• Freshwater sediment;
  • Microbial community analysis;
  • 16S rRNA;
  • Lipid biomarker;
  • Colorless sulfur bacterium;
  • Holophaga/Acidobacterium group


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

The aim of this review is to interpret recent studies in which molecular methods were used to identify and characterize prokaryotes in lake sediments and related habitats. In the first part studies based on the phylogenetic diversity of prokaryotes found in lacustrine habitats are summarized. The application of various cultivation-independent methods for the characterization of distinct groups of sediment bacteria is exemplified with morphologically conspicuous, colorless sulfur bacteria in the second part of this review. Finally, traditional and recently developed methods are described which could be used for linking the function of microbial populations with their identification. The potential of these approaches for the study of lake sediments is discussed in order to give a perspective for future studies in this habitat.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

Freshwater lakes are important reservoirs of water and food in some regions of the Earth. Nutrient cycles in these environments are largely influenced by metabolic processes localized in the surface layers of sediments. Most freshwater sediments are quite heterogeneous ecosystems which give rise to many different environmental niches even on a millimeter scale. They therefore harbor highly complex microbial communities with regard to species composition and metabolic activity. The interplay between the deposition of organic matter from the overlying water column and decomposition processes leads to the formation of sediment layers which differ in carbon content and available electron acceptors. The complexity of this environment is often increased by mixing of the sediment by currents or the activity of invertebrates.

Initially, most work on the microbiology of sediments was done by measurement of decomposition processes and isolation of microorganisms catalyzing various forms of carbon mineralization. The retrieved sediment bacteria were usually cultured using selective media and their abundance was quantified with dilution-based most-probable-number (MPN) techniques (e.g. [1]). The improvement of traditional microbial methods by the use of fine-scale microsensors and tracer techniques [2] or newly designed selective media [3] enables still new discoveries in this field.

Nevertheless, it has also become evident in recent years that an analysis of the population structure is one prerequisite for understanding microbial processes in aquatic habitats, comparable to ecological studies of higher eukaryotes which were based mainly on population analyses. Prokaryotes are too small for a morphological classification and have to be put in axenic culture before characterization. Depending on the ecosystem only 0.001–15% of the total number of visible cells can be retrieved by isolation [4], a fact which is now often designated ‘the great plate count anomaly’[5]. Hence, traditional microbiological methods are not suitable for the study of the full microbial diversity. Only with the introduction of molecular methods based on sequencing small-subunit ribosomal RNA (SSU rRNA) genes has a more complete analysis of microbial diversity become possible [6]. The retrieval of 16S rRNA genes directly from the environment has made it possible to estimate the phylogenetic diversity of a natural bacterial community without preceding cultivation steps. In addition, the phylogenetic information obtained by applying the rRNA approach to a microbial community may often be helpful in the directed isolation or identification of novel microorganisms from the respective ecosystem. Studies based on a combination of SSU rRNA-based molecular techniques and traditional methods, like the analysis of lipid biomarkers, will be of growing importance in microbial ecology in order to get a clue of the complex interactions in natural microbial ecosystems.

This paper is dedicated to reviewing recent results on the identification and characterization of ecologically significant prokaryotes in freshwater sediments. Microorganisms can be ecologically significant either by their large number in a distinct habitat or because they take part in a key nutrient cycle, such as morphologically conspicuous, colorless sulfur bacteria. Readers interested in a general overview of the diversity of prokaryotes in sediments are referred to the review of Nealson [7]. Compilations of currently used methods for the characterization of microbial communities, on the other hand, can be found in the reviews of Madsen [8], Green and Scow [9], and Theron and Cloete [10].

2Phylogeny-based studies of prokaryotes in freshwater sediments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

2.1Analyses of the phylogenetic composition of microbial communities

Several studies in which a culture-independent phylogenetic approach was compared to a cultivation-dependent analysis of microbial diversity have clearly demonstrated that the culturable fraction of a bacterial population is neither quantitatively nor qualitatively representative of the total microbial community in the respective environment [11–13]. These results exclude the possibility that the fraction of non-cultivable cells is composed of bacteria which cannot be grown in the laboratory because they are dead or temporarily in a metabolically inactive state. In contrast, it seems that during the isolation procedure distinct strains become selected which are usually not typical or numerically dominant in the habitat. Therefore, cultivation-independent population studies are essential to the description of microbial community structures.

An important characteristic of freshwater lake sediments is that they are not spatially separated from adjacent habitats, but are part of complex ecosystems. The composition of microbial communities in freshwater sediments may therefore be largely influenced by interactions with surrounding aquatic and terrestrial habitats. An autochthonous input is possible by the primary production of the overlying water body, whereas the runoff of rivers can contribute an allochthonous input [7]. In this respect it is of great interest to compare the composition of bacterial populations in surface sediments and the overlying water column, in order to reveal what sort of bacteria thriving in freshwater sediments are indeed restricted to this habitat.

A rough estimate of the phylogenetic diversity of microbial communities is feasible by the PCR-assisted cloning and sequencing of SSU rRNA genes. Several studies have used this approach for the analysis of bacterial communities in lacustrine habitats. Although the studied habitats differ widely in their characteristics, e.g. trophic status, some general conclusions can be drawn from the results, which are summarized in Table 1.

Table 1.  Analyses of bacterial populations in various lacustrine habitats by retrieval of 16S rRNA genes
  1. aHA, Holophaga/Acidobacterium; CFB, Cytophaga/Flexibacter/Bacteroides; AB, Actinobacteria; LGC, low-G+C Gram-positives; VE, Verrucomicrobia; PL, Planctomycetes; CY, Cyanobacteria and chloroplasts; ?, unaffiliated sequences and sequences belonging to candidate divisions or divisions less important in freshwater.

  2. bFrequency of sequences affiliated to a phylogenetic group in percent of the total number of analyzed sequences: ◯, 0%; •, 1–9%; •?, 10–29%; •?•, >29%.

  3. cSequences were retrieved from extracted rRNA of the same site.

Ecosystem and referenceNumber of sequencesBacterial classesa
Adirondack Mountain Lakes [14]108•?b•?••?•?
Lake Constance ‘lake snow’[15]69•?•?••?•?
Lake Constance sediment [16]44•?•?••?•?
Priest Pot sediment [17]76•?•?c•?•
Rainbow Bay sediment [18]35•?•?•?•?
Sources of the cloned sequences were epilimnetic water samples [14], aggregates of organic material floating in the water (‘lake snow’) [15], profundal sediment [16,17], and littoral sediment [18].

Sequences affiliated to the β-subclass of Proteobacteria were frequently recovered from freshwater sediments and the overlying water body. In the water column of some freshwater habitats bacteria belonging to this phylogenetic group could be independently identified as a dominant fraction of the microbial community by fluorescence in situ hybridization (FISH) [19]. In contrast, sequences of δ-Proteobacteria were more frequently retrieved from sediments than the water column, indicating that the occurrence of several representatives of this group is restricted to benthic environments. This effect may be due to an oxygen gradient between the usually aerated water body and the anoxic sediment and would therefore be less distinct when meromictic lakes are examined, which are characterized by a permanently anoxic layer of water above the sediment surface [20]. Finally, it has been found that the diversity of bacteria belonging to previously unknown phylogenetic groups containing no or very few cultured species, like the recently defined Holophaga/Acidobacterium lineage [21], is quite high in freshwater sediments.

Although population analyses based on the retrieval and amplification of rRNA genes enable a more comprehensive view of bacterial diversity, the results should be interpreted with caution due to the limitations of the used methods. Several steps of such studies, including extraction of DNA, in vitro amplification and cloning, can cause a bias towards certain phylotypes. Thus, the current PCR-dependent rRNA approach for population studies has some pitfalls, which have been reviewed by von Wintzingerode et al. [22]. In addition, it should be stressed that in most studies the number of analyzed clones is rather low compared to the expected diversity of a clone library derived from a natural environment. Nevertheless, it seems unlikely that major phylogenetic lineages could completely escape molecular detection by the biases of culture-independent methods, whereas cultivation-dependent methods often discriminate or select for certain phylogenetic groups.

Another weak point of PCR-based population studies is the use of DNA as template. It has been argued that genomic DNA of dead cells can be very stable, surviving for long periods of time in the environment. Hence, there have always been attempts to use extracted RNA or intact ribosomes as templates for in vitro amplification [23,24]. It is assumed that the rRNA content of bacterial cells is to some extent correlated with their activity so that clone libraries using rRNA as starting material would give a better impression of the active microbial population by avoiding the background of dead or inactive cells. In one study dealing with freshwater sediment this problem was studied by comparing clone libraries constructed with extracted DNA and RNA, respectively. The extracted rRNA served as a template for reverse transcription in cDNA which then was PCR-amplified and cloned [17]. The results indicate some differences in the two clone libraries, although these are not really significant because the total number of compared clones was too low. Interestingly, sequences affiliated to the recently defined Holophaga/Acidobacterium group were found in the rRNA-based clone library. This clearly demonstrates that bacteria belonging to a diverse and widespread phylogenetic group, which has been mainly defined on 16S rRNA sequence data, participate actively in the microbial processes of this freshwater sediment.

2.2Localization and quantification of distinct phylogenetic groups

The extent of phylogenetic diversity, as revealed by the retrieval of SSU rRNA sequences, says only little about the quantitative composition of a microbial community, even if it may correlate well with the species richness of an ecosystem. An analysis of the population structure is, however, necessary to identify important members and evaluate their role in the microbial community. Approaches currently used to study the abundance of distinct types of microorganisms include the use of specific PCR primers and dot/slot-blot hybridization of extracted RNA. These methods are currently widely used to detect and quantify certain populations of prokaryotes in freshwater sediments. In situ hybridization with fluorescently labeled probes has the additional advantage of showing the spatial distribution of sediment bacteria at a single cell level.

However, in a typical freshwater sediment many of the microorganisms are not detectable by FISH, since the rRNA contents of cells may be below the detection limit or the cell envelopes are impermeable to fluorescently labeled probes. The situation can be different in some nutrient-rich environments with large populations of highly active bacteria. In one study of a marine sediment rich in organic material over 70% of cells could be detected by FISH, allowing a rough estimate of the population structure [25].

In several studies, which will be discussed below, the significance of distinct bacterial populations in freshwater sediments could be studied by using specific PCR primers and/or quantitative slot-blot hybridization of extracted RNA.

2.2.1Sulfate-reducing bacteria

Li et al. [26] extracted rRNA from the profundal sediment of a mesotrophic freshwater lake and hybridized it with probes specific for some major phylogenetic groups of sulfate-reducing bacteria. A positive correlation was found between the relative abundance of extracted SSU rRNA originating from members of the genus Desulfobulbus and the rate of sulfate reduction in this sediment. It was concluded that this genus plays a major role in the sulfur cycle, whereas the genera Desulfovibrio and Desulfobacterium were apparently less active members of the sulfate-reducing population. In a previous study on this sediment no correlation was found between the culture-dependent counting of sulfate-reducing bacteria using the MPN method and the rate of sulfate reduction, indicating a higher sensitivity of the hybridization assay as compared to the culture-based approach.

2.2.2Ammonia-oxidizing bacteria

Some bacteria which occur in freshwater sediments in numbers too low for detection by FISH or quantitative hybridization of extracted RNA can nevertheless play a major role in the cycling of important nutrients due to a high substrate turnover rate. Autotrophic ammonia-oxidizing bacteria, for instance, are part of the nitrogen cycle and largely responsible for the oxidation of ammonia to nitrite, but occur only in low densities in many freshwater environments. For their detection an alternative approach was developed based on the in vitro amplification of environmental DNA with a set of diagnostic PCR primers. In the studies by Hastings et al. [27] and Whitby et al. [28] a nested PCR technique was applied to analyze the distribution and seasonal dynamics of members of the Nitrosospira and Nitrosomonas europaea-N. eutropha lineages in freshwater lakes. In a first step bacterial SSU rRNA genes were amplified from extracted environmental DNA using universal bacterial primers. The resulting PCR products then served as templates for a second amplification step using Nitrosospira- or N. europaea-N. eutropha-specific primers. The fidelity of the resulting amplification products was verified by hybridization to an internal oligonucleotide probe complementary to the SSU rRNA genes of most ammonia oxidizers among the β-subclass of Proteobacteria. In littoral and profundal sediments of a eutrophic lake MPN counts of ammonia oxidizers were in the range of only 100–700 cells g−1 (dry weight), and direct application of the specific PCR primers to extracted DNA yielded no amplification products. In contrast, it was possible to retrieve amplification products by applying the nested PCR approach with Nitrosospira-specific primers, but not with the N. europaea-N. eutropha-specific primers, indicating a higher abundance of Nitrosospira in this eutrophic lake [27]. However, as the same approach was applied to an oligotrophic lake it was possible to amplify also nitrosomonad SSU rRNA genes, but during the summer months only [28]. The N. europaea-N. eutropha 16S rRNA genes obtained were further analyzed and two sequence clusters related to either N. europaea or N. eutropha became apparent. Sequences of the N. europaea cluster were found exclusively at littoral sediment sites, whereas N. eutropha sequences were detected only at profundal sediment sites, leading to the conclusion that representatives of both groups have adapted to different ecological niches.


A combination of the recovery of SSU rRNA sequences and a quantitative hybridization of extracted RNA was applied to determine the distribution of archaea in Lake Michigan sediment [29]. Surprisingly, the results of the quantitative RNA hybridization revealed two major peaks of archaeal rRNA in the oxic and suboxic zones of this environment. From previous culture-dependent studies it would be expected that most archaea in temperate sediments will be represented by anaerobic methanogens, phylogenetically affiliated to the Euryarchaeota. Therefore, the existence of a population of hitherto uncultured archaea adapted to oxic environmental conditions was proposed. To evaluate this hypothesis, archaeal SSU rRNA genes were amplified from oxic zones of the sediment with specific PCR primers. Four of the retrieved sequences could be affiliated to a group of environmental sequences within the archaeal kingdom Crenarchaeota. Sequences related to this group were initially discovered in temperate subsurface ocean waters [30] and later also recovered from other ecosystems, e.g. freshwater lake sediments in the USA [31] and Germany [16]. The detection of crenarchaeotal sequences in moderate or cold environments was unexpected since all cultured representatives of the Crenarchaeota are thermophilic. It could be shown by quantitative hybridization of extracted RNA using a specific oligonucleotide probe that representatives of such mesophilic crenarchaeota are really active in the Lake Michigan sediment. Most of the crenarchaeotal rRNA was found in the oxic zone of the sediment. However, it contributed only to 10% of the archaeal rRNA or 1% of the total extracted SSU rRNA, indicating the presence of additional novel archaea in this sediment.

2.3Enrichment and isolation of typical sediment bacteria based on their phylogenetic identification

Compared to the success of SSU rRNA-based studies in detecting novel lineages of prokaryotes in natural environments only slow progress has been made in the complementary isolation and characterization of representatives of these taxa. The main reason might be that the directed cultivation of distinct microorganisms demands time-consuming and laborious trial-and-error experiments with an unpredictable outcome, which in consequence prevents a rapid accumulation of data in this field. Bearing in mind the large diversity of microorganisms, it is not very likely that in the near future a universal approach will be developed which could significantly increase the total fraction of culturable bacteria. On the other hand, specially adapted techniques or new combinations of methods have to be developed for the isolation of novel bacteria because traditional plate-count and dilution techniques are in most cases not very efficient. The efficiency of a directed isolation of novel prokaryotes from a natural environment can be increased by the phylogenetic screening of pure cultures or enrichments. The potential of these cultivation strategies will, however, still depend largely on the ability to reconstitute the micromilieu required for growth by the target cells.

Several studies are available which describe the isolation of phylogenetically distinct bacteria from natural environments or mixed cultures. Kane et al. [32] were the first who used whole-cell hybridization with fluorescent oligonucleotide probes to monitor the enrichment of target cells and their subsequent isolation as pure cultures. In another study a collection of pure cultures retrieved from activated sludge was screened using oligonucleotide probes to detect strains which had been shown to be abundant in this environment by FISH [33]. Huber et al. [34] used ‘optical tweezers’ for the physical separation and cloning of a phylogenetically identified coccoid archaeon from an enrichment culture dominated by filamentous cells.

Examples of the isolation or enrichment of ecologically relevant bacteria from freshwater sediments using SSU rRNA-based cultivation approaches are presented below.

2.3.1Phylogenetic identification of novel β-Proteobacteria

We used traditional dilution culture techniques to retrieve the most abundant culturable bacteria from littoral and profundal sediments of two large freshwater lakes in southern Germany (Lake Constance and Lake Chiemsee). A phylogenetic screening of these isolates by partial sequencing of 16S rRNA genes revealed that most strains belonged to the β-subclass of Proteobacteria (S. Spring, unpublished results). A phylogenetic comparison of a collection of β-Proteobacteria isolated from sediments of Lake Constance with cloned 16S rRNA sequences retrieved from ‘lake snow’ (suspended organic matter) or profundal sediment from this environment showed no tight correlation in most cases. Nevertheless, most of the isolated strains could be considered novel at least at the species level, having similarity values below 97% to 16S rRNA sequences of known cultured species [35].

One strain, which was obtained from the 10−7 dilution of littoral sediment from Lake Chiemsee, could be phylogenetically affiliated to a cluster of sequences retrieved from ‘lake snow’ of Lake Constance. The phylogenetic position of the newly isolated strain CS-K2 among representatives of the β-Proteobacteria is shown in Fig. 1. The 16S rRNA gene of this isolate has a similarity value of 98.6% with the sequence LST3091 representing a cluster of numerous partial ‘lake snow’ sequences within the β2-group of Proteobacteria [15]. The abundance of sequences belonging to this cluster in clone libraries constructed from ‘lake snow’ varied with the depth of the sample. In a clone library constructed from material collected in a water depth of 15 m, over 20% of the sequences were affiliated to this cluster, whereas in a sample from 25 m depth these sequences were found to represent less than 1% of the obtained clones, which would suggest that the littoral is the preferred habitat of these bacteria. The closest cultured relative of strain CS-K2 was found to be Burkholderia cepacia, but the similarity value of the two 16S rRNA sequences is only 92.7%. Thus, ecological and phylogenetic evidence indicates that strain CS-K2 probably belongs to a new genus of β-Proteobacteria indigenous to certain freshwater habitats.


Figure 1. Phylogenetic tree of the β-subclass of Proteobacteria based on almost complete 16S rRNA sequences. The positions of strain CS-K2, newly isolated from littoral sediment of Lake Chiemsee, and environmental sequences retrieved from ‘lake snow’ aggregates or profundal sediment of Lake Constance are shown. This tree is based on a distance matrix analysis including only positions which are occupied by identical residues in at least 50% of all sequences from this subclass. The sequence of Escherichia coli was used as an outgroup (not shown). The bar indicates 10% estimated sequence divergence.

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In view of these results it appeared worth while to study the phenotypic characteristics of this strain. The data obtained support the phylogenetic analysis and suggest a localization in a novel genus. Ecologically significant traits of strain CS-K2 are the formation of aggregates in liquid culture and the ability to oxidize thiosulfate chemolithoheterotrophically to sulfate (Spring et al., unpublished results).

One might speculate that bacteria belonging to this cluster form part of the microbial population in littoral sediments, which is brought into this habitat together with settling organic material from the overlying water column, as has been previously shown for marine coastal sediments [36]. In summary, the usefulness of phylogenetic screening of environmental isolates for the identification of ecologically relevant bacteria could be clearly demonstrated.

2.3.2Probe-assisted enrichment of members of the Holophaga/Acidobacterium group

Several studies [12,13] and our own results have shown that the phylogenetic screening of newly isolated pure cultures obtained with traditional cultivation techniques will in most cases lead to the identification of representatives of novel species or genera, but only very rarely to the retrieval of microorganisms which represent recently discovered phylogenetic lineages. To achieve the targeted cultivation of bacteria which have no close cultured relative the monitoring of enrichment cultures using whole-cell hybridization with specific oligonucleotide probes offers several advantages compared to the screening of isolates. Firstly, a large number of different artificial media can be screened in a reasonable time for the presence of target cells since laborious isolation procedures are avoided. Secondly, the purification of some bacteria into axenic culture may be impossible due to a dependence on conditions provided by other microorganisms which are also enriched together with the target cells.

We successfully used a probe-assisted enrichment strategy for the cultivation of members of the Holophaga/Acidobacterium group from freshwater lake sediments. We focused on this phylum for several reasons: SSU rRNA sequences of these bacteria are widely distributed and abundant in clone libraries of environmental DNA from various locations. In addition, the phylogenetic depth of this lineage is comparable to that of the Proteobacteria [21], suggesting a similar degree of metabolic versatility. In contrast to the large number of diverse 16S rRNA sequences retrieved from the environment only three representative species are available in pure culture (Holophaga foetida, Geothrix fermentans and Acidobacterium capsulatum), which clearly demonstrates the need for new cultivation strategies to isolate members of this phylogenetic group. One prerequisite for the successful cultivation of phylogenetically identified bacteria is that they are indeed active members of the microbial community and not only present as DNA. Recently, it was possible to detect active cells in ‘lake snow’, freshwater sediment, and soil by applying various cultivation-independent techniques [17,21,37–39].

Whole-cell hybridization and the oligonucleotide probe Irog 1, specific for subcluster A of the Holophaga/Acidobacterium lineage [21], were used to monitor the enrichment of members of this phylogenetic group in artificial media. The screening of a large number of different cultures inoculated with serially diluted freshwater sediment samples led only in a few exceptional cases to the detection of target cells. However, the enrichment efficiency could be significantly increased by employing a mechanical filtration step prior to the inoculation of media, thereby excluding all microorganisms larger than 0.8 μm from the inoculum. Screening various enrichment cultures by whole-cell hybridization enabled a further improvement in the medium composition and hence the development of a successful enrichment strategy which worked reproducibly [16]. Thereupon, positive enrichment cultures could be obtained from sediment samples of Lake Baikal and Lake Constance, which survived several transfers without loss of the target cells. It was now possible to reveal the diversity of enriched representatives of the Holophaga/Acidobacterium phylum by applying the rRNA approach to several different mixed cultures. The 16S rRNA sequences so obtained which could be affiliated to this novel phylum were compared with environmental sequences of this group, allowing the design of more specific probes targeting the enriched cells. Representatives of several distinguishable phylogenetic clusters could be identified in the enrichment cultures by whole-cell hybridization with the newly designed probes. In Fig. 2 an in situ hybridization of a typical enrichment culture obtained from Lake Baikal sediment is shown. Interestingly, bacteria which became enriched along with the target cells were often identified as α-Proteobacteria by hybridization to an oligonucleotide probe specific for this phylogenetic group.


Figure 2. Whole-cell hybridization of an enrichment culture obtained from Lake Baikal sediment. For details of the enrichment procedure see [16]. Scale bar represents 10 μm. Three oligonucleotide probes targeting different phylogenetic groups were used simultaneously. Left: Epifluorescence micrograph. Cells labeled in green bound probe Irog1 specific for subcluster A of the Holophaga/Acidobacterium phylum [21], cells labeled in blue hybridized with a probe specific for the α-subclass of Proteobacteria, and cells hybridizing simultaneously with a probe specific for a distinct phylogenetic group of enriched target cells and probe Irog1 are shown in orange. Colors were generated digitally by a confocal laser scanning microscope. Right: The same field viewed in phase contrast.

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The simultaneous occurrence of representatives of the Holophaga/Acidobacterium group with metabolically active bacteria belonging to the Beijerinckia group of the α-subclass of Proteobacteria has been previously observed in acidic soils from various locations [37–39]. Furthermore, the results of one of these studies indicate the assimilation of methanol by both types of microorganisms, although it could not be completely excluded that metabolic products or intermediates of primary users of this substrate were utilized by tightly associated non-primary users [39]. An assumed interaction of the two types of bacteria in several habitats and enrichment cultures could be explained by an adaptation to the same ecological niche, utilization of similar growth substrates or even syntrophism among representatives of two divergent phylogenetic groups. With regard to the design of future isolation strategies it may be worth while to consider that a tight coupling of the metabolism of distinct α-Proteobacteria and members of the Holophaga/Acidobacterium group could prevent the isolation of the latter into pure culture.

3Cultivation-independent studies on morphologically conspicuous sulfur bacteria

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

The cells of most microorganisms indigenous to freshwater sediments are very small, having an average size of around 0.5 μm [40]. The majority of these bacteria can be reliably detected and counted only after staining with 4′,6-diamidino-2-phenylindole or other nucleic acid stains. Exceptions are several colorless sulfur bacteria, which are common in many sediments and display a range of conspicuous morphotypes, including filamentous forms, calcite-containing ovoid cells (Achromatium oxaliferum) and magnetotactic bacteria.

Large bacterial filaments, which are occasionally found in certain freshwater sediments, in most cases represent colorless sulfur bacteria belonging to the genera Thiothrix, Thioploca or Beggiatoa. These microorganisms are most abundant in sulfide-rich marine sediments or activated sludge but are ecologically less significant in natural freshwater habitats. Studies on pure cultures or enrichments of these bacteria have revealed that most strains are mixotrophic or lithoautotrophic sulfide oxidizers. Beggiatoa and Thiothrix have adapted to a life in overlapping gradients of sulfide and oxygen or nitrate, whereas marine Thioploca spp. are presumed to shuttle between spatially separated pools of sulfide and oxygen or nitrate [41].

In contrast to the filamentous sulfur bacteria, A. oxaliferum still resists all isolation attempts. Moreover, only very few isolates of magnetotactic bacteria have successfully been grown as pure cultures. With the advent of culture-independent methods it has become possible to study these groups of bacteria in more detail. Recent results, which are discussed below, indicate that Achromatium spp. and magnetotactic bacteria may play an important role in the sulfur biogeochemistry of freshwater sediments similar to the more thoroughly studied filamentous sulfide-oxidizing bacteria in marine sediments.

3.1Magnetotactic bacteria

Magnetotactic bacteria are frequently found in freshwater or marine sediments. They are most abundant in the oxic–anoxic transition zone (OATZ) of sediments but have also been detected in stratified water columns or soil. Due to the synthesis of intracellular crystals consisting of magnetic iron minerals (magnetosomes) they are able to align and swim along magnetic field lines. The presence of large sulfur inclusions could be demonstrated in most but not all representatives of magnetotactic bacteria.

Magnetotactic bacteria represent a diverse group with respect to phylogeny, morphology and physiology [42]. Common features are a Gram-negative cell wall structure, motility by means of flagella, and intracellular crystals of magnetic iron oxide or iron sulfide minerals. They prefer microoxic or anoxic niches in the environment showing a negative tactic and/or growth response to atmospheric concentrations of oxygen [43]. The only available and validly described pure cultures of magnetotactic bacteria belong to the genus Magnetospirillum within the α-subclass of Proteobacteria [44]. Representatives of this genus apparently do not play a major role in most studied habitats and have a chemoorganotrophic metabolism. In contrast, coccoid magnetotactic bacteria and several other morphotypes not available in pure culture were found to be locally abundant in the OATZ of various freshwater sediments [45–47]. Much of the phylogenetic and morphologic diversity within this group of microorganisms could be revealed by applying the rRNA approach to samples of magnetically collected bacteria from natural environments. To date magnetotactic prokaryotes have been affiliated to two major bacterial lineages, the Proteobacteria and the newly defined Nitrospira phylum. Among the Proteobacteria representatives are found within the á- and ä-subclasses [42]. Most cells of magnetotactic bacteria directly retrieved from freshwater sediments are characterized by large sulfur inclusions, but up to now no representative could be put into pure culture. However, the isolation of a marine magnetotactic coccus was reported that contained sulfur globules when grown in artificial sulfide–oxygen gradients [48]. Studies of this strain demonstrated for the first time the ability of magnetotactic bacteria to grow chemolithoautotrophically with sulfide or thiosulfate as electron and energy source. In addition, it was possible to analyze the magnetotactic response of a sulfide-oxidizing magnetotactic bacterium under reproducible conditions in the laboratory [49]. It turned out that the mechanism of magnetotaxis may be more complicated than originally thought and implies two different redox states in these cells. According to this hypothesis an oxygen concentration lower than that preferred results in a reduced state in the cell, whereas a higher than optimal oxygen concentration leads to an oxidized state. The internal redox state may be sensed by the NADH/NAD ratio in the cell, as was proposed for Escherichia coli[50]. In the oxidized state cells swim parallel to the magnetic field (i.e. northward in the northern hemisphere). When cells switch to an alternative reduced state, they reverse the flagellar rotation and swim antiparallel to the magnetic field lines (i.e. southward in the northern hemisphere). Due to the inclination of the magnetic field lines cells are directed downwards when they are in the oxidized state and upwards when they are in the reduced state, thereby enabling them to shuttle between different redox layers. This tactic behavior may therefore be referred to as a magnetically guided redoxtaxis. It differs fundamentally from the aerotactic response of microaerophilic bacteria, which use a temporal sensory mechanism to determine changing oxygen concentrations during swimming. The magnetically guided redoxtaxis, in contrast, offers the advantage of quickly finding favorable microniches in the environment without the need for continuous orientation to chemical gradients which are often very heterogeneous in sediments. One may speculate that magnetotaxis has evolved in some bacteria to enable them to utilize efficiently the separated pools of reduced sulfur species in deep layers and oxygen in upper layers of their habitat, and that this is comparable to the vertical migration of certain Thioploca spp. in sheaths [51].

This model would fit especially well for an unusual, large magnetotactic rod, frequently found in the sediment of some freshwater lakes. This as yet non-cultivable magnetotactic bacterium is phylogenetically affiliated to the Nitrospira phylum and was given candidatus status due to its distinctive phenotypic traits [46]. Cells of Candidatus Magnetobacterium bavaricum have dimensions of 1–1.5×6–9 μm, and are characterized by large sulfur inclusions and up to 1000 hook-shaped magnetosomes consisting of magnetite (Fig. 3A,B). The number of magnetosomes observed in this morphotype is much higher than in most other magnetotactic bacteria and exceeds the number necessary for efficient orientation in the Earth's magnetic field. Consequently, it was speculated that the stored iron may play a role in the physiology of this organism [46]. In order to study the abundance and distribution of Candidatus M. bavaricum littoral sediments of a freshwater lake in Southern Germany (Lake Chiemsee) were transported to the laboratory and stored in aquaria. The highest abundance was found at the OATZ of the sediment and was determined to be up to 7×105 live cells cm−3. The total cell number in this sediment layer was found to be 1.1×108±0.4×108 cells cm−3. Due to the large size of this bacterium (average volume ca. 25.8±4.1 μm3) it was estimated that it could account for approximately 30% of the microbial biovolume in this layer of the sediment. Significant numbers were also found in the anoxic regions of the habitat, but not in the oxic layers. The immersion of microscopic slides into the sediment allowed a study of the distribution of the Candidatus M. bavaricum in situ by epifluorescence microscopy using a specific oligonucleotide probe complementary to a discriminative region of its 16S rRNA. Although the use of glass slides in sediments has some limitations, because they represent only an artificial support, it could be demonstrated that these bacteria apparently attach to sites which were located at the OATZ, where they form microcolony-like structures (Fig. 3C,D). Around the microcolonies, precipitates of iron oxide minerals and sheaths of filamentous bacteria resembling members of the genus Leptothrix in appearance were visible. A novel Leptothrix strain (L. mobilis) was in fact subsequently isolated from the sediment slides [52]. However, a positive effect of L. mobilis on the growth of Candidatus M. bavaricum in artificial gradients could not be obtained.


Figure 3. Morphology of Candidatus M. bavaricum and its spatial distribution in a freshwater sediment. A: Phase contrast micrograph of live cells showing yellowish inclusions of sulfur. B: Electron micrograph of Candidatus M. bavaricum displaying hundreds of magnetosomes arranged in several chains oriented parallel to the long axis of the cell (courtesy of N. Petersen). Scale bars represent 10 μm in panel A and 1 μm in panel B. C, D: In situ detection of Candidatus M. bavaricum on microscope slides grown over with sediment bacteria. C: Phase contrast micrograph. D: Same field viewed with epifluorescence microscopy using a specific fluorescently labeled oligonucleotide probe. Scale bar represents 100 μm.

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The large amount of stored sulfur and iron suggests the hypothesis that Candidatus M. bavaricum may gain energy from the anaerobic reduction of Fe(III) with sulfide or other substrates as electron donor [46]. The sulfur so formed would be stored and could be efficiently oxidized with oxygen in the upper sediment layers. In addition, oxygen may be necessary for the precipitation of magnetite required for magnetosome formation [43]. In the studied Lake Chiemsee sediment the sulfide concentration was very low (<10 μmol) and due to a rapid chemical oxidation in the upper layers sulfide is probably only available in deep anoxic zones of the sediment. Therefore, a magnetically guided redoxtaxis may help this bacterium to migrate easily over long distances in order to find sites rich in sulfide in the deeper layers of the sediment or sites which are microoxic in the upper layers. In order to avoid the waste of energy by constant movement along gradients, cells would attach to preferred microniches until they reach an unfavorable internal redox state that triggers a magnetotactic response either parallel or antiparallel to the geomagnetic field lines. This hypothetical model is presented in Fig. 4.


Figure 4. Proposed model of magnetically guided redoxtaxis in Candidatus M. bavaricum inhabiting freshwater sediments of the northern hemisphere. Cells are guided along the geomagnetic field lines depending on their internal redox state either downward to the reduced zone or upward to the microoxic zone, enabling a shuttling between separate pools of sulfide or other reduced substrates and oxygen. Cells are supposed to attach to solid phases when they have reached a balanced redox state. High levels of oxygen would switch cells immediately to an oxidized state provoking the north-seeking motility typical of magnetotactic bacteria viewed under the microscope in the northern hemisphere.

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3.2Achromatium spp.

A. oxaliferum represents a morphotype common in the surface layers of certain freshwater or brackish sediments. This bacterium contains characteristic intracellular calcite and sulfur inclusions and belongs to the largest known prokaryotes, reaching more than 80 μm in length and 20 μm in width [53].

For a long time it was not clear whether microorganisms resembling the morphological description given for A. oxaliferum do indeed belong to a single species. The application of a full cycle rRNA approach to natural communities of Achromatium revealed phylogenetically heterogeneous populations of this currently non-cultivable bacterium which have been assigned to a deep branch of the γ-subclass of Proteobacteria [54–56]. In one study divergent sequences retrieved from various habitats could be correlated to morphologically distinct organisms by whole-cell in situ hybridization with fluorescently labeled oligonucleotide probes, suggesting the existence of diverse species adapted to different ecological niches [56].

The occurrence of Achromatium in freshwater habitats seems to be restricted to organically rich littoral sediments, characterized by an oxic top layer and low sulfate reduction rates in the range of 10–300 nmol cm−3 day−1[57]. However, the environmental factors which favor the dominance of Achromatium populations over other morphologically conspicuous sulfur bacteria are not exactly known. The distribution and abundance of Achromatium in the sediment of a freshwater lake was analyzed and correlated with chemical gradients by Gray et al. [53]. The highest density was found with around 106 cells ml−1 in the microoxic zone of this environment. Low numbers of cells also occurred in the anoxic layers of the sediment. Although in terms of cell numbers A. oxaliferum constituted only around 1% of the bacterial population, they constituted 90% or more of the bacterial biovolume in the microoxic zone due to their extremely large size. The results of this study further indicated that these organisms can react to changing chemical gradients in the sediment by vertical migration and are able to tolerate anoxic conditions for at least short periods of time.

Since the discovery of A. oxaliferum it was supposed that this organism may gain energy from the oxidation of reduced sulfur species which would lead to the accumulation of large sulfur globules typical for cells of this bacterium. Recently, ecophysiological evidence for the first time allowed a verification of the proposed metabolic type for at least some representatives of the Achromatium morphotype [53]. It was found that the pore water sulfate concentration was correlated with the frequency of Achromatium cells, both reaching a maximum in the microoxic zone of sediment cores. In sediment samples containing active Achromatium cells, which were treated with sodium molybdate to inhibit the bacterial reduction of sulfate, the accumulation of sulfate was observed. The amount of produced sulfate increased linearly with the time of incubation and the number of added cells. In addition, the uptake of reduced sulfur species by Achromatium could be shown using microautoradiography with 35S-labeled sulfate [58]. The authors assumed that the labeled sulfate was converted by sulfate-reducing bacteria to sulfide which was subsequently taken up and detected by microautoradiography in Achromatium cells. The major fraction of sulfide oxidized was obviously intermediately stored as sulfur within these cells. The same technique was applied to study the uptake of organic substrates or bicarbonate labeled with 14C. It turned out that the substrate spectrum of cells in natural communities of Achromatium spp. differed considerably. Most cells took up labeled acetate or protein hydrolysate, but not glucose. The potential of autotrophy demonstrated by the utilization of bicarbonate differed in natural populations of Achromatium from two different sites. In one pond around 50% of the cells used bicarbonate for carbon assimilation whereas this ability was not detectable in cells from another pond, indicating the existence of physiologically diverse species of Achromatium adapted to different habitats. It was concluded that natural communities of Achromatium may consist, on the one hand, of chemolithoautotrophic or mixotrophic species and, on the other hand, of chemoorganoheterotrophic or chemolithoheterotrophic species. One way to prove this assumption would be the combination of microautoradiography with FISH using species-specific probes as described later in this review.

In contrast to the stored sulfur, the function of the calcite inclusions still remains unclear. Although there is no direct evidence, it is tempting to speculate that the specific weight of this organism is controlled by the amount of intracellular calcite, thereby facilitating a positioning in the sediment column. Some early observations may support this hypothesis. Lauterborn [59] noted that cells rich in sulfur had stored low amounts of calcite whereas cells containing less sulfur were densely packed with calcite. In another early report it was noted that cells suffering from oxygen deficiency are almost free from calcite [60]. These observations could be interpreted in such a way that cells increase their specific weight by accumulating calcite in order to be guided to deeper layers of the sediment for taking up sulfide, whereas cells in need of oxygen for sulfur oxidation would reduce the amount of calcite to increase their buoyancy in order to reach the oxic top layer of the sediment.

Thus, Achromatium spp. would use gravity, sulfur-oxidizing magnetotactic bacteria the magnetic field and Thioploca spp. vertical migration by gliding in order to shuttle between different redox zones. It has been suggested that the large diversity of colorless sulfur bacteria may have evolved as an adaptation to increasing levels of atmospheric oxygen in the history of the Earth which forced sulfide oxidizers to overcome the problem of their being spatially separated pools of electron donors and acceptors [41].

4Linking the function of microbial populations with their identification

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

Apart from a few exceptions, the phylogenetic identification of microbial populations using cultivation-independent methods does in most cases not provide any information about their possible role in metabolic processes in the respective environment. Hence, it is of great interest to couple the taxonomic identification of currently non-cultivable microorganisms with an analysis of their function in the environment. Numerous molecular techniques have therefore been developed to estimate the activity of microorganisms in their natural environment in order to assess their ecological significance. Some of these methods have been tested only on a few selected habitats, so that not much is known about their practical use for the study of freshwater sediments. However, the introduction of such approaches will probably lead to a better understanding of the microbial ecology of many ecosystems, including freshwater sediments.

4.1Analyses of microbial communities based on lipid biomarkers

Membrane lipids and their associated fatty acids are essential components of every living cell. Among these, a wide variety of glycerolipids, either ester- or ether-linked, is present in bacteria, archaea and eukaryotes. Phospholipid fatty acids (PLFA) are assumed to be suitable measures of viable microbial biomass in sediments [9,61]. Upon cell death, hydrolysis of their phosphodiester linkage by exogenous and endogenous phospholipases is rapid. Hence, the ratio of diglyceride fatty acids to phospholipid fatty acids provides an estimate of the proportion of non-viable to viable bacterial biomass [62]. Employing the appropriate conversion factors (average bacteria and archaea contain 100 μmol PLFA (g dry weight)−1[63]), concentrations of PLFA in natural environments can be used to estimate the abundances of certain groups of microorganisms. It is important to note, however, that the values given for conversion factors vary between 11 and 197 μmol PLFA (g dry weight)−1[64]. This wide range may partly be due to the various methods which have been used for the calculation of conversion factors. The validity of such calculations was recently discussed by Green and Scow [9].

4.1.1Distribution of lipid biomarkers among prokaryotes

Since PLFA are structurally diverse and exhibit a relatively high degree of biological specificity, they are employed as biomarkers for the identification of well studied groups of microorganisms (Table 2). Whereas some of the PLFA occur in all bacteria tested (e.g. palmitate, C16:0), a few PLFA may serve as specific biomarkers for selected groups of bacteria (e.g. 10me18:0 for Actinomycetes).

Table 2.  Compilation of signature lipids for bacteria and their distribution
Saturated FA16:0Bacteria, Eukaryotes[65]
Polyenoic FA18:2ω6cFungi, algae, protozoa[67]
 18:3ω3cMicroeukaryotes, fungi, marine algae[68]
 18:3, 20:3, 20:4Mycorrhizae[69]
 20:5ω3Psychrophilic Shewanella spp.[70]
 20:3ω6, 20:4ω6, 20:5ω3Microeukaryotes, diatoms[68]
 Polyunsaturated >C20Eukaryotes[66]
 22:6ω3Colwellia psychrerythraea[72]
Monoenoic FA16:1ω7c, 16:1ω7t, 16:1ω5c, 17:1ω6, 17:1ω9, 18:1ω9c, 18:1ω7c, 18:1ω7t, cy17:0, cy19:0Primarily Gram-negative bacteria[73]
 16:1ω7, 18:1ω7 predominantThio-oxidizing bacteria[74]
 16:1ω7, 18:1ω7, 18:1ω9Eukaryotes[66,75]
 16:1ω8c, 16:1ω6c, 16:1ω5t, 16:1ω5c predominantType I methanotrophic bacteria (e.g. Methylomonas, Methylococcus)[76]
 16:1ω13tPhotosystem I[68]
 15:1ω6, 17:1ω6Desulfobulbus spp.[77]
 18:1ω8c predominantType II methanotrophic bacteria (e.g. Methylosinus, Methylocystis)[76]
Terminally branched FAi14:0, i15:0, a15:0, i16:0, i17:0, a17:0Arthrobacter spp. and other Gram-positive bacteria, Gram-negative sulfate-reducing bacteria (e.g. Desulfovibrio spp.)[68]
  Cytophaga, Flavobacterium[64]
Mid-chain branched FA10me16:0Desulfobacter spp.[78]
 10me16:0, 10me18:0Actinomycetes[79]
Branched monoenoic FAi17:1ω7cDesulfovibrio spp.[80]
  Syntrophobacter wolinii, Syntrophobacter pfennigii[81]
Hydroxy FAUsually 3-OHPrimarily Gram-negative bacteria[66]
Mycolic acidβ-OH, α-branchedMycobacterium, Nocardia[82]
Isoprenoid glycerol etherCaldarchaeolHyperthermophilic archaea, few methanogens[83]
 ArchaeolMethanogens, extreme halophiles[83]
 SitosterolHigher plants[63]
HopanoidsBacteriohopanetetrol, aminobacteriohopanetriolBacteria[86]
BenzoquinonesUbiquinones, coenzyme QAerobic Gram-negative bacteria[87]
NaphthoquinonesMenaquinones, dimethylmenaquinoneAerobic Gram-positive bacteria, anaerobic Gram-negative bacteria, extreme halophiles[87]
Nomenclature: ‘:’, number of unsaturations; ‘ω’, distance of first unsaturation from methyl end; ‘c’ or ‘t’, cis or trans geometric isomers of unsaturation; ‘i’ or ‘a’, iso or antiiso branching; ‘cy’, cyclopropyl moiety; ‘me’, position of the methyl group.

In addition to glycerolipids, eukaryotic cell membranes contain sterols which regulate membrane fluidity. The sterol analogues in bacteria are hopanoids, although three steroid-synthesizing species of bacteria are known (Methylococcus capsulatus, Nannocystis exedens, Polyangium sp.). Hopanoids occur in about 30% of all tested bacterial species, but do not seem to occur in archaea. Their occurrence within the bacteria is rather scattered and erratic [86] and thus appears not to be suitable for taxonomic investigations. However, the presence of derivatives of bacterial hopanoids in geological sediments and crude oil has implications for the origin of old organic carbon [86].

4.1.2Lipid biomarkers as tools for profiling active microbial communities

Besides their use in the taxonomy of isolated microorganisms in pure culture, profiling of fatty acids can provide information on the overall structure of active microbial communities and the biomass of certain groups of microorganisms. Therefore, analysis of fatty acid profiles has been advocated as a rapid and inexpensive alternative for describing complex microbial communities. Complete PLFA patterns have been used as a fingerprint for the comparison of different microbial communities [63,66,81] or to follow changes in the composition of a single community stimulated by variations of environmental conditions [68]. Macalady et al. [88] used this approach to analyze the sediment microbial community structure in a eutrophic freshwater lake polluted with mercury. They found that the microbial community structure was strongly related to mercury methylation potential, sediment organic carbon content, and lake location. In contrast, inorganic mercury concentration appeared to have no effect on community structure. It has to be noted, however, that changes of PLFA patterns may not only indicate different compositions of microbial communities, but may also reflect a change of the overall physiological state of a microbial community. The effects of elevated temperatures or physiological stress on the PLFA composition of bacteria are well known and have been analyzed in several laboratory studies [89–92], but in most cases it is not known to what extent these effects alter PLFA profiles under natural conditions.

Ratios of some functional groups of fatty acids have been used to deduce the occurrence of subgroups of bacteria. Typical indicators comprise the polyenoic PLFA (eukaryotes), monoenoic PLFA (mostly Gram-negative bacteria), or terminally branched saturated PLFA (Gram-positive bacteria and anaerobic Gram-negative bacteria) [93]. The highest level of resolution is achieved by analyzing specific fatty acids that act as biomarkers of certain functional groups or species of microorganisms (e.g. methanotrophs).

In the study of Macalady et al. [88] the specific analysis of lipid biomarkers characteristic of sulfate-reducing bacteria indicated that representatives of the Desulfobacter group were highly active and abundant at a site with high mercury methylation potential, leading to the conclusion that members of this group could be important mercury methylators in this sediment.

It is evident from Table 2 that most lipid biomarkers occur in microorganisms which belong to different taxonomic and phylogenetic groups; the 16:1ω8c and 18:1ω8c biomarkers of methanotrophic bacteria or hydroxyarchaeol of methanogenic archaea representing notable exceptions [94–96]. Because of this low specificity, proportions of the different lipid biomarkers are mostly used to elucidate the composition of microbial assemblages [63], which renders PLFA analysis of entire microbial communities rather complex. This is demonstrated by the fact that the mid-chain branched fatty acid 10me16:0 can serve as a biomarker for Desulfobacter spp. only if not accompanied by 10me18:0 (which is found in Actinomycetes) (Table 2).

Additionally, phylogenetically closely related species may differ in their PLFA profiles as a result of adaptation, as exemplified under laboratory conditions by the presence or absence of eicosapentaenoic acid (20:5ω3) in Shewanella spp. [70]. These observations point to a more serious complication, namely the fact that information on the occurrence of individual markers in defined groups of microorganisms can only be derived from the analysis of isolated pure cultures. Consequently, the PLFA approach is not really culture-independent as is often assumed. In conclusion, the analysis of the taxonomic composition of microbial communities must be viewed with caution until the knowledge of the quantitative and qualitative distribution of lipid biomarkers among microorganisms and their variability within the same strain is more extensive. Obviously, the PLFA approach is not suitable to distinguish most of the individual bacterial species in complex communities. In fact, a recent comparison of PLFA profiles with 16S rRNA dot-blot hybridizations for the detection of sulfate reducers and Syntrophobacter sp. in granular sludge demonstrated that the PLFA analysis alone cannot be very accurate, at least in this habitat [81]. Compared to the 16S rRNA approach, the analysis of lipid biomarkers provides rapid and efficient information on biodiversity of the active microbial community, albeit at a lower resolution and restricted to groups of previously studied isolates.

4.1.3Isotope labeling of lipid biomarkers

PLFAs are not only suitable for the analysis of community composition, but have also been used as indicators of substrate usage of a few defined groups of bacteria.

One approach is based on the measurement of the stable carbon isotope abundance in signature lipid biomarkers by isotope ratio mass spectrometry coupled to gas chromatography by a combustion interface [97]. Whereas stable isotope ratios have been used extensively to study organic carbon sources of animals, this approach has been used only recently to infer the carbon sources of microorganisms and to trace carbon flow in sediments [98].

The biomass of heterotrophic microorganisms has a stable carbon isotope ratio similar to their growth substrate. Generally, lipids are depleted in 13C (by 3–6‰) as compared to total bacterial biomass due to the discrimination of carbon isotopes by pyruvate decarboxylase during the oxidation of pyruvate to acetyl coenzyme A [99]. In order to elucidate carbon sources of microbial growth in natural samples, the isotopic fractionation between substrate and PLFA must be known and be constant. For most microorganisms investigated, the isotope ratios in fatty acids seem to be independent of the growth stage of the cells, with methane oxidizers representing a notable exception to this rule [97,100]. The δ13C pattern is not uniform for the different fatty acids of a given microbial species. Tetradecanoic acid (C14:0) is generally depleted in 13C compared to palmitic acid (C16:0) while octadecanoic acid (C18:0) is enriched. Also, the glycolipid fractions are often depleted in 13C as compared to the phospholipid fractions. Depending on the type of growth substrate, the same fatty acid may be depleted in 13C for some species, but enriched for others [97].

Consequently, the investigation of bacterial substrate usage appears most promising only for those bacterial groups or species which form unique lipids or fatty acids (Table 2).

One approach takes advantage of the fact that biologically produced CH4 is strongly depleted in 13C in contrast to other carbon sources enabling the identification of prokaryotes utilizing CH4 by a significant depletion of signature lipids in 13C. An investigation of archaea in sediments from a methane seep involved δ13C analysis of archaeol and 2-hydroxyarchaeol, and provided evidence for anaerobic methane consumption, rather than methane production by a group of prokaryotes probably related to methanogenic archaea as revealed by a complementary 16S rRNA analysis [94].

Another strategy involves the identification of lipid biomarkers by the addition of 13C- or 14C-labeled substrates. This offers the advantage of using more complex organic substrates like acetate or xenobiotic compounds.

Based on the δ13C value of their specific PLFA biomarkers, it could be shown that the Gram-positive Desulfotomaculum acetoxidans (and not the Gram-negative Desulfobacter spp.) oxidizes acetate in estuarine and brackish sediments, and that type I methanotrophic bacteria dominate methane oxidation in a freshwater sediment [101].

In a second approach, PLFAs serve as indicators of substrate usage after radiolabeling of selected bacteria. The incorporation of 14C-labeled substrates into microbial lipids generates fingerprints of metabolically active microorganisms and, depending on the specificity of the PLFA markers used, may provide information about the type of microorganisms that metabolize organic substrates in complex microbial communities [102]. As for the 13C studies mentioned above, the radiolabeling technique has mainly been applied to the study of methane-oxidizing bacteria. Thus, 14CH4 addition to agricultural soil was mainly assimilated in C16 fatty acids and thus by type I methanotrophs. Although type II methanotrophs were detected in the same soil by PLFA analysis, they did not seem to assimilate a significant fraction of the added CH4. Conversely, and in another study, analysis of radiolabeled PLFA biomarkers indicated that a new sort of type II methanotroph was responsible for methane oxidation in various forest soils [95,96]. These results were supported by the detection of a new, phylogenetically distinct methane monooxygenase gene sequence which clustered with the type II methanotrophs. Potentially, the radiolabeling of group-specific lipids thus provides more insight into the physiologically active fraction of microbial communities.

4.2Stable-isotope probing and separation of chromosomal DNA

A labeling of the chromosomal DNA by stable-isotope-labeled substrates offers the advantage of isolating an entire copy of the genome of metabolically active microorganisms, which allows not only the phylogenetic identification by SSU rRNA analysis but also the detection and cloning of functional genes. In a recent report the application of this approach for the analysis of a methanol-utilizing microbial community in forest soil was described [39]. The utilization of 13CH3OH by microorganisms leads to the synthesis of 13C-labeled DNA which could be purified from unlabeled DNA due to a higher buoyant density which could be resolved by density gradient centrifugation. Subsequent phylogenetic analyses of the obtained fraction of ‘heavy’ chromosomal DNA by PCR-assisted sequence retrieval allowed the identification of two phylogenetic groups apparently responsible for the methanol consumption in this habitat. Interestingly, besides bacteria belonging to the α-subclass of Proteobacteria members of the Holophaga/Acidobacterium group could be identified. Thus, the authors concluded that non-cultivable representatives of this newly defined phylum can perform the same function as bacteria from other well established phylogenetic lineages. A limitation of this technique could be the long incubation periods required to label cells of slow-growing bacteria, which are expected to dominate the microbial population in complex habitats like soil or sediments. Hence, metabolic intermediates or products synthesized by the primary users of the labeled substrate may lead to an incorporation of the 13C label by non-primary, accompanying or syntrophic organisms. Secondly, prolonged incubations with high concentrations of a labeled substrate can lead to a shift in the population structure, biasing the obtained results. Thirdly, the application of such an approach will be restricted to a small number of suitable substrates.

4.3Combination of microautoradiography and FISH

The use of radiolabeled substrates in combination with microautoradiography has been applied in many ecological studies to measure activities of microalgae, autotrophic and heterotrophic prokaryotes [103], however, its major limitation has been its inability to correlate the detected activity with the corresponding organism. This problem was recently overcome by combining it with whole-cell hybridization using fluorescently labeled oligonucleotide probes [104]. The combination of these two methods enables a microscopic in situ analysis of both the identity and the specific substrate uptake profile of different individual bacterial groups or cells within complex microbial communities under different incubation conditions. The information obtained from this method can provide further valuable hints for the development of new isolation strategies of hitherto unculturable bacteria and, in combination with the whole rRNA approach, of more specific gene probes. It has to be mentioned that the method is semiquantitative and can only address those ecological questions for which suitable radiolabeled substrates and/or gene probes are available.

Possible applications of this method for the study of freshwater sediments could include the in situ analysis of the metabolic capabilities of sediment bacteria enriched in artificial media or of conspicuous uncultured bacteria easily separable from the sediment.

4.4Reverse transcriptase PCR (RT-PCR) of mRNA

Another promising strategy for revealing the activity of distinct microorganisms in their habitat is the specific amplification and characterization of mRNA transcripts extracted from the environment. This approach is based entirely on the characterization of nucleic acids and offers the advantage that no labeled substrates have to be introduced into the environment avoiding the inherent limitations of such procedures like long incubation periods and growth on metabolic intermediates. Suitable targets for RT-PCR include transcripts of genes encoding enzymes with a key function in bacterial metabolism (e.g. ammonia monooxygenase [105] or dissimilatory sulfite reductase [106]) or involved in the catabolic degradation of xenobiotic compounds (e.g. naphthalene dioxygenase [107]). The usefulness of RT-PCR for the measurement of microbial activity is based on the high turnover rates of mRNA transcripts in cells. However, it has to be noted that the transcription of DNA into mRNA does not under all circumstances result in the expression of a functional protein. The sequence diversity of genes which encode homologous proteins in different taxonomic groups can be used for an affiliation of the retrieved transcripts to distinct phylogenetic groups.

A protocol based on RT-PCR was recently applied to detect and characterize naphthalene dioxygenase mRNA transcripts in groundwater contaminated with coal tar waste [107]. In this study diverse dioxygenase transcripts could be retrieved from indigenous groundwater bacteria thereby documenting the in situ expression of these genes by a variety of microorganisms. The majority of dioxygenase sequences were found to be most similar to homologous genes of either Burkholderia or Pseudomonas strains.

Critical steps inherent to this method are the extraction of mRNA in a quantity and of a quality which is adequate for RT-PCR and the availability of suitable primer sets to perform amplification of stretches of the targeted transcripts which are of sufficient length and variability. In addition, horizontal transfer of genes encoding functional proteins is rather common in prokaryotes and often prevents the reliable affiliation of a distinct sequence to a certain phylogenetic assemblage.

5Conclusions and outlook

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

Due to the limitations of traditional culture-dependent methods the use of molecular techniques has become of growing importance for the study of microbial communities in various ecosystems including freshwater lake sediments. Above all, the rRNA approach was successfully applied to reveal the existence of several novel lineages of hitherto unknown prokaryotes in freshwater sediments leading to a broadening of our view on microbial diversity in this habitat. It has to be stressed, however, that cultivation-independent, PCR-based methods also have inherent biases preventing a reliable assessment of the structure of bacterial populations which may in some cases lead to a misinterpretation of the abundance of certain phylogenetic groups. Such pitfalls may be avoided by hybridizing whole cells or extracted rRNA from the studied habitat with specific oligonucleotide probes in order to verify the initial results. Furthermore, the retrieval of a novel 16S rRNA sequence reveals very little about the phenotypic traits of the respective organism and its metabolic activity. Only when the retrieved sequence can be clearly affiliated to a monophyletic lineage characterized by a common phenotypic trait, e.g. ammonia oxidation or sulfate reduction, can some conclusions be drawn about the function of the corresponding microorganism. In most cases, however, the simple knowledge of phylogenetic diversity in an environment helps very little in understanding the interacting metabolic processes and the factors which control them. Nevertheless, a molecular approach can help in the identification of sediment bacteria which are ecologically relevant because of their high abundance or activity. These microorganisms can then be the subject of detailed studies or a target of directed cultivation experiments.

Cultivation may not even be necessary for the analysis of the function of certain phylogenetically identified bacteria if they are morphologically distinctive and can be easily separated from the bulk of sediment bacteria, as has been demonstrated with Achromatium spp.

However, the majority of prokaryotes living in natural environments are rather inconspicuous so that several molecular techniques were developed in order to overcome the lack of information about the function of bacteria identified by cultivation-independent methods. Despite the progress which has been made in linking the identification of distinct microorganisms with their functions in situ, it will still be necessary to isolate or enrich novel bacteria to reveal their metabolic potential under various environmental conditions.

The results presented in this review should demonstrate that experimental strategies based on the combination of molecular techniques with traditional cultivation-dependent methods have great potential in revealing some of the hidden complexity of natural microbial ecosystems.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References

We are indebted to the DFG for continuous support of our work during the last six years by Grants Schl 120/10-2, -4, and -5. The model of the magnetotactic behavior of Candidatus Magnetobacterium bavaricum presented here was significantly improved by critical comments of D.A. Bazylinski, Ames, IA, USA.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Phylogeny-based studies of prokaryotes in freshwater sediments
  5. 3Cultivation-independent studies on morphologically conspicuous sulfur bacteria
  6. 4Linking the function of microbial populations with their identification
  7. 5Conclusions and outlook
  8. Acknowledgements
  9. References
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