Prokaryotic life in a potash-polluted marsh with emphasis on N-metabolizing microorganisms

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Abstract

Prokaryotic life along the salt gradient of the potash marsh resulting from mining waste at Schreyahn, Northern Germany, was screened for the distribution of total prokaryote (assessed by the 16S rRNA gene) and of N2-fixing (nifH gene), denitrifying (nosZ) and nitrifying (amoA) microorganisms. Information on prokaryotes was retrieved from the different soil sites (a) by culturing in conventional media, (b) by isolating the DNA, amplifying the target genes by PCR followed by sequencing, (c) by employing the recently developed computer program (TReFID [Rösch, C., Bothe, H., 2005. Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Applied and Environmental Microbiology 71, 2026–2035]) based on tRFLP data. New sequences were obtained as well as ones that were almost identical to those found at far distant locations. Whereas the distribution of plants strictly follows the salt gradient, this is apparently not the case with prokaryotes. Bacteria of hypersaline areas coexist with salt-non-tolerant species. The recently developed TReFID program is successfully applied to characterize a prokaryote community structure.

Introduction

In saline habitats, halophilic organisms occur which are strictly dependent for their growth on a high salt concentration. Others can resist salt stress and are confined to salt marshes due to their low competitiveness elsewhere. This is true for both microorganisms (Oren, 1999) and higher plants (Ellenberg, 1988). The latter show impressive belt formations dependent on the soil salt concentration at all marshes throughout the world. In Western and Continental Europe, Salicornia europaea L. (glasswort or marsh samphire) can cope with the highest salt concentration and often forms monocultures in high salt soils. The next most tolerant belt is formed by Puccinellia spp., being P. maritima (Huds.) Parl. (common salt marsh grass) on the Atlantic coast, P. distans (Jacq.) Parl. (reflexed salt marsh grass) on German inland salt marshes and P. limosa (Schur) E. Holmb (swamp salt marsh grass) on the Hungarian plain. Soils with lower salt contents are often dominated by Aster tripolium L. (salt aster), which, however, shows a somewhat broader range of distribution because it can thrive on wetter and drier stands with higher or lower salt contents (Ellenberg, 1988). As known in plant sociology, these belt formations are independent of the salt type, thus irrespective of NaCl, Na2SO4 or K2CO3 dominating in soils. Due to the capacity of the ions to bind water, salt marsh soils have an extremely negative water potential, and halophytes have to endure extended periods of drought. As noted early by botanists (Stocker, 1928), stress caused by drought rather than by the salt itself and its type is the major cause of belt formation of halophytes in salt marshes.

The characterization of prokaryotic life in hypersaline environments has recently been a centre of interest in microbial ecology (Oren, 2002, Øvreås et al., 2003) and has revealed a high diversity of organisms from various taxonomic affiliations (see Section 4). It has, however, not yet been examined whether the distribution of prokaryotes follows that of plants and is also dictated by the gradient in the salt concentration (or better in the water potential) in salt marshes. The current study aims at characterizing prokaryotic life in the soil around a potash mine at Schreyahn, Wendland, in Northern Germany. This heap originates from the residuals of below-ground mining performed between 1905 and 1926 (Horst and Redel, 1977). The soil surrounding this heap supports the typical belt formation of plants dependent upon the salt concentration, with a surprisingly high richness of different halophytes. Soil samples were taken from the roots of the belt indicators S. europaea, P. distans and A. tripolium, from the plant-free, central area and from the heap itself. Prokaryotic life in the samples was analysed by several methods. (a) Bacteria were grown in conventional media, and their DNA was extracted for PCR amplifications of the 16S rRNA gene (for total bacterial community) and of genes coding for characteristic enzymes of the nitrogen cycle: nifH (nitrogenase reductase), nosZ (N2O-reductase of denitrifiers) and amoA (ammonium monooxygenase of nitrifiers). (b) DNA was extracted directly from the soil samples for generating PCR-amplicons of the above-mentioned target genes. This method provided a clone library, and sequencing revealed information on new prokaryotes which grouped mainly next to uncultured microorganisms. However, due to the high diversity of approximately 104 ribotypes (∼different bacteria) in soils (Torsvik et al., 1990), such groups of independent sequences provided by methods a and b do not permit, by any means, to comprehensively assess the bacterial community structure. (c) DNA extracted from the soil samples was, therefore, used to generate tRFLP profiles for identifying prokaryotes utilizing the recently developed TReFID program (Rösch and Bothe, 2005). By using up to 13 restriction enzymes, DNA fragments labelled with a fluorescence dye (tRFs) were generated from the soil sample. The computer assignment tool TReFID screens for the presence of a pattern specific for any bacterium among the current 22,145 entries in the TReFID database in the collection of the tRFs of the soil. The search for organisms retrieved from the TReFID database was then extended to all soil samples taken from the zone indicator plants or from the bulk soil to assess the distribution of prokaryotes in dependence on the salt load.

Section snippets

Soil sampling and soil parameters

Soil samples from the Schreyahn potash marsh (52°55′53″ N, 11°04′36″ E) were taken from the upper 10 cm of the middle part of the A. tripolium, P. distans or S. europaea vegetation zones (about 1 kg per zone which was enough to represent each zone). Additionally, samples from the central, plant free mud zone and from a potash heap were taken to obtain information on the microbial community structure along a transect from lowest to highest salt load. There was no litter or biofilm on top of the soil

Parameters of soil samples taken from the potash marsh

The pH values of the soil samples from the Schreyahn potash mine varied between 7.3 and 8.4 (Table 1). Since the pH of the non-polluted soils in the vicinity of the Schreyahn village was between 5 and 6, the high values in the surrounds of the K2CO3 heap had presumably resulted from the out-washings of the salt by rainfall. Among land plants, S. europaea is known to endure the highest salt loads (Ellenberg, 1988), indicated also at Schreyahn by the high electric conductivity (EC) data (Table 1

Discussion

Most alkaliphilic sites are confined to dry areas with high evaporation rates and have pH values around 10 (Sorokin and Kuenen, 2005) and organisms that colonize such sites must be adapted to these harsh conditions. In contrast, the Schreyahn salt marsh is located in the temperate zone, with generally non-limiting rain supply during the year. The dominating salt is K2CO3, the pH value is only around 8.0, and the different plants serve as indicators of the horizontal salt gradient in the soil.

Conclusions

Approaches based on culturing of bacteria or on sequencing of PCR-products of DNA from small clone libraries yield data of relatively few bacteria of a soil community due to its extreme species richness. In spite of this, the molecular analysis of bacteria occurring at the polluted site resulting potash mining provided information on a lot of new, not yet deposited sequences but also on some bacteria that had been described to occur at far distant locations.

The recently developed TReFID

Acknowledgements

The authors are indebted to Dr M.G. Yates, formerly at the Unit of Nitrogen Fixation in Brighton, UK, for carefully commenting and improving the English. The excellent technical expertise of Mirela Stecki and Karin Otto is also gratefully acknowledged.

References (57)

  • S. Benlloch et al.

    Prokaryotic genetic diversity throughout the salinity gradient of a coastal salar saltern

    Environmental Microbiology

    (2002)
  • J. Borneman et al.

    Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation

    Applied and Environmental Microbiology

    (1997)
  • J. Borneman et al.

    Molecular microbial diversity of an agricultural soil in Wisconsin

    Applied and Environmental Microbiology

    (1996)
  • A. Buchan et al.

    Dynamics of bacterial and fungal communities on decaying salt marsh grass

    Applied and Environmental Microbiology

    (2003)
  • D.H. Buckley et al.

    Exploring the Biodiversity of Soil—A Microbial Rain Forest

  • D.G. Burns et al.

    Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable

    Applied and Environmental Microbiology

    (2004)
  • B.P. Burns et al.

    Microbial diversity of extant stromatolites in the hypersaline marine environment of Shark Bay, Australia

    Environmental Microbiology

    (2004)
  • T. Dalsgaard et al.

    N2 production by the anammox reaction in the anoxic water column of Golfo Dulce, Costa Rica

    Nature

    (2003)
  • M. Demba Diallo et al.

    Phylogenetic analysis of partial bacterial 16S rDNA sequences of tropical grass pasture soil under Acacia tortilis subsp. raddiana in Senegal

    Systematic and Applied Microbiology

    (2004)
  • S.P. Donachie et al.

    The Hawaiian archipelago: a microbial diversity hotspot

    Microbial Ecology

    (2004)
  • J. Dunbar et al.

    Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning

    Applied and Environmental Microbiology

    (1999)
  • J. Dunbar et al.

    Empirical and theoretical bacterial diversity in four Arizona soils

    Applied and Environmental Microbiology

    (2002)
  • H. Ellenberg

    Vegetation Ecology of Central Europe

    (1988)
  • Felsenstein, J., 2004. PHYLIP: Phylogeny Inference...
  • L.A. Francis et al.

    Diversity of amoA genes across environmental gradients in Chesapeake Bay sediments

    Geobiology

    (2003)
  • N. Galtier et al.

    SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny

    Computer Applications in Biosciences

    (1996)
  • H. Haeupler et al.

    Bildatlas der Farn- und Blütenpflanzen Deutschlands

    (2000)
  • K. Horst et al.

    Salzpflanzen und salzliebende Pflanzengesellschaften bei Schreyahn - ein schutzwürdiges Refugium im Hannoverschen Wendland

    Jahresheft des Heimatlichen Arbeitskreises Lüchow-Dannenberg

    (1977)
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