Research Article
07 June 2004

Stable Carbon Isotopic Fractionations Associated with Inorganic Carbon Fixation by Anaerobic Ammonium-Oxidizing Bacteria

ABSTRACT

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. Similar results were obtained for the anammox bacterium Candidatus “Scalindua sorokinii,” which thrives in the anoxic water column of the Black Sea, suggesting that different anammox bacteria use identical carbon fixation pathways, which may be either the Calvin cycle or the acetyl coenzyme A pathway.
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. 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. 1, structure I).
FIG. 1.
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.

Anammox samples.

Anammox bacterial cells were grown in enrichment cultures in an anaerobic sequencing batch reactor as described previously (17). Candidatus “Brocadia anammoxidans” strain Delft was enriched from an anaerobic wastewater treatment plant in Rotterdam, The Netherlands. Particulate organic matter for lipid analyses was collected in the western basin of the Black Sea (site 7605 [42°30.71′N, 30°14.69′E] and site 7620 [42°55.56′N, 30°03.65′E]) during the R/V Meteor cruise in December 2001. Material was collected at several depths by in situ filtration of large volumes (∼1,000 liters) of water through 292-mm-diameter precombusted glass fiber filters (nominal pore size, 0.7 μm) with in situ pumps. Water samples for dissolved inorganic carbon (DIC) analysis were obtained by a pumpcast-CTD system and were killed off with HgCl2.

Enzyme assay conditions.

Candidatus “Brocadia anammoxidans” cells from either fluidized bed reactors (two samples) or from a sequencing batch reactor (also two samples) were washed twice with a solution containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, and 2 mM MgCl2. They were then suspended anaerobically inside a glove box (95% N2, 5% H2) in 4 ml of buffer and passed four times through a French pressure cell at 4°C. Unbroken cells and debris were removed by centrifugation in gas-tight tubes at 40,000 × g. The supernatant was used as cell extract and contained about 12 mg of protein per ml. Assays were carried out at least in triplicate (standard deviation, less than 15%) in anaerobic quartz cuvettes as described previously (5, 6, 12), with enzymes or extracts of the appropriate reference organisms used as controls (Table 1).
TABLE 1.
TABLE 1. Enzyme activities in cell extracts of Candidatus “Brocadia anammoxidans” and control organisms
Enzyme (EC no.)Candidatus “Brocadia anammoxidans” sp act (nmol/min/mg of protein)Control sp act (nmol/min/mg of protein)Control sourcea
CO dehydrogenase (EC 1.2.99.2)<1230UASB sludge
  3,780Methanotrix soehngenii
ATP citrate (pro-3S) lyase (EC 3.1.2.16)<1130Desulfobacter
Ribulose bisphosphate carboxylase (EC 4.1.1.39)<165Nitrosomonas europaea
3-Hydroxy propionate dehydrogenase (EC 1.1.1.59)<17,800Lactate dehydrogenase
Malate dehydrogenase (EC 1.1.1.37)52160Paracoccus denitrificans
Nitrate reductase (EC 1.7.99.4)34120Paracoccus denitrificans
a
UASB, upflow anaerobic sludge blanket.

Lipid and isotope analysis.

Lipids were analyzed by gas chromatography (GC), GC-mass spectrometry (MS), and GC-isotope ratio-monitoring MS (irmMS) as described previously (15).
Analysis of the δ13C of total DIC in the medium was performed by headspace analysis of 0.5 to 1 ml of water that had reacted with H3PO4 for at least 1 h at room temperature. The headspace was subsequently analyzed 10 times with a ThermoFinnigan Gas Bench II coupled to a DeltaPLUS irmMS system, with typical standard deviations of 0.1‰. Stable carbon isotope ratios were determined relative to lab standards calibrated with NBS-18 carbonate (International Atomic Energy Agency [IAEA]). Analysis of the δ13C of total DIC in the Black Sea water column was performed by headspace analysis of 2 ml of water after the addition of 100 μl of H3PO4 and equilibration at 40°C for 2 h. The headspace was subsequently analyzed six times by using a Multiflow system connected to an Isoprime irmMS system, with typical standard deviations of 0.1 to 0.2‰. Stable carbon isotope ratios were determined relative to lab standards calibrated on NBS-19 carbonate and CO-8 (IAEA).
The δ13C values of the biomass were determined by elemental analysis-irmMS using a Carlo Erba Flash elemental analyzer coupled to a ThermoFinnigan DeltaPLUS irmMS system, with reproducibility ranging from 0.1 to 0.7‰. Stable carbon isotope ratios were determined by using lab standards calibrated on NBS-22 oil (IAEA).

Enzyme activities.

Strictly anaerobically prepared cell extracts of Candidatus “Brocadia anammoxidans” were used to determine the key enzymes of the four known CO2 fixation pathways (Table 1). However, all assays showed activity of less than 1 nmol per min per mg of protein in the extracts, while the activity of control organisms was at least 65 nmol per min per mg of protein under the same conditions. A standard housekeeping enzyme like malate dehydrogenase showed a good level of activity (52 nmol per min per mg of protein). The enzyme potentially involved in providing reduced equivalents to the CO2 fixation pathway, nitrate/nitrite oxidoreductase, was present at 34 nmol per min per mg of protein, indicating that cell extract preparations did not compromise the observed activities. Furthermore, the CO2 fixation rate should have been on the order of 4 nmol per min per mg of protein (17), which should have been detectable in concentrated extracts. This lack of CO2-fixing enzyme activities in cell extracts prompted us to investigate the stable isotopic fractionation patterns of anammox bacteria.

Isotope analysis.

In an initial experiment, an enrichment culture of the anammox bacterium Candidatus “Brocadia anammoxidans” was grown for 34 days. DIC and biomass analyses showed a large difference in 13C contents, with the cell material being depleted of 13C by 26‰ compared to the inorganic carbon, suggesting a large isotopic fractionation by the anammox bacterium (Table 2). To further investigate this finding, an enrichment culture consisting of about 80% of the anammox bacterium Candidatus “Brocadia anammoxidans” was grown for 86 days (Table 2). The DIC present in the medium remained constant at −24.4‰ ± 0.2‰, but the collected cell material was isotopically more variable and ranged from −47.9 to −56.8‰, with no clear visible trend (Table 2). These results show that, similar to our initial experiment, the cell material is strongly depleted in 13C compared to CO2; i.e., the isotopic fractionation between cell material and CO2, expressed as ε, ranges between 11 and 22‰. This fractionation is similar to the range observed for organisms when either the Calvin cycle or the acetyl-CoA pathway is used but is much larger then fractionations observed for organisms when the reverse tricarboxylic acid cycle or the 3-hydroxypropionate pathway is used (2, 19, 20, 21).
TABLE 2.
TABLE 2. Stable carbon isotope data and fractionation factors obtained from biomass of Candidatus “Brocadia anammoxidans” strain Delft and particulate organic matter from the Black Seaa
 δ13C (‰ vs VPDB)ε (‰ vs VPDB)c
DICCO2aBiomassIIIbIIIIVVbiomass-IICO2-IICO2-IV
Reactor 1 (days)           
    34−27.5−35.6−53.5NDNDNDNDNDNDNDND
Reactor 2 (days)           
    1−24.3−32.4−48.7−66−65−67NDND1734 
    8−24.2−32.3−52.6−74−73ND−75ND224447
    58−24.6−32.7−47.9−70−63−70−74−69163245
    70−24.6−32.7−54ND−64−75−76−69113446
    86NDND−56.8−76−71−73−74−7015NA 
Black Sea (m depth)           
    90−1.3d−12.3NAND−58NDNDNDNA49ND
    95−1.4d−12.4NAND−58NDNDNDNA49ND
    100−1.4−12.4NAND−55NDNDNDNA45ND
a
Calculated according to an equation described previously (10). The temperatures were 35°C in cultures and 8.3°C in the Black Sea. For structures of anammox lipids (I to V), see Fig. 1. ND, not determined; NA, not applicable, VPDB, Vienna Pee-Dee Belemnite.
b
Since ladderane fatty acids are known to disintegrate during GC analysis, all the different fatty acid methyl esters containing cyclobutane rings were summed.
c
ε biomass-ladderanes = {[(1,000 + δ13C biomass)/(1,000 + δ13C ladderanes)] − 1} × 1,000.
d
Inferred from δ13C DIC measured at 80- and 100-m depths.
Confirmation of the large 13C fractionation by anammox bacteria was obtained by analyzing lipids (Fig. 1) known to be exclusively derived from these bacteria (17) (Table 2). Values that were highly 13C depleted were observed for anammox lipids compared to CO2, showing that indeed the anammox bacterium strongly fractionates against 13C. This result suggests that this anammox bacterium uses either the Calvin cycle or the acetyl-CoA pathway. However, the large difference in 13C contents of ladderane lipids compared to those of CO2 (up to 47‰) (Table 2) is beyond the maximum range of fractionations (ca. 40‰) (4, 11, 15) observed for lipids biosynthesized by organisms using the Calvin cycle. This tentatively suggests that anammox bacteria may use the acetyl-CoA pathway, which is known to sometimes yield larger fractionations in 13C than the Calvin cycle (11). The absence of CO dehydrogenase/acetyl-CoA synthase in cell extracts of Candidatus “Brocadia anammoxidans” indicates that the assay for this enzyme still has to be optimized, although great care was taken to ensure the strict anaerobic conditions generally necessary to detect this enzyme (5).
To investigate the isotopic fractionation of anammox bacteria under natural conditions, we analyzed the isotopic compositions of ladderane lipids (Fig. 1, structure II) present at several depths in the anoxic water column of the Black Sea (9). Molecular investigations have shown that these lipids originate from a different anammox bacterium, provisionally named Candidatus “Scalindua sorokinii,” which has a 12.4% sequence difference in the 16S rRNA gene compared to Candidatus “Brocadia anammoxidans” (9). As observed for the enrichment culture of Candidatus “Brocadia anammoxidans,” the lipids of Candidatus “Scalindua sorokinii” are strongly depleted in 13C (Table 2), and the fractionation compared to CO2 is even larger (up to 49‰) than that observed for the same lipids in the Brocadia enrichment culture (up to 47‰). This result suggests not only that anammox bacteria also strongly fractionate against 13C under natural conditions but also that the autotrophic carbon fixation pathways within the diverse group of anaerobic ammonium-oxidizing bacteria are similar.

Conclusions.

Our study of the stable carbon isotopic compositions of lipids and the biomass of anaerobic ammonium-oxidizing bacteria shows that they possess a carbon fixation pathway which strongly fractionates against 13C. The results are consistent with either the use of the Calvin cycle or perhaps more likely, based on the large 13C depletions, the acetyl-CoA pathway.

Acknowledgments

We thank L. van Niftrik and B. Maas for laboratory cultivation of anammox biomass. C. Hanfland and the AWI (Bremerhaven) provided the in situ pumps, G. Lavik (MPI) provided assistance with the pumps, and G. Klockgether (MPI) assisted with extractions.
We thank the crew of the R/V Meteor for collaboration and the Romanian and Turkish authorities for access to their national waters.

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cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 70Number 6June 2004
Pages: 3785 - 3788
PubMed: 15184193

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Received: 28 November 2003
Accepted: 4 March 2004
Published online: 7 June 2004

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Authors

Stefan Schouten [email protected]
Department of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research, 1790 AB Den Burgh
Marc Strous
Department of Microbiology, Faculty of Science, University of Nijmegen, 6500 GL Nijmegen, The Netherlands
Marcel M. M. Kuypers
Max Planck Institute for Marine Microbiology, D-28359 Bremen, Germany
W. Irene C. Rijpstra
Department of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research, 1790 AB Den Burgh
Marianne Baas
Department of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research, 1790 AB Den Burgh
Carsten J. Schubert
Limnological Research Center, Swiss Federal Institute for Environmental Science and Technology, CH 6047 Kastanienbaum, Switzerland
Mike S. M. Jetten
Department of Microbiology, Faculty of Science, University of Nijmegen, 6500 GL Nijmegen, The Netherlands
Jaap S. Sinninghe Damsté
Department of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research, 1790 AB Den Burgh

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