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Keywords:

  • anammox;
  • hot springs;
  • ladderane lipids;
  • 16S rRNA gene;
  • microorganisms

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g−1 sediment were detected in five hot springs analyzed with temperatures up to 65 °C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5–99.8% sequence identity) in three hot springs with temperatures up to 52 °C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Terrestrial hot springs provide suitable habitats for a variety of photoautotrophic and chemolithoautotrophic microorganisms (Castenholz, 1984; Ward et al., 1998). Temperature and hydrogen sulfide content among others seem to be important factors, which affect the distribution and diversity of these organisms in hot springs (Ward et al., 1998; Purcell et al., 2007). These environments can contain dense microbial mats with complex communities, which exhibit a high biodiversity, including phototrophic organisms, mostly cyanobacteria and filamentous green nonsulfur bacteria (Ward et al., 1987), thermophilic denitrifying bacteria (Hollocher & Kristjansson, 1992) and archaea (Barns et al., 1996; Kvist et al., 2005;Meyer-Dombard et al., 2005; Huang et al., 2007). Recently, thermophilic autotrophic crenarchaeal nitrifiers were enriched from terrestrial hot springs (de la Torre et al., 2008; Hatzenpichler et al., 2008) and were found to play an active role in the nitrogen cycle of these hot ecosystems (Reigstad et al., 2008; Zhang et al., 2008). Inspired by these findings, we started the present survey of various hot spring ecosystems in order to find indications of other microorganisms that may play a role in the conversion of nitrogen compounds in these thermophilic environments with a special focus on anaerobic ammonium oxidizing bacteria.

Anammox, the anaerobic ammonium oxidation to dinitrogen gas with nitrite as electron acceptor, is a key process in the global nitrogen cycle (see Arrigo, 2005; Brandes et al., 2007; Francis et al., 2007 for recent reviews), which constitutes a novel route to convert fixed inorganic nitrogen to gaseous N2. The anammox process is linked to one group of organisms forming a distinct phylogenetic group related to the Planctomycetes (Strous et al., 1999a; Schmid et al., 2007). Anammox bacteria contain a special intracellular compartment called the anammoxosome, where anammox catabolism is assumed to take place (Sinninghe Damstéet al., 2002; van Niftrik et al., 2004, 2008). The membrane of this ‘organelle’ consists of unusual ladderane lipids forming a dense barrier, which reduces the permeability of the membrane to small molecules, for example protons or the toxic intermediate hydrazine of the anammox reaction, which can easily permeate less dense bacterial membranes (Sinninghe Damstéet al., 2002). The ladderane core lipid contains three or five linearly concatenated cyclobutane rings either ester or ether bound to the glycerol backbone, which is unprecedented in nature. Anammox has been found in a range of environments, i.e. anoxic water columns (Dalsgaard et al., 2003; Kuypers et al., 2003), marine and estuarine sediments (Dalsgaard & Thamdrup, 2002; Trimmer et al., 2003; Schmid et al., 2007), freshwater lakes (Schubert et al., 2006) and also polar marine sediments and sea ice (Rysgaard & Glud, 2004a; Rysgaard et al., 2004b). The group of anammox bacteria is currently associated with at least five genera, Candidatus Brocadia, Candidatus Kuenenia, Candidatus Anammoxoglobus, Candidatus Jettenia and Candidatus Scalindua (Strous et al., 1999a; Kartal et al., 2007; Schmid et al., 2007; Quan et al., 2008). So far, available 16S rRNA gene sequences from marine environments were found to be closely related to Candidatus Scalindua sp. (Kuypers et al., 2003, 2005; Schubert et al., 2006; Schmid et al., 2007; Woebken et al., 2008). Although the anammox process is well known in mesophilic environments, little is known about its importance in thermophilic and hyperthermophilic environments. Recently, Byrne et al. (2008), however, found indications for anammox activity at 60–85 °C in deep-sea hydrothermal vent areas, where temperatures as high as 153 °C are observed.

To investigate the occurrence of anammox bacteria in high-temperature environments, we studied microbial mats and sediments in five hot springs from California and Nevada with temperatures between 36.1 and 65.2 °C, where previously, in similar hot springs, archaeal amoA genes and 16S rRNA genes related to crenarchaeaota were also detected (Huang et al., 2007; Zhang et al., 2008). The samples were analyzed using different molecular techniques for the detection of anammox bacteria, i.e. ladderane biomarker lipid analysis and 16S rRNA gene-based phylogeny.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Sampling

Samples were collected from five locations in the Great Basin hot springs of Northern California and Western Nevada in February 2007, at the source and further downstream of the spring (Fig. 1). The hot springs were selected to represent a range of temperatures and chemistries (Table 1). At each site, temperature, pH and oxygen were measured in situ in the overlying water before collection of bacterial mats and sediments. Temperature was determined with an Ama-Digit ad 20 th digital thermometer. The oxygen concentration was measured using a WTW Oxi 315i digital meter. The pH was determined with a WTW pH 315i digital meter. Water samples for nutrient analysis were poisoned with HgCl2, transported to the NIOZ after being cooled and measured spectrophotometrically using an autoanalyzer system (Bran and Luebbe TRAACS 800+). Mat and sediment samples were stored frozen at −20 °C until used for further analysis.

image

Figure 1.  Map of hot spring locations in California and Nevada.

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Table 1.   Physical and nutrient data for overlying waters and lipid data for microbial mats and sediments of California and Nevada hot springs
SiteLocationSample descriptionTemperature (°C)pHO2 (%)*NH4+ (μM)NOx (μM)NO2 (μM)C18-[5] (ng g−1 sediment)C18-[3] (ng g−1 sediment)C20-[5] (ng g−1 sediment)C20-[3] (ng g−1 sediment)Total (ng g−1sediment)NL5
  • *

    % Air saturation.

  • ND, not detected; NA, not analyzed.

1.1Leonard's hot spring, CAMat (source)52.47.7500.70.60.21.90.45.64.912.80.75
1.3 Sediment (effluent)36.18.4830.00.90.10.30.050.40.21.00.57
2.1Ray's hot spring, CAStreamers(source)89.18.2NA0.00.70.2NDNDNDNDND
2.4 Mat+sediment (effluent)58.18.7NA2.36.96.0NDNDND1.81.8
2.5 Mat+sediment (effluent)37.18.9NA0.85.81.90.20.080.70.71.70.76
3West Valley warm spring, CASediment (source)23.78.5540.127.60.10.3NDNDND0.3
4Patua-Hazen hot spring, NVSediment (source)51.37.9NA13.37.10.825.0ND19.87.352.00.44
5.1Lee's hot spring, NVSediment (source)96.68.2452.30.60.5NDNDNDNDND
5.2 Sediment (effluent)65.28.7850.41.00.70.03ND0.20.150.40.88

Ladderane lipid analysis

Samples of about 50 mg–13 g of freeze-dried and homogenized material were ultrasonically extracted five times using a dichloromethane–methanol mixture (2 : 1 by volume). The extracts were combined and the bulk of the solvent was removed by rotary evaporation under vacuum, and the extract was further dried over a Na2SO4 column. An aliquot of the lipid extract was saponified with aqueous 1 N KOH in methanol for 2 h at 100 °C. Nonsaponifiable lipids (neutral lipids) were extracted out of the basic solution (pH>13) using dichloromethane. Fatty acids were obtained by acidifying the residue to pH 3 and subsequent extraction with dichloromethane. The fatty acid fraction was methylated by adding diazomethane (CH2N2) to convert fatty acids into their corresponding methyl esters (FAMEs). The excess CH2N2 was removed by evaporation. To remove very polar components, aliquots of the FAMEs were eluted with ethyl acetate over a small column filled with silica. Polyunsaturated fatty acids were removed by eluting the aliquots with ethyl acetate over a small AgNO3 (5%) impregnated silica column, yielding a saturated fatty acid fraction. The fatty acid fractions were dissolved in acetone and then filtered through a 0.45-μm, 4-mm-diameter polytetrafluoroethylene (PTFE) filter.

These fractions were analyzed by HPLC coupled to positive ion atmospheric pressure chemical ionization tandem MS (HPLC/APCI-MS/MS) in selective reaction monitoring mode as described Hopmans et al. (2006) with some modifications described by Rattray et al. (2008). Specifically, separation was achieved using a Zorbax Eclipse XDB-C8 column (3.0 × 250 mm, 5 μm; Agilent) and a flow rate of 0.18 mL min−1 MeOH. The source settings were vaporizer temperature 475 °C, discharge current 2.5 μA, sheath gas (N2) pressure 30 (arbitrary units), auxiliary gas (N2) pressure 5 (arbitrary units), capillary temperature 350 °C, source collision-induced dissociation (CID) −10 V. Argon pressure was maintained at 1.5 mTorr in the second quadrupole. Quantification of ladderane lipids was carried out using an external calibration curve using standards of isolated methylated ladderane fatty acids containing the [3]- and [5]-ladderane moieties (Fig. 2, structures I–IV) (Sinninghe Damstéet al., 2002; Hopmans et al., 2006; Rattray et al., 2008). A detection limit (defined by a signal to noise ratio of 3) of 30–35 pg injected was achieved with this technique.

image

Figure 2.  Partial selective reaction monitoring traces of four ladderane core lipids obtained by HPLC/(APCI)-MS/MS analysis of a sediment sample from Leonard's Hot Spring, and their corresponding structures, (I) C18-[5]-ladderane FAME, (II) C18-[3]-ladderane FAME, (III) C20-[5]-ladderane FAME and (IV) C20-[3]-ladderane FAME. Fatty acids were analyzed as methyl esters (FAME).

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Molecular techniques and phylogenetic analysis

High molecular weight DNA isolation and hot-start PCR were performed according to Schmid et al. (2000, 2005). The DNA from each site was used as a template in a PCR amplification with primer set Pla46F and 1390R. The products of these PCRs were used in a second nested PCR with primer sets AMX368F–AMX820R (codes with gray background, Fig. 3) or Pla46F–AMX820R (other codes). Products of the second PCR amplifications were subsequently cloned with the TOPO TA cloning kit (Invitrogen, Breda, the Netherlands) according to the manual of the manufacturer and sequenced. The 16S rRNA gene sequences of the clones were compared with their closest relatives in the GenBank database by blastn searches (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and with the rdp classifier tool (http://rdp.cme.msu.edu/classifier/). Further phylogenetic and molecular evolutionary analyses were performed with the mega 4.1 program (Tamura et al., 2007).

image

Figure 3.  Phylogenetic tree of 16S rRNA gene sequences determined by Neighbor-Joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length =2.69043569 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (pairwise deletion option). There were a total of 636 positions in the final dataset. Phylogenetic analyses were conducted in mega4(Tamura et al., 2007). Sequences were identified using primer sets AMX368F–AMX820R (codes with gray background) or Pla46F–AMX820R (other codes).

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Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Occurrence and distribution of ladderane lipids in California and Nevada hot springs

To search for the presence of anammox bacteria, we first analyzed ladderane biomarker lipids in microbial mats and sediments from several hot springs in California and Nevada, either from the source or further downstream (Table 1). We specifically targeted four ladderane core lipids (see structures in Fig. 2) that are present in nearly all currently known anammox genera (Rattray et al., 2008). Ladderane core lipids were detected in almost all hot spring samples with concentrations between 0.3 and 52 ng g−1 sediment (total concentration of all four ladderane lipids) and temperatures between 24 and 65 °C (Table 1). Highest concentrations of ladderane lipids were detected at Patua-Hazen hot spring (52 ng g−1 sediment), followed by Leonard's hot spring source mat (12.8 ng g−1 sediment). At all other sites more distant from the hot spring source and at lower in situ temperatures only low concentrations between 0.3 and 1.8 ng g−1 sediment of ladderane lipids were found (Table 1). The overall low concentration (0–0.4 ng g−1 sediment) of the C18-[3]-ladderane fatty acid can be explained by the fact that this specific lipid is only synthesized by marine anammox bacteria belonging to the ‘Scalindua’ genus (Rattray et al., 2008). No ladderane lipids were detected in the sediment at the source of Lee's hot spring and in the streamers of Ray's hot spring where temperatures of 96.6 and 89.1 °C were recorded. These temperatures are probably too high for any anammox activity and only specific prokaryotes can thrive there. The high ladderane lipid concentration at Patua-Hazen hot spring coincides with substantially higher NH4+ and NOx concentrations compared with other hot springs that may promote anammox activity (Table 1). Although oxygen concentrations in the waters overlying the mats were high (Table 1), the mats themselves are most likely anoxic as indicated by the occurrence of wax ester lipids specific for green nonsulfur-like bacteria (GNSLB), which are anoxygenic phototrophs (S. Schouten et al., unpublished data). Furthermore, Zhang et al. (2007) also found 16S rRNA gene sequences falling in the phylogenetic cluster of the GNSLB in hot spring mats from Nevada and California, indicating that these mats are anoxic or at least contain anaerobic microsites where anaerobic bacteria such as anammox can thrive.

Further evidence for the in situ production of ladderane lipids in anammox bacteria was derived from the relative distribution of ladderane lipids. Anammox bacteria have recently been found to adapt to changing temperatures by modifying their membrane composition. The amount of shorter chain ladderane fatty acids increases relative to the amount of longer chain fatty acids at lower temperatures and vice versa. The index of ladderane lipids with five cyclobutane rings, termed NL5, has been proposed to quantify this relative change (Rattray, 2008). The NL5 of the ladderane lipids in the hot spring samples varied between 0.44 and 0.88 (Table 1), indicating relatively more production of the longer chain fatty acids at elevated temperatures (>25 °C) at the investigated sites. Especially the high NL5 value of 0.88 calculated for Lee's hot spring (site 5.2) indicates adaptation of the membrane lipid composition to high temperatures, and further indicates that anammox bacteria might be thriving in these hot spring environments.

16S rRNA gene analysis of California and Nevada hot springs

Ladderane core lipids are derived from both living and fossil anammox bacterial biomass, and might not necessarily be indicators for active cells. We, therefore, also analyzed the 16S rRNA gene sequences from several of these environments. Amplification of 16S rRNA genes using a nested PCR approach with primers specific for anammox bacteria resulted in the detection of several 16S rRNA gene sequences from selected hot spring sites where ladderane core lipids were detected, i.e. Leonard's hot spring, Ray's hot spring, Patua-Hazen hot spring and Lee's hot spring, suggesting the presence of active anammox bacteria at these sites with temperatures ranging between 36.1 and 52.4 °C. Phylogenetic analysis of the 16S rRNA gene sequences showed that some of them were related to known anammox bacteria (Fig. 3). At the source of Leonard's hot spring mat, clones were closely related to Candidatus Kuenenia stuttgartiensis, while most downstream sequences were more closely related to both Candidatus Brocadia fulgida and C. Kuenenia stuttgartiensis. At Ray's hot spring at 37.1 °C sequences were found that are most closely related to Candidatus Brocadia anammoxidans. At Patua-Hazen hot spring, where highest concentrations of the ladderane core lipids were observed, clones were found which did not seem to fall in the known anammox clusters, but were positioned phylogenetically between Planctomycetes and anammox bacteria. This could mean that these organisms are also capable of anaerobic ammonium oxidation and also produce ladderane lipids such as anammox bacteria, or the ladderane lipids detected at this site are not produced autochthonously, which seems rather unlikely in view of the high ladderane lipid concentrations.

Four clones closely related to Dictyoglomus thermophilum were found in sediments of Patua-Hazen and Lee's hot spring at temperatures of 51.3 and 65.5 °C. Dictyoglomus thermophilum is an extremely thermophilic bacterium deeply rooted near the base of the order Thermotogales (Patel et al., 1987). Finally, some of the retrieved 16S rRNA gene sequences detected in Ray's hot spring (HRH874) and Patua-Hazen hot spring (HRH876, 877, 879) were related to uncultivated bacteria falling in the cluster of Planctomycetes (Fig. 3).

Implications

Our combined results of ladderane lipids and 16S rRNA gene sequences suggest that anammox bacteria are present in hot springs at temperatures of at least up to 52 °C. This is higher than the current temperatures at which anammox bacteria are found to be thriving. Candidatus Kuenenia and Candidatus Brocadia are genera present in wastewater treatment systems, and active at temperatures up to 43 °C (Strous et al., 1999b), while only members of the Candidatus Scalindua genus have so far been found in oxygen-limited marine environments at generally lower ambient temperatures (Schmid et al., 2007; Woebken et al., 2008). Thus, the fact that the sequences detected in the hot springs ranging in temperature between 36 and 52 °C are more closely related to Kuenenia and Brocadia rather than Scalindua is in agreement with the generally higher optimal growth temperatures (c. 35 °C in bioreactors vs. in general much lower environmental temperatures of 12–15 °C) of these species. Very recently, anammox activity was observed at 60–85 °C in samples obtained from hydrothermal vent areas at the Mid-Atlantic Ridge (Byrne et al., 2008). In that study, 16S rRNA gene sequences that were also more closely related to Kuenenia sequences were retrieved.

The results of the Patua-Hazen hot spring are intriguing, i.e. this spring contains abundant ladderane lipids, but the 16S rRNA gene sequences detected form a cluster that does not fall into the currently known anammox bacteria, but is more related to deep-branching Planctomycetes as was recently also reported for a hot spring in Oklahoma (Elshahed et al., 2007). Future enrichment culture work may reveal whether these organisms are indeed capable of catalyzing the anammox reaction. Nevertheless, our results indicate that anammox bacteria are present in hot springs, and may form an important and as yet undiscovered link in the nitrogen cycle of these hot spring environments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We want to thank Francien Peterse, Marcel van der Meer and Henry Boumann for assistance in sampling the hot springs. This study was supported by grants (853.00.031 and 853.00.032) to J.S.S.D. and M.S.M.J. from the Research Council for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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