One important topic of aquatic microbial ecology has been the physiological properties of total microbial assemblages, in particular of the metabolic and respiratory processes that drive biogeochemical transformations (13, 53, 142). In contrast, traditional microbiological research is mainly centered on the physiological properties of bacterial strains in pure culture (109, 127). In between these two levels of analysis there is a conspicuous gap of information. Little is known about the growth-related characteristics of single microbial populations that are realized at environmental conditions in the presence of competitors, predators, viruses, substrate heterogeneity, chemical gradients etc. For example, the genome of the marine planctomycte Rhodopirellula baltica strain 1 contains >100 different sulfatases (90), but it is unclear which ecological advantage is associated with this feature (it might, e.g., reflect in situ growth on a complex mix of substrates such as sulfated polysaccharides). In order to understand the stability or fluctuations of a particular biogeochemical process, it would be important to distinguish if it is mediated by a single, physiologically highly versatile microbial population, or if it is carried out by several bacterial groups that may provide a greater functional redundancy (318).
With the exception of stable isotope analysis (236) or pulse-labeling of nucleic acids (302), physiological information is typically lost by methods that use DNA or rRNA extracts for microbial identification. By contrast, physiological properties can be readily related to a particular genotype at the single cell level. FISH by itself may already provide some information about the physiological state of a population, because the signal intensity of hybridization is proportional to cellular ribosome content. The ribosome content of marine isolates tends to increase with growth rate (138). However, rRNA concentration is a parameter that may sometimes be difficult to interpret. Some marine bacteria may maintain high numbers of ribosomes even during periods of prolonged starvation (68). This is probably essential to rapidly respond to changes in growth conditions in a patchy environment (212). Thus, ribosome content may allow a limited assessment of bacterioplankton “activity” at the community level (52, 80), but it needs to be interpreted with caution if single populations are to be compared. In addition, cell identification by FISH can be combined with a number of other methods that visualize particular physiological properties of individual cells, e.g., substrate uptake, DNA synthesis, respiration (47, 196, 213), and even with stable isotope probing (198).
Microautoradiography.
One of the most powerful approaches to study physiological activities in mixed microbial assemblages dates back to the 1960s. The uptake of radiolabeled tracer substrates into individual cells can be visualized by a photographic technique termed microautoradiography (30, 31) (Fig. 6a). In combination with cell identification by FISH, this approach allows us to assess the partitioning of substrate consumption between different microbial populations in mixed assemblages. Microautoradiography-FISH was first used to determine organic and inorganic substrate uptake of ammonia-oxidizing bacteria and of other groups in activated sludge (154), but has since then been adapted for microbes in lakes and in the marine water column (4, 47, 98, 169, 199, 296, 306). Recently, protocols have been developed that integrate the superior CARD-FISH staining with microautoradiography for the analysis of bacterial substrate uptake in environments such as the mesopelagic zone (4, 296). It has even been attempted to add a quantitative aspect to microautoradiography by estimating the amount of incorporated radiolabel from the number of grains that are formed around active cells (46, 168, 170).
Without wanting to diminish the potential of microautoradiography, one should be aware of some limitations. Currently, there exist a variety of more or less time-consuming protocols for microautoradiography-FISH of water column bacteria, and some protocols likely cause a high loss of bacterial cells (47, 199). Moreover, it appears rather difficult to standardize some aspects of the procedure (the exposure time and photographic development) to an extent that would meaningfully allow us to quantitatively compare results from different studies. Finally, the choice of adequate tracer substrates may not be trivial. Comparatively little is known about the composition of the dissolved organic matter pool and about the turnover and the concentrations of specific organic carbon compounds in the water column. Sometimes the environmentally relevant substrates are not commercially available and have to be laboriously custom synthesized, e.g., chitin and proteins (47), or the marine algal osmolyte dimethylsulfonopropionate (DMSP) (306). In freshwater systems such as bog lakes much of the dissolved organic matter consists of a complex mix of high molecular weight substances (e.g., humic acids) (299). In these habitats it might be difficult to decide on appropriate model substrates.
Fluorescent activity tracers and flow sorting
In order to study particular aspects of growth in individual microbial populations there are technically less demanding alternatives to microautoradiography. Pelagic bacteria with an active electron transport system of the respiratory chain reduce tetrazolium salts to water-insoluble crystals (322). Such formazan grains are deposited intracellularly, and they can be detected both microscopically (247) and by flow cytometry (54). In combination with FISH cell respiration can thus be visualized in single microbial populations, as was shown for filamentous bacteria from activated sludge (196). However, the tetrazolium reduction method appears to be a rather insensitive means to distinguish between growing and non-growing (or dead) bacteria in the plankton. Accordingly, bacteria with visible formazan deposits are regarded as the most highly active fraction within a larger set of growing cells (268). While there may be good reasons to identify such highly active bacterial populations, no investigation has combined tetrazolium reduction and FISH to study microbes in the water column of natural aquatic systems.
Bromodeoxyuridine is a halogenated nucleotide analogue of thymidine that is incorporated into newly synthesized DNA of bacteria and eukaryotes (213, 245, 302). It provides a non-radioactive alternative to microautoradiography with tritiated thymidine (207) and it allows us to quantify growth rates at the single-cell level (116). Bromodeoxyuridine incorporation has been combined with CARD-FISH to visualize de novo DNA synthesis in different freshwater and marine bacterial populations (209, 213, 310) (Fig. 6b). This offers a sensitive means to detect changes of growth rates in single microbial populations in situ. During bottle incubations of filtered seawater, a rise in the numbers of bromodeoxyuridine incorporating Alteromonas sp. cells clearly preceded cell multiplication (213). Significant seasonal differences and short-term variability of growth rates were observed in members of the Roseobacter spp. and NOR5 lineages from coastal North Sea picoplankton (209). High bromodeoxyuridine incorporation by actinobacteria in mountain lakes indicated that these bacteria were not passively introduced from soils, but autochthonously growing members of the bacterioplankton community (310).
Flow cytometry may provide an alternative means of detecting activity or substrate uptake in single microbial cells. It allows the physical sorting of particular bacterial populations of interest for further analyses (22, 72). So far, microbial cells from plankton samples have been mainly sorted by phenotypic features, e.g., cell volume or cellular DNA or protein content (72, 150, 267, 326). Sorted bacteria have been analyzed by molecular methods (21, 323) and for radiotracer incorporation (151, 327). In contrast to microautoradiography, the tracer uptake rates of specific cell populations can be readily quantified by this approach.
Direct sorting of microbial cells after FISH staining has first been shown in highly productive wastewater treatment systems (284). Recently this approach has also been adapted for bacterial cells from coastal marine bacterioplankton, taking advantage of the superior signal intensities of CARD-FISH staining (262). Such a combination of cell identification and flow sorting potentially offers the ability to quantitatively investigate substrate uptake of single populations in natural samples (327). Moreover, it might eventually provide a means of obtaining functional genes or larger genome fragments from phylogenetically coherent groups of microbes directly sorted from environmental samples.
Distribution and Dynamics of Different Populations
For unknown reasons, heterotrophic aquatic microbes form large populations in particular habitats or at particular seasons, and are rare at other locations or time points (35, 63, 136, 144, 218, 280). One challenge of population ecology is to explain the observed distribution patterns of different bacterial taxa from their physiological properties and from their interactions with other organisms. Admittedly this may appear a rather far-fetched goal for a discipline that has just started to understand which microbes are frequent in different aquatic environments. However, the ability of macroecology to understand the role of individual plant and animal species is to a large extent based on an understanding of their distribution patterns and population dynamics at various environmental conditions. So far, only a few studies have investigated abundance changes of particular microbial taxa in the water column in a context of physicochemical parameters or food webs (63, 132, 136, 147, 223, 278).
For the purpose of gaining ecological insight from spatial or temporal distribution patterns, binary information about the presence or absence of a particular bacterial group, as provided by cloning or fingerprinting techniques, is probably insufficient. Such approaches allow us to detect fundamental differences between communities, e.g., between marine and freshwater habitats (180), along rivers (264), or in mesocosm (244, 253), but they can hardly distinguish if a set of environmental variables is more or less favorable for a specific population. A too-coarse division of aquatic microbial assemblages, e.g., into subphyla of Proteobacteria by FISH with the respective probes (171), also suffers from drawbacks, since different populations with potentially contrasting dynamics might be put into ecologically meaningless categories (Fig. 2). Such studies may contribute to our understanding of large biogeographic divisions, e.g., between marine and freshwater habitats (89), and of basic discontinuities in the composition of microbial assemblages, e.g., along estuaries (28). However, both the qualitative community analyses by fingerprinting and investigations on large taxonomic units by FISH should be regarded as intermediate steps towards the quantitative study of ecologically coherent and phylogenetically more tightly-defined populations.
Experimental Enrichments in the Field
For decades, aquatic microbial ecologists have complemented descriptive studies on the distribution of microbes in various habitats with a range of simple field experiments. These have been referred to as “bottles, bags and buckets” (203), or more respectfully, as micro- to mesocosms, limnocorrals, enclosures, etc. Typically, various volumes of water are taken directly from the environment studied and this water is incubated at more or less in situ conditions after manipulation of, e.g., substrates and nutrients (61, 152, 177, 202), or of particular functional groups of the food web (16, 133, 293, 317). Depending on the container size, the duration of such experiments ranges from days to weeks. Short-term incubations share a basic logic with tracer uptake experiments: a measurable response to a manipulation should allow a deduction about the original state of the assemblage or some of its members. Sometimes dialysis bags with defined pore sizes may provide a more advanced alternative to bottles (80, 172, 278). Such bags allow readier exchange of dissolved substances if exposed directly in the water column. Even so, some features of the original environment are probably irreversibly destroyed, in particular the assumed continuum between the dissolved and the particulate organic matter (12). Larger containers may provide a useful means to artificially induce blooms of specific phytoplankton groups (244) or to manipulate metazooplankton densities (129, 223).
Such investigations are often plagued by the mysterious “bottle effect,” a hard-to-define concept that reflects the worry of whether phenomena observed in confined assemblages are nonspecific consequences of the confinement rather than a result of the planned manipulation. Nevertheless, experimental mesocosms are among the few tools available to microbial ecologists that go beyond a purely descriptive analysis of aquatic microbial assemblages. A number of well-defined hypotheses about microbial populations have been successfully addressed by such approaches (16, 61, 132, 223, 244, 278, 293).
Defined Laboratory Communities and Pure Culture Studies
Experimental systems such as the Winogradsky column (316) have fundamentally shaped our understanding of environmental microbiology. However, concomitant with the rise of cultivation-independent methods, laboratory investigations on experimental communities have somewhat suffered from a lack of popularity among microbial ecologists. This may have been in parts a consequence of Thomas Brock's harsh words about studies on “mixed cultures of unknown provenance … at some ill-defined state” (32). It may also be related to the realization that many laboratory investigations have been performed on microorganisms that are readily culturable (112, 212) but that are rare in the water column (62, 312). These drawbacks may no longer apply. For one, an increasing number of aquatic microbes have been isolated during the past years that also form large populations in situ (63, 110, 238) and that thus represent adequate model organisms for laboratory investigations (27, 109). Second, 16S rRNA-based molecular tools now provide new means to precisely follow the population dynamics of different bacteria in mixed experimental assemblages (16, 221). An increasing wealth of genomic information from isolated environmental bacteria may eventually even allow us to link the performances of individual members of such model assemblages to the expression of particular genes in the context of a well-defined experimental setup.
Thus, laboratory investigations on bacterial isolates or model communities add another important dimension to the understanding of microbial population ecology. For example, particular physical structures within aquatic habitats cannot be preserved in experimental approaches in the field. Laboratory systems can artificially produce aggregated organic matter in natural water samples (101) and trap single flocs in a three-dimensional flux field (230). Such designs provide the adequate physical and chemical environment to study microbial activities and successions on organic aggregates and particles (100-102).
Laboratory studies may also provide a better control over parameters that might mask the hypothesized relationship between the studied population and the variable of interest. Continuous-cultivation systems, not necessarily chemostats (126), stabilize the composition of mixed assemblages by enforcing a minimal growth rate and by providing constant temperature, illumination, and input of substrates and nutrients. This allows us to experimentally sustain transient ecological phenomena over prolonged periods of time, e.g., the rise of particular predator populations (112, 221, 232, 234, 250, 279), bacterial-viral interactions (181), or the breakdown of cyanobacterial blooms (304).
Investigations on pure cultures offer the possibility to distinguish between phenotypic and physiological properties of different aquatic bacteria, e.g., in their interactions with algae (29, 99, 146), their chemotactic responses (15, 23), motility patterns (140), grazing sensitivity (27, 114), or cell filamentation (115, 273). This provides the opportunity to test hypotheses about the ecological relevance of such features.
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