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

Isotopic analyses of Candidatus "Brocadia anammoxidans," a chemolithoautotrophicbacterium that anaerobically oxidizes ammonium (anammox), showthat it strongly fractionates against ¹³C; i.e., lipids aredepleted by up to 47 ${per thousand}$ versus CO₂. Similar results were obtainedfor the anammox bacterium Candidatus "Scalindua sorokinii,"which thrives in the anoxic water column of the Black Sea, suggestingthat different anammox bacteria use identical carbon fixationpathways, which may be either the Calvin cycle or the acetylcoenzyme A pathway.

INTRODUCTION

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Introduction

Anaerobic ammonium oxidation (anammox) is the oxidation of ammoniumwith nitrite as the electron acceptor and dinitrogen gas asthe product (for reviews, see references 7 and 8). The processis mediated by obligately anaerobic chemolithoautotrophic bacteriathat form a monophyletic cluster inside the Planctomycetales,one of the major divisions of the Bacteria. So far, four specieshave been detected and enriched from the biomass of sewage treatmentplants: Candidatus "Brocadia anammoxidans" (18), Candidatus"Kuenenia stuttgartiensis" (13), Candidatus "Scalindua wagneri,"and Candidatus "Scalindua brodae" (14). Candidatus "Scalinduasorokinii" was detected in the anoxic water column of the BlackSea (9), providing the first direct evidence for anammox bacteriain the natural environment. Anammox bacteria have a cell compartmentknown as the anammoxosome, which is the site of anammox catabolism.The lipid bilayer membrane surrounding this anammoxosome containsunusual lipids, so-called "ladderane" lipids, concatenated cyclobutanemoieties that are either ether and/or ester linked to the glycerolbackbone or occur as free alcohols (e.g., Fig. 1, structuresII to IV) (16). The other membranes of anammox bacteria containlipids typical for planctomycetes in general: iso, normal, andmid-chain methyl hexadecanoic acids (e.g., Fig. 1, structureI).

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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 pathwaythey use. There are currently four known pathways for CO₂ fixationin microorganisms (see, e.g., references 2 and 3). The Calvincycle, with ribulose bisphosphate carboxylase as a key enzyme,is operative in many organisms. The 3-hydroxypropionate pathwayhas 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 phototrophicbacteria. Finally, the acetyl coenzyme A (acetyl-CoA) pathway,with carbon monoxide dehydrogenase/acetyl-CoA synthase as theindicative enzyme, is detected in many anaerobic microorganisms.In addition to enzyme activities, stable carbon isotopic compositionsof total cell material and individual lipids, carbohydrates,and amino acids are often used to infer these biosynthetic pathwaysin organisms, as the fractionation from the inorganic carbonsource to the autotrophic biomass in ¹³C depends on the biosyntheticpathway used (1, 19, 20, 21).

Here, we determined enzyme activities and studied the stablecarbon isotopic fractionations of Candidatus "Brocadia anammoxidans"to investigate its carbon fixation pathway. Since this bacteriumcan be grown only in enrichment cultures, and bulk cell materialis thus not solely derived from anammox bacteria, we also determinedthe isotopic compositions of the specific lipids of this bacterium.Furthermore, the isotopic compositions of ladderane lipids derivedfrom Candidatus "Scalindua sorokinii" growing in the anoxicwater column of the Black Sea (9) were determined in order toexamine the ¹³C fractionation patterns of anammox bacteria undernatural conditions.

Anammox samples.

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Anammox samples.

Anammox bacterial cells were grown in enrichment cultures inan anaerobic sequencing batch reactor as described previously(17). Candidatus "Brocadia anammoxidans" strain Delft was enrichedfrom an anaerobic wastewater treatment plant in Rotterdam, TheNetherlands. Particulate organic matter for lipid analyses wascollected 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 wascollected at several depths by in situ filtration of large volumes( $~$ 1,000 liters) of water through 292-mm-diameter precombustedglass fiber filters (nominal pore size, 0.7 µm) with insitu pumps. Water samples for dissolved inorganic carbon (DIC)analysis were obtained by a pumpcast-CTD system and were killedoff with HgCl₂.

Enzyme assay conditions.

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Enzyme assay conditions.

Candidatus "Brocadia anammoxidans" cells from either fluidizedbed reactors (two samples) or from a sequencing batch reactor(also two samples) were washed twice with a solution containing50 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 aFrench pressure cell at 4°C. Unbroken cells and debris wereremoved by centrifugation in gas-tight tubes at 40,000 x g.The supernatant was used as cell extract and contained about12 mg of protein per ml. Assays were carried out at least intriplicate (standard deviation, less than 15%) in anaerobicquartz cuvettes as described previously (5, 6, 12), with enzymesor extracts of the appropriate reference organisms used as controls(Table 1).

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TABLE 1. Enzyme activities in cell extracts of Candidatus "Brocadia anammoxidans" and control organisms

Lipid and isotope analysis.

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Lipid and Isotope analysis.

Lipids were analyzed by gas chromatography (GC), GC-mass spectrometry(MS), and GC-isotope ratio-monitoring MS (irmMS) as describedpreviously (15).

Analysis of the ${delta}$ ¹³C of total DIC in the medium was performedby headspace analysis of 0.5 to 1 ml of water that had reactedwith H₃PO₄ for at least 1 h at room temperature. The headspacewas subsequently analyzed 10 times with a ThermoFinnigan GasBench II coupled to a Delta^PLUS irmMS system, with typical standarddeviations of 0.1 ${per thousand}$ . Stable carbon isotope ratios were determinedrelative to lab standards calibrated with NBS-18 carbonate (InternationalAtomic Energy Agency [IAEA]). Analysis of the ${delta}$ ¹³C of total DICin the Black Sea water column was performed by headspace analysisof 2 ml of water after the addition of 100 µl of H₃PO₄and equilibration at 40°C for 2 h. The headspace was subsequentlyanalyzed six times by using a Multiflow system connected toan Isoprime irmMS system, with typical standard deviations of0.1 to 0.2 ${per thousand}$ . Stable carbon isotope ratios were determined relativeto lab standards calibrated on NBS-19 carbonate and CO-8 (IAEA).

The ${delta}$ ¹³C values of the biomass were determined by elemental analysis-irmMSusing a Carlo Erba Flash elemental analyzer coupled to a ThermoFinniganDelta^PLUS irmMS system, with reproducibility ranging from 0.1to 0.7 ${per thousand}$ . Stable carbon isotope ratios were determined by usinglab standards calibrated on NBS-22 oil (IAEA).

Enzyme activities.

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Enzyme activities.

Isotope analysis.

Conclusions.

References

Strictly anaerobically prepared cell extracts of Candidatus"Brocadia anammoxidans" were used to determine the key enzymesof the four known CO₂ fixation pathways (Table 1). However,all assays showed activity of less than 1 nmol per min per mgof protein in the extracts, while the activity of control organismswas at least 65 nmol per min per mg of protein under the sameconditions. A standard housekeeping enzyme like malate dehydrogenaseshowed a good level of activity (52 nmol per min per mg of protein).The enzyme potentially involved in providing reduced equivalentsto the CO₂ fixation pathway, nitrate/nitrite oxidoreductase,was present at 34 nmol per min per mg of protein, indicatingthat cell extract preparations did not compromise the observedactivities. Furthermore, the CO₂ fixation rate should have beenon the order of 4 nmol per min per mg of protein (17), whichshould have been detectable in concentrated extracts. This lackof CO₂-fixing enzyme activities in cell extracts prompted usto investigate the stable isotopic fractionation patterns ofanammox bacteria.

Isotope analysis.

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Enzyme activities.

Isotope analysis.

Conclusions.

References

In an initial experiment, an enrichment culture of the anammoxbacterium Candidatus "Brocadia anammoxidans" was grown for 34days. DIC and biomass analyses showed a large difference in¹³C contents, with the cell material being depleted of ¹³C by26 ${per thousand}$ compared to the inorganic carbon, suggesting a large isotopicfractionation by the anammox bacterium (Table 2). To furtherinvestigate this finding, an enrichment culture consisting ofabout 80% of the anammox bacterium Candidatus "Brocadia anammoxidans"was grown for 86 days (Table 2). The DIC present in the mediumremained constant at –24.4 ${per thousand}$ ± 0.2 ${per thousand}$ , but the collectedcell material was isotopically more variable and ranged from–47.9 to –56.8 ${per thousand}$ , with no clear visible trend (Table2). 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 ${varepsilon}$ , ranges between 11 and 22 ${per thousand}$ . This fractionationis similar to the range observed for organisms when either theCalvin cycle or the acetyl-CoA pathway is used but is much largerthen fractionations observed for organisms when the reversetricarboxylic acid cycle or the 3-hydroxypropionate pathwayis used (2, 19, 20, 21).

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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 Sea^a

Confirmation of the large ¹³C fractionation by anammox bacteriawas obtained by analyzing lipids (Fig. 1) known to be exclusivelyderived from these bacteria (17) (Table 2). Values that werehighly ¹³C depleted were observed for anammox lipids comparedto CO₂, showing that indeed the anammox bacterium strongly fractionatesagainst ¹³C. This result suggests that this anammox bacteriumuses either the Calvin cycle or the acetyl-CoA pathway. However,the large difference in ¹³C contents of ladderane lipids comparedto those of CO₂ (up to 47 ${per thousand}$ ) (Table 2) is beyond the maximum rangeof fractionations (ca. 40 ${per thousand}$ ) (4, 11, 15) observed for lipids biosynthesizedby organisms using the Calvin cycle. This tentatively suggeststhat anammox bacteria may use the acetyl-CoA pathway, whichis known to sometimes yield larger fractionations in ¹³C thanthe Calvin cycle (11). The absence of CO dehydrogenase/acetyl-CoAsynthase 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 anaerobicconditions generally necessary to detect this enzyme (5).

To investigate the isotopic fractionation of anammox bacteriaunder natural conditions, we analyzed the isotopic compositionsof ladderane lipids (Fig. 1, structure II) present at severaldepths in the anoxic water column of the Black Sea (9). Molecularinvestigations have shown that these lipids originate from adifferent anammox bacterium, provisionally named Candidatus"Scalindua sorokinii," which has a 12.4% sequence differencein the 16S rRNA gene compared to Candidatus "Brocadia anammoxidans"(9). As observed for the enrichment culture of Candidatus "Brocadiaanammoxidans," the lipids of Candidatus "Scalindua sorokinii"are strongly depleted in ¹³C (Table 2), and the fractionationcompared to CO₂ is even larger (up to 49 ${per thousand}$ ) than that observedfor the same lipids in the Brocadia enrichment culture (up to47 ${per thousand}$ ). This result suggests not only that anammox bacteria alsostrongly fractionate against ¹³C under natural conditions butalso that the autotrophic carbon fixation pathways within thediverse group of anaerobic ammonium-oxidizing bacteria are similar.

Conclusions.

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Enzyme activities.

Isotope analysis.

Conclusions.

References

Our study of the stable carbon isotopic compositions of lipidsand the biomass of anaerobic ammonium-oxidizing bacteria showsthat they possess a carbon fixation pathway which strongly fractionatesagainst ¹³C. The results are consistent with either the useof the Calvin cycle or perhaps more likely, based on the large¹³C depletions, the acetyl-CoA pathway.

ACKNOWLEDGMENTS

We thank L. van Niftrik and B. Maas for laboratory cultivationof anammox biomass. C. Hanfland and the AWI (Bremerhaven) providedthe in situ pumps, G. Lavik (MPI) provided assistance with thepumps, and G. Klockgether (MPI) assisted with extractions.

We thank the crew of the R/V Meteor for collaboration and theRomanian and Turkish authorities for access to their nationalwaters.

FOOTNOTES

* Corresponding author. Mailing address: Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, P.O. Box 59, 1790 AB Den Burgh, The Netherlands. Phone: 31 222 369565. Fax: 31 222 319674. E-mail: schouten{at}nioz.nl.

REFERENCES

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