence microscopy of representative cells from enrichment culture B on day 1. The cells were stained with DAPI (A) or hybridized with the archaea-specific probe Arc915 (B) and then analyzed by CARD-FISH. Same field of view; the scale bar represents 10 μm.

Figure 2

Cell numbers, ammonium and nitrite concentrations (A) as well as CO₂ fixation rates, and the archaeal fraction (B) in triplicate enrichment cultures (termed A, B and C). Error bars show the standard deviations of cell counts and nitrite and ammonium concentrations from cultures grown in triplicate.

Figure 3

(A) Growth curve of enrichment culture G, showing ammonium consumption, total cell numbers, and the fraction of archaeal cells. ¹⁵N-labeled ammonium was added on day 40. The combined ¹⁴NH⁺₄ and ¹⁵NH⁺₄ concentration is plotted together with the calculated concentration of ¹⁴NH⁺₄ based on the percentage of labeling. Ratios of ¹³C vs. ¹²C (B) and ¹⁵N vs. ¹⁴N (C) enrichment in single cells was determined using NanoSIMS. Error bars show the standard deviation among triplicate samples. The data point of day 0 corresponds to unlabeled cells from enrichment E.

Figure 4

Cell-specific CO₂ fixation rates calculated from archaeal cell numbers and bulk CO₂ fixation rates during the growth of enrichment cultures A, B, and C. The exponential phase is shown in gray.

Figure 5

Neighbor-joining trees showing the phylogenetic placement of the enrichment based on the cloned, nearly full-length 16S rRNA gene (A) and amoA gene (B) sequences. The scale bar represents 10 iterations per 100 nucleotides (A) or amino acids (B).