Stable carbon isotopic Fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria

TL;DR: Isotopic analyses of Candidatus “Brocadia anammoxidans,” a chemolithoautotrophic bacterium that anaerobically oxidizes ammonium (anammox), show that it strongly fractionates against 13C; i.e., lipids are depleted by up to 47‰ versus CO2.

Abstract: Anaerobic ammonium oxidation (anammox) is the oxidation of ammonium with nitrite as the electron acceptor and dinitrogen gas as the product (for reviews, see references 7 and 8). The process is mediated by obligately anaerobic chemolithoautotrophic bacteria that form a monophyletic cluster inside the Planctomycetales, one of the major divisions of the Bacteria. So far, four species have been detected and enriched from the biomass of sewage treatment plants: Candidatus “Brocadia anammoxidans” (18), Candidatus “Kuenenia stuttgartiensis” (13), Candidatus “Scalindua wagneri,” and Candidatus “Scalindua brodae” (14). Candidatus “Scalindua sorokinii” was detected in the anoxic water column of the Black Sea (9), providing the first direct evidence for anammox bacteria in the natural environment. Anammox bacteria have a cell compartment known as the anammoxosome, which is the site of anammox catabolism. The lipid bilayer membrane surrounding this anammoxosome contains unusual lipids, so-called “ladderane” lipids, concatenated cyclobutane moieties that are either ether and/or ester linked to the glycerol backbone or occur as free alcohols (e.g., Fig. ​Fig.1,1, structures II to IV) (16). The other membranes of anammox bacteria contain lipids typical for planctomycetes in general: iso, normal, and mid-chain methyl hexadecanoic acids (e.g., Fig. ​Fig.1,1, structure I). FIG. 1. Structure of anammox lipids, i.e., branched fatty acids (I), ladderane fatty acids (II), ladderane glycol ether (III), ladderane glycerol ether (IV), and hop-17(21)-ene (V), present in the enrichment culture of Candidatus “Brocadia anammoxidans.” ... Anammox bacteria have been shown to be chemoautotrophic organisms (17), but it is still unclear which carbon fixation pathway they use. There are currently four known pathways for CO2 fixation in microorganisms (see, e.g., references 2 and 3). The Calvin cycle, with ribulose bisphosphate carboxylase as a key enzyme, is operative in many organisms. The 3-hydroxypropionate pathway has been observed in Chloroflexus aurantiacus and some archaea. The reverse citric acid cycle, with citrate lyase as a key enzyme, has been found in some sulfate-reducing bacteria and phototrophic bacteria. Finally, the acetyl coenzyme A (acetyl-CoA) pathway, with carbon monoxide dehydrogenase/acetyl-CoA synthase as the indicative enzyme, is detected in many anaerobic microorganisms. In addition to enzyme activities, stable carbon isotopic compositions of total cell material and individual lipids, carbohydrates, and amino acids are often used to infer these biosynthetic pathways in organisms, as the fractionation from the inorganic carbon source to the autotrophic biomass in 13C depends on the biosynthetic pathway used (1, 19, 20, 21). Here, we determined enzyme activities and studied the stable carbon isotopic fractionations of Candidatus “Brocadia anammoxidans” to investigate its carbon fixation pathway. Since this bacterium can be grown only in enrichment cultures, and bulk cell material is thus not solely derived from anammox bacteria, we also determined the isotopic compositions of the specific lipids of this bacterium. Furthermore, the isotopic compositions of ladderane lipids derived from Candidatus “Scalindua sorokinii” growing in the anoxic water column of the Black Sea (9) were determined in order to examine the 13C fractionation patterns of anammox bacteria under natural conditions.