Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem

Internat. Rev. Hydrobiol. 93 2008 4–5 606–623

DOI: 10.1002/iroh.200711044

ROLAND PSENNER*, 1, ALBIN ALFREIDER1 and ASTRID SCHWARZ2

1Universität Innsbruck, Institut für Ökologie, Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria; e-mail: [email protected] 2TU Darmstadt, Institut für Philosophie, Schloss, D-64283 Darmstadt, Germany

Review Paper Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem. What’s Next?

key words: philosophy and history of science, bacteria, ecological concepts

Abstract

Aquatic microbial ecology aims at nothing less than explaining the world from “ecological scratch”. It develops theories, concepts and models about the small and invisible living world that is at the bottom of every macroscopic aquatic system. In this paper we propose to look at the development of Aquatic Microbial Ecology as a reiteration of classical (eukaryotic) limnology and oceanography. This was conceptualized moving historically from the so-called water desert to microcosm to ecosystem. Each of these concepts characterizes a particular historical field of knowledge that embraces also practices and theories about living beings in aquatic environments. Concerning the question of “who is there”, however, Aquatic Microbial Ecology historically developed in reverse order. Repetition, reiteration and replication notwithstanding, Aquatic Microbial Ecology has contributed new ideas, theories and methods to the whole field of ecology as well as to microbiology. The disciplining of Aquatic Microbial Ecology happened in the larger field of plankton biology, and it is still attached to this biological domain, even conceiving of itself very self-consciously as a discipline of its own. Today, Aquatic Microbial Ecology as a discipline is much broader than plankton ecology ever was, for it includes not only oceans and freshwaters but also benthic, interstitial and groundwater systems. The success of Aquatic Microbial Ecology is expressed by its influence on other fields in ecology. The challenge is to further develop its theoretical and methodological features while at the same time contributing to current pressing problems such as climate change or the management of global water resources.

And then it may not be fanciful to suppose that even in the year nineteen hundred and nineteen a great number of minds are still only partially lit up by the cold light of knowledge. It is the most capri- cious illuminant. They are still apt to ruminate, without an overpowering bias to the truth, whether a kingfisher’s body shows which way the wind blows; whether an ostrich digests iron; whether owls and ravens herald ill-fortune; and the spilling of salt bad luck; what the tingling of ears forebodes, and even to toy pleasantly with more curious speculations as to the joints of elephants and the politics of storks, which came within the province of the more fertile and better-informed brain of the author (1919) VIRGINIA WOOLF from the essay “Reading”, In: LEONARD WOOLF (ed.), 1950: The Captain’s Death Bed and Other Essays, – London: Hogarth Press, p. 157.

1. Introduction

In the April 2005 issue of Aquatic Microbial Ecology, the editors DOLAN et al. asked whether the most cited articles during the first decade of this journal (1995–2005) presented

* Corresponding author

“... evidence for gradualism or punctuated equilibrium?” Their answer was quite clear: although the use of molecular tools was evident, especially for describing the presence of taxonomically distinct populations, these new instruments had not (yet) been adapted to quantify fluxes or to study the control of fluxes. For DOLAN and coauthors, the questions formulated by POMEROY in 1974, regarding “ … the identity of metabolically active microorganisms and their role in the food web”, were still not resolved. Consequently they proposed that changes in the field might better be described by the term “gradualism” rather than “punctuation”. However already in 1961, A. E. KRISS had said as much by stating that “The next endeavor [of marine microbiology] … must be the study of the dynamics of microbial populations regarding seasonal zooplankton migration and phyto plankton blooms. Thus microbiological surveys must be intensified by all available means …” otherwise we would never be able to tackle oceanographic problems and, eventually, make use of the observed processes (KRISS, 1961). So have we been replicating the same questions in aquatic microbial ecology for the last 50 years? And is aquatic microbial ecology nothing else than plankton biology under the worst possible conditions – to paraphrase an old saying about the relationship between ecology and physiology? To discuss these questions in a broader context we turned towards the history and philosophy of ecology or, more specifically, to the epistemology of aquatic ecology. We propose to compare the body of knowledge of limnology and oceanography of the late 19th through the first half of the 20th century with current knowledge in aquatic microbial ecology. As a discipline, Aquatic Microbial Ecology did not start until 1983 with the often-cited paper of FAROOQ AZAM and coworkers (AZAM et al., 1983), who emphasized the ecological role of microbes in the water-column and coined the concept “microbial loop” which he later transformed into “microbial food web”. This article had an extremely high impact on subsequent research in the field: to date it has been cited 1800 times since its publication and has won the JOHN MARTIN Award of the American Society of Limnology and Oceanography that was presented to FAROOQ AZAM and co-authors at the ASLO meeting in Victoria, Brit- ish Columbia, in June 2006.1 The subject “aquatic microbial ecology” has come of age, as shown in Figure 1, but it seems to be more appealing than ever. In the following we want to pursue the dynamics as well as the stabilizing factors of the field and we propose to discuss the following two hypothesis: H1) Aquatic microbial ecology is based on a well-established concept regarding the fundamental importance of bacteria in biogeochemical cycles and – consequently – on global climate2. H2) Microbial ecologists have only recently begun to discover the actors in the game, the species and communities. The order of development was thus inverse to that of limnology and oceanography in general, where (i) the species and communities had first been described, (ii) then they were recognized to inhabit and create particular places and (iii) finally their ecological significance was studied. We think that looking back into the past of scientific enterprises such as aquatic microbial ecology helps us also to illuminate present problems. And if theories and methods of the history and philosophy of science contribute to clarify scientific reasoning by referring to the past, we might well expect that history and philosophy of science have a say on future developments in the field of aquatic microbial ecology. However, we cannot develop this in depth, because it would require a much more detailed analysis of the case studies presented here concerning the relation between theory and practice, instruments and methods,

1 The JOHN MARTIN Award was given for the first time in 2006 (Oceanography and Limnology Bulletin 15: 29). 2 The authors of The new science of metagenomics: Revealing the secrets of our microbial planet put it bluntly: “Microbes run the world. It’s that simple.” (http://www.nap.edu/catalog/11902.html).

400

300

200 Number of Articles

100

0 1960 1970 1980 1990 2000 Year

Figure 1. Increase of scientific articles dealing with aquatic microbial ecology from 1960 to 2006. Source: ISI Web of Science (Science Citation Index Expanded including 5,900 major journals). Search criteria: Boolean Search (titles only) based on the following key words: (aquatic OR freshwater OR limnic OR lake OR lakes OR river OR rivers OR pond OR ponds OR marine OR oceanic OR sea OR groundwater) AND (microbial OR microorganisms OR microbes OR bacteria OR bacterial OR archaea OR archaeal OR prokaryotes OR prokaryotic OR viruses OR protists OR flagellates OR ciliates).

institutions and publishing habits. Despite these shortcomings we hope to show that looking back into the past might reveal important, perhaps even crucial aspects of present scientific practice and reasoning.

2. Plankton Ecology

The theoretical background of our considerations is presented in the book “Water Desert, Microcosm, Ecosystem. A History of the „Conquest“ of Aquatic Space” (SCHWARZ, 2003) where the author describes the history of aquatic ecology from its very beginnings. It starts with the argument that the depth of lakes and oceans was perceived as a desert, which meant at the time lifeless and hostile to any human activity. Accordingly, EDWARD FORBES (1815–1854) considered the ocean bottom below 540 m depth as “azoic” (FORBES, 1844). Things began to change when biologist GEORGES AIMÉ BONPLAND (1773–1858), involved in the technological project of installing and checking deep-sea cables, realized to his surprise that the benthic fauna in front of the Algerian coast extended to a depth of 1800 m. These first spatial classifications were further differentiated by LOUIS AGASSIZ (1807–1873), a Swiss naturalist who emigrated to the US and worked at Harvard University. He thought that the distribution of alpine flora could serve as an analogy to describe the depth structure of the marine fauna. Later on CHRISTIAN EHRENBERG (1795–1876) and THOMAS H. HUXLEY (1825–1895) helped to convince the skeptics that life is present in all parts of the ocean. Dredging of large animals from the deep sea in the second half of the 19th century soon convinced the scientific community that there was no lifeless zone at the bottom of the sea. However, it was much more difficult to study planktonic life forms, especially the

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem 609 microscopic ones. Hence the concept of the water desert persisted but it shifted now to the pelagic zone. A more recent analogue to the “azoic” depths and water “deserts” would be, for instance, the deep subsurface, Antarctic ice, but also cloud droplets – all of them are sites that are now considered to be active microbial habitats. Life is, thus, extending in both directions, i.e. into the atmosphere, even into outer space, and towards the inner spaces of Earth. The concept of life is so successful that the “azoic” regions seem to shrink with each new sampling effort. These findings of active communities in ice, snow and the deep subsurface (PSENNER et al., 2003; AMEND and TESKE, 2005) have a crucial significance for a limnologist (RP) who, in his first lecture, gave numbers such as “97% of the water on Earth is oceanic, and 99% of all freshwater is stored in ice and in the underground”. From this perspective, limnology had neglected the two largest freshwater reservoirs, and it was microbial ecology that provided new contributions to the study of the ignored aquatic realms. This diagnosis gives rise to the question, why today’s omnipresence of life in hostile spaces such as ice and snow couldn’t be perceived for such a long time compared to the “vitalization” of so many terrestrial places – even deserts. And what might have been the reasons for this blindness? As we have already noted, it was not only ice, but also plain water, that was considered too diluted for life and therefore considered an authentic desert. New insight came from JOSEPH DALTON HOOKER (1817–1911) who coined the expression “invisible vegetation” when he stated that in the southern polar ocean diatoms keep the balance between fauna and flora, implying a kind of nutrient cycling. Already one century earlier, DAVID HÜMLIN had observed lake blooms but considered them not as the result of organisms living in the lake, but material blown in by wind or developing on the ground of the lake, that was actually considered to be situated “below” the lake. Finally it was the surroundings and the tributaries that gave life to the lake, for instance by connecting it to the sea. Thus, from the 18th century on there existed geomorphological and hydro- lo gical descriptions of rivers, of limited regions of the Sea and also of lakes3. But it was not until about 1870 that the lake was widely recognized as being inhabited by a plethora of planktonic and benthic organisms – as a microcosm. Other conceptualizations of the lake at the time were offered, making the lake an “organism”, an “island” or a “geographic individual”. One of the most influential conceptualizations was proposed by the Swiss naturalist FRANÇOIS-ALPHONSE FOREL (1841–1912). It became crucial for turning limnology into a scientific discipline (1886) as well as for the construction of a theoretical framework of a systemic understanding of the lake. Forel depicted the lake as a laboratory model for large-scale phenomena happening “in the immensity of the ocean” and therefore not accessible to scientific analysis: „It is much easier to study a lake than an ocean“ (FOREL, 1896). FOREL paved the way for the consideration of the lake as an experimental system, that is, to look at the lake as a system in which organisms interact, certain functions are processed and an exchange and cycling of material happens (FOREL, 1896). To get an idea of discussions at the time we may recall the dispute, 100 years later, about free-living ciliates: do we follow Finlay’s (FINLAY, 2002) concept of ubiquity (if the environment fits, we will find certain organisms almost everywhere in the world), or should we follow the concept of biogeography proposed by FOISSNER (2006): allopatric populations in distant “islands” will soon develop specific traits, but we need sophisticated methods to distinguish them from another? At the turn of the 19th to the 20th century, taxonomy and data

3 A neat hydrological description of Lac Leman was already provided in 1779 by the famous Swiss naturalist HORACE-BÉNEDICT DE SAUSSURE (1799), and of Lake Constance by DAVID HÜMLIN in 1783. Rivers were also object to geomorphological and hydrological measurements at the time (SCHWARZ 2003, pp. 116). Several expeditions were launched at that time by the colonial nations to take stock of the depth of the oceans but also for the discovery of other scientific and economic data and resources, for instance the British and French ships Racehorse, Carcass and Boussole, Astrolabe.

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com 610 R. PSENNER et al. collection were at the centre of interest, followed by more systematic plans and projects to detect seasonal differences in the appearance of organisms and the reasons thereof. By around 1900 the concept of the water desert had changed and assumed an image of richness. But what was the decisive step from a lake that is conceptualized as a “microcosm” (with explicitly reference to the tradition of natural history) to the more functional understanding of the lake as a “ecosystem”? According to SCHWARZ (2003) the implemen- tation of three conceptual templates she called niche, microcosm and energy, was decisive for the establishment of ecology as a scientific discipline. Concepts and theories about population, competition, the transport of nutrients and organic matter were developed and models designed to simulate for instance productivity (phytoplankton) and predation (zooplankton) in lakes and oceans. Thereby “bacteria” were treated as an entity fulfilling the role

Table 1. Summary of the three basic concepts (SCHWARZ, 2003) applied to aquatic microbial ecology. The associated research style revolves around the concept of niche, microcosm and ecosystem.

Water desert Microcosm Ecosystem

Bacteria are decom- Bacteria are active under in Bacteria are an important part of the food posers, they recycle situ conditions and take up web, reshuffling DOC into the grazer inorganic nutrients substrates at the nanomolar food web; they may, thus, be seen as (N and P) from detri- level: radiolabelled substances “primary producers” at the base of the tus and dissolved can be used to measure bac- food chain: dissolved organic matter (also material terial activity under in situ from terrestrial vegetation) has only one conditions way back to the food chain, i.e., via bacteria; bacteriophages or viruses may “dis- sipate” ~ 20% of bacterial production Bacteria occur at low Bacteria are not only decom- Uptake rates of certain bacterial strains densities (as demon- posers of organic matter but or groups by protists can be measured strated by plate counts, also food for micro zoo- by epifluorescence microscopy and FISH MPN etc.) plankton methods; some grazers can feed effi- ciently down to ~ 106 ml–1 of free-living bacteria Bacteria have high Bacteria can be distinguished Bacteria can be resistant to grazing by numbers if observed phylogenetically by their 16S morphological and other responses where- with fluorescent dyes rRNA genes – it is not neces- by specific taxonomic groups behave in the microscope, sary to cultivate them in order differently but … to do taxonomy … most bacteria are Major groups of bacterial Most bacteria (even those with thick cell inactive or dormant, cells can be identified in their walls and low numbers of ribosomes) can thus … natural environment by Fluo- be identified in their natural environment rescent In situ Hybridisation by CARD-FISH at the genus, species or at the group level without population level. cultivation: they can be followed in space and time … 99% of all bacteria An increasing number of Bacteria can be identified in situ and cannot be cultured bacteria from natural environ- their individual overall activity can be and, consequently, not ments is characterized by full measured at the same time (MAR-FISH, be identified, for they genome sequencing SIMS-FISH); specific activities (gene do grow only if sub- expression by mRNA detection) of single strate concentrations cells can be measured at the same time are high (millimolar)

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem 611 of decomposers and mineralizers, a role described already in 1872 by FERDINAND COHN4 who argued that plant and animal life would come to an end without the action of bacterial decomposers. Plankton ecology models, for instance that developed by the Plankton Ecology

Figure 2. Visualization of the aquatic (pelagic and benthic) food web in Cedar Bog Lake by LINDE- MAN (1942).

4 Die gesammte Naturordnung ist darauf gegründet, daß die Körper, in denen das Leben erloschen, der Auflösung anheimfallen, damit ihre Stoffe wieder neuem Leben dienstbar werden können. Denn die Masse des Stoffes, welche sich zu lebenden Wesen gestalten kann, ist auf der Erde beschränkt; immer die nämlichen Stofftheilchen müssen in ewigem Kreislauf von einem abgestorbenen in einen lebenden Körper übergehen; ist auch die Seelenwanderung eine Mythe so ist die Stoffwanderung eine naturwissenschaftliche Thatsache. Gäbe es aber keine Bacterien, so würden die in einer Generation der Thiere und Pflanzen verkörperten Stoffe auch nach deren Ableben gebunden bleiben, wie es die chemischen Verbindungen in den Felsgesteinen sind; neues Leben könnte sich nicht entwickeln, weil es ihm an Körperstoff fehlen müßte. Indem die Bacterien in rascher Fäulniß jeden abgestorbenen Leib zu Erde werden lassen, machen sie allein das Hervorsprießen neuen Lebens, und damit die Fortdauer der lebendigen Schöpfung möglich.“

Group, PEG (SOMMER et al., 1986), worked perfectly without bacteria and bacterivores until the 1970s when limnologists began to recognize the “other” role of bacteria, i.e., their function as producer of biomass at the base of the microbial food web. It took another decade to realize the presence of viruses and some more years to quantify their impact on bacteria (BERGH et al., 1989).

3. Aquatic Microbial Ecology

Why did it take so long to recognize the role of bacteria in aquatic environments? We did not see them. The mesh sizes of our nets were too coarse, cultivation methods too selective, microscopes too shortsighted, dyes to faint, and physiological methods too far from natural conditions. While net fishing worked for cladocerans, algae and certain protozoa, it does not work for small flagellates, bacteria and viruses. This explanation is not different from that given e.g., for the identification of the “nanoplankton” in 19195. On the other hand, there was already in the 19th century some interesting activity regarding microbes in aquatic systems, especially in the applied field of drinking water and waste water management but also in a more theoretical setting: It is again FOREL who pointed to the importance of microorganisms as “antagonists of the animal kingdom concerning respira- tion” in a lake (1874: 143). The increasing importance assigned to “bacteria” in the early 20th century was mirrored by lively and controversial discussions that coalesced around a visual depiction of the circulation of matter. The “bacteria-box” moved from a marginal position to the centre and was finally filled with “OOZE and bacteria” (Fig. 2) in LINDEMAN (1942) where terrestrial soil, water and ooze served as model for the lacustrian “ooze”. Thus, the attention devoted to bacteria in aquatic systems had substantially changed. A more detailed historical and conceptual analysis of this important shift is still missing.

LIGHT CO2 NH4

D i s Phytoplankton DOC s o l v Autolysis products e N, P compounds d Bacteria

P a r Ciliates t i c u Cladocera l Rotifers a t e Uptake Release Fish

Figure 3. Early visionary representation of a pelagic microbial food web from around 1973. Dis- solved … use dissolved nutrients and organic matter; particulate … use particulate organic matter. Redrawn and translated from PSENNER (1976)

5 The term “Teichnanoplankton” was coined by EINAR NAUMANN denoting organisms of a size between 5 and 10 μm, today they cover a range between 2 and 20 μm.

One of the earliest figures showing a microbial food web was conceived in 1973 in a Ph.D. thesis (PSENNER, 1976). Here we present the drawing in English translation (Fig. 3.). How blind were we 30 years ago? No so-called “heterotrophic nanoflagellates” (only ciliates), no viruses (but autolysis), no “autotrophic picoplankton” (just phyto plankton) etc. Bacteria, however, were definitely described as link rather than as sink and as an integral component of the food web, substantiated by in situ experiments where bacterivores (with the focus on ciliates) were excluded. In retrospect we may regard it as anticipation of the coming age of microbial food web studies, but at that time the appropriate methods to prove the depicted interactions between bacteria, protists and metazooplankton were rather primitive. In contrast to microbial ecology that emerged in the late 1970s, aquatic microbiology began earlier, i.e., with the shift from “hygienic” methods (plate counts) in the 1960ies (BUCK and CLEVERDON, 1960) to the direct observation of bacteria. The possibility of direct observation was already explored by KUSNETZOW and KARZINKIN (1931) and by BERE (1933). It was boosted – more than one generation later – by the use of fluorescent staining of DNA and RNA (LEHNER and NOWAK, 1957; BELL and DUTKA, 1972; ZIMMERMANN and MEYER-REIL, 1974; PORTER and FEIG, 1980) and the application of membrane filters (review by KARL, 2007). Already in 1965 RODINA published a textbook of methods in aquatic micro biology, and around this time appeared the first papers about heterotrophic activity of pelagic bacteria (KUSNETZOW and ROMANENKO, 1966; WRIGHT and HOBBIE, 1966; OVERBECK, 1970). This activity was studied by measuring uptake rates of labeled organic compounds, such as glucose. The dispute whether bacterial growth rates and biomass production can reliably be measured by the use of radiolabeled nucleotides or amino acids, generally thymidine and leucine (FUHRMAN and AZAM, 1980; KIRCHMAN et al., 1985), has still not been settled but there is a general agreement that the bulk activity of heterotrophic bacteria can be estimated by the addition of biomolecules labeled with tritium and 14C isotopes in “natural”, i.e., nanomolar concentrations. Although the first successful attempts to grow bacteria in lake water were undertaken in the 1960ies (MELCHIORRI-SANTOLINI and CAFARELLI, 1967) we still live with the dogma that only about 1% of all aquatic micro organisms can be grown in pure cultures in the lab. Recently, however, several groups tried to overcome these obsta- cles, for instance by isolating single cells with flow cytometry (ZENGLER et al., 2002) or by way of cultivation in diffusion chambers (BOLLMANN et al., 2007). There have been several revivals and adaptations of plating methods (for instance by the group of OVERBECK in Plön during the 1970ies) to study microorganisms living in dilute suspensions, but the great shift occurred in 1986 (PACE et al.) with the introduction of rRNA sequence analysis to determine the species composition within complex microbial communities and ten years later with the publication of a FISH (fluorescent in situ hybridization) protocol for the quantitative detection and identification of planktonic bacteria (GLÖCKNER et al., 1996; ALFREIDER et al., 1996) that was further improved by PERNTHALER et al. (2007). It is based on the fact that ribosomal RNA genes in bacteria show a tremendous diversity and specificity for covering different taxonomical levels. Bacterial cells contain large numbers of ribosomes: so, if a specific seg- ment of the ribosomal RNA can be targeted with even a single fluorescent molecule, the corresponding cell becomes visible in a microscope or a flow cytometer. The worldwide application of this method showed that not only bacteria but also Archaea (WOESE and FOX, 1977) play a pivotal role in oceans (KARNER et al., 2001) and freshwaters (GLÖCKNER et al., 1999). The detection that one liter of water may contain an order of magnitude more viruses than bacteria, i.e., around 10 billion (BERGH et al., 1989), added to the growing knowledge of microbial food webs. With the FISH method, researchers felt that they became independent of isolation and plating methods, the backbone of classical microbiology which is today regarded as a selection rather than as detection method. Scientists were now able to start from a genetic profile of the microbial populations present in the water, design a general or specific rRNA probe and target the organisms without any previous cultivation step. It marked the beginning of a new

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com 614 R. PSENNER et al. era in microbial ecology, i.e., the chance to follow the wax and wane of pelagic microbes trough time (PERNTHALER et al., 1998) more or less the same way as previous ecologists studied phytoplankton and zooplankton species, affording a resolution and reliability that matched those of classical eukaryotic plankton studies. So what had been regarded as the major impediment of bacterial studies, i.e., their small size and morphological similarity, turned out to be the greatest advantage for the application of phylogenetic methods. Com- bining FISH with other analytical tools, such as the uptake of radiolabeled amino acids (MAR-FISH, LEE et al., 1999; STAR-FISH, OUVERNEY and FUHRMAN, 1999) or the analysis of stable isotopes (ORPHAN et al., 2001) makes it a powerful tool for determining identity and activity of single cells at the same time. Moreover, eukaryotic microbiology is profit- ing from the improvements of FISH methods to enhance taxonomic reliability and direct observation of ciliates under in situ conditions (FRIED et al., 2002). These advances in detection and classification of aquatic bacteria without previous cultivation allowed us to follow also the fate of certain groups in the food chain, for instance uptake and digestion of bacteria within food vacuoles of protists (JEZBERA et al., 2005), which shed new light on the question of prey density and survival of predators. At the moment, microbial ecology is moving rapidly to develop or apply new methods to enhance genetic microbial community profiling (NOCKER et al., 2007). HUBER et al. (2007) examined the sequences of more than 900,000 microbial small-subunit ribosomal RNA amplicons by massive parallel sequencing, with the ultimate target to describe “all” microbial populations – or at least their genes. So while our knowledge of “what is there” is increasing fast, we can now also have a glimpse of “what they can do”6 by analyzing the whole genome of organisms of interest (GLÖCK- NER et al., 2003) and – possibly – by microbial community genomics (DELONG, 2005), a knowledge that is increasing with each microbial genome sequenced (600 up to now, but growing rapidly). Nevertheless, we still do not have a reliable answer to the question “what are they actually doing”, except if we look at either a specific strain/process or the overall effect, such as nitri- fication or oxygen consumption. Combining phylogenetic methods with microscopic observations of the uptake of radioactive compounds by single cells (LEE et al., 1999; OUVERNEY and FUHRMAN, 1999) can tell us, which group of bacteria is “active”, and this can be done in principal for every single cell. Another approach to identify microbes responsible for distinct environmental processes is based on RNA based stable isotope probing (MANEFIELD et al., 2002). The old concept of OHLE and EINSELE who focussed on microbial processes, e.g., in the cycling of phosphorus, iron and sulphur, by measuring their metabolites in the water, was revived in a recent paper by HUPFER et al. (2007) who combined phylogenetics, instrumental analysis and the measurement of in situ processes. The smallest predator prey system on Earth, i.e., protistan bacterivores and bacteria, has existed for at least a billion years. It may thus be the most sophisticated example of co-evolution (PERNTHALER, 2005) where all strategies of capture and avoidance have been played by many billions of generations. So did aquatic microbial ecology eventually finally catch up with plankton ecology? Or did it even go beyond the possibilities of classical plankton ecology? An interesting aspect of microbial ecology definitely distinguishes microbial ecology from classical ecology (and evolution) is mentioned by COHAN (2002) who states that the basic concepts for the classification of organisms, especially the species concepts, do not apply to bacteria: what is generally regarded as a bacterial species may actually be a genus, and we may better use the term “ecotype”. It is well known that many Daphnia species reproduce parthenogenetically, thus forming clones instead of populations, but switch to sexual reproduction under certain conditions. Bacteria, however, do not “switch” from clonal to sexual reproduction but use mobile genetic elements for lateral gene transfer quite regularly, depending, among other things, on density (i.e., encounter rate) and phyloge-

6 As we will see below, this pair of question was introduced very effectively by DUBILIER (2007).

Table 2. Classical plankton ecology vs. aquatic microbial ecology.

Classical plankton ecology Aquatic microbial ecology

Eukaryotes Prokaryotes (and eukaryotes) Planktonic life is, by definition, restricted “Aquatic” bacteria will live wherever liquid water exists to the water column (from –30º to 130 ºC), even if it forms only a thin layer; the realm of “aquatic” microbial ecology thus stretches from the deep subsurface to the stratosphere The species – a recombining group of Asexual species or ecotype – where divergence is not irreversibly separated organisms with the constrained by sexual reproduction but by natural selec- major cohesive force of sexual reproduction; also ecotypes are evolutionary lineages that are irre- tion that hinders genetic divergence versibly separated, whereby sexual isolation is irrelevant (but: Daphnia clones, hybrids) to evolve permanent divergence in bacteria (but: named “species” contain many ecotypes)

Why are there so many species? How many species/ecotypes are there? (the plankton paradox[on]) (16S rRNA, genomic DNA) How abundant is this species at that spe- Are sequences and patterns found in DNA extracts cific site for a certain period of time? synonymous with species or ecotypes or metagenomic chimeras? What is this organism doing there? Does the presence of bacterial cells indicate living – or “active” – organisms? (MAR-FISH, mRNA, “single cell physiology”) Why do certain species or entire groups, Why are bacterial numbers in planktonic environments such as “algae” or simply chlorophyll a, so extremely constant in space and time? show such enormous oscillations? (top-down control by bacterivorous protists) (bottom-up and top-down control) A species occurs only in its particular Everything is everywhere. Small unicellular species are habitat or niche! ubiquitous if the niche fits, BUT “identical 16S rRNA species” may have different responses to temperature and possibly to other environmental factors, so a bacterial species is more like a genus and may consist of several ecotypes If we know the environmental conditions Knowing the full genome, we can do predictive ecology, (such as pH, temperature etc.) we can i.e., we can – in theory – anticipate the environmental predict the occurrence of certain species constraints of certain species, but how to deal with ver- or genera satile bacteria that can do “everything”? So we may better strive to identify “active” genes in real cells that can be identified and quantified! netic relatedness (i.e., lateral gene transfer decreases exponentially with taxonomic distance; FRASER et al. 2007). LINDELL et al. (2007) point at the role of viruses in gene swapping in one of the most numerous cells on Earth, the cyano bacterium Prochlorococcus. Notwith- standing several years of dispute, ecologists – but also some taxonomists – seem to be reluctant to use the new terms, especially when they question the very basis of evolution (WIESER, 2007), although COHAN (2002) points at a potential solution for this dilemma by introducing the term “force of cohesion” that may apply to plants, animals and prokaryotes. For another example, the dispute whether the term “biogeography” makes sense in aquatic microbiology is still raging on (FINLAY, 2002; FOISSNER, 2006; MARTINY et al., 2006 ). Such terminological issues may not be solved solely by the development of more sophisticated

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com 616 R. PSENNER et al. phylogenetic methods, but by a deeper understanding of sources, dispersal and sinks of microbial cells (TELFORD et al., 2006).

4. Perspectives and Crises

At the turn of the year 2002, the editors of the journal Environmental Microbiology asked a number of experts what they think about the future development of the field. RUDI AMANN, after looking into his “crystal ball”, wrote: “... did we ever see more than the tip of the iceberg? Did we ever get deeper than the ‘proof of principle’? We are still far from a good description of those microorganisms that catalyse essential parts of the biogeochemical cycles. On a global scale, which microbial species contribute how much, in tons per year, to the fixation of nitrogen or to methane oxidation? Which are responsible, that a certain chemical persists in one and is degraded in another site?” These observations point at the importance of microbes in the stability/fragility of global cycles – an issue that can hardly be overstated at the moment but that reminds us at the same time of the arguments of KRISS (1961) and POMEROY (1974) forty years ago (see Introduction). The fact that AMANN requests more funding for such “fundamental” questions is not astonishing. While some authors recommend to make use of the knowledge present in large databases (DE LORENZO, 2002), others point out that we need fresh data and a focus on the “empty spaces in metabolic maps” (WACKET, 2002) by referring to JOSEPH CONRAD’s dictum “The most interesting places are the empty spaces on a map”. Five years later, a fresh look into the “crystal ball” reveals the fear of an emerging data flood, and CURTIS (2007) comes up with a plea for “theory”. Now, this is far from being a new idea – one might even say that calling for “more theory” is an equally familiar and ambiguous move in the history of science. It might be read as a syndrome of a coming crisis that appears when a particular research program weakens. This can be caused by either internal or external influences, for instance through the introduction of new instruments, methodologies, model organisms, or entire experimental systems. Those changes have been described in different philosophical languages that apply to different fields and incidents: they are referred to as an epistemic break or rupture, as a “scientific revolution”, or also a move to a pluralism of competing theories. The most general problem of these philosophical terms is that they were developed while drawing on case studies mostly from physics. However, the past decades have insistently shown that biology and of course also ecology have their own philosophically relevant features that still need to be developed with great care and in more detail. To cite a skeptic, DUBILIER (2007) thinks that we do not know very much even of the least diverse habitats. Impressed, however, by the advances in detection speed and depth achieved by SOGIN et al. (2006) she calls for the application of high speed techniques to sequence also the low-abundance populations in more complex ecosystems such as the ocean. Do more sequences provide more information or simply new questions? NELSON (2003) argued that “... regardless of all the progress that we are making as environmental microbiologists, we are still presented with the situation where at the individual microbe level close to 40% of each genome remains as hypothetical or conserved hypothetical proteins” – an assumption confirmed by “Venter’s expedition” which discovered that the sequenced DNA encodes more than 6 million hypothetical proteins, doubling the number of known proteins in a single stroke (RUSCH et al., 2006). Most of the new proteins, however, “... are of unknown function and a quarter of them has no similarity to any known proteins” (BOHANNON, 2007). This makes us think that “old-fashioned” methods, such as cultures and isolations may some- times provide more insight into new pathways and processes, epitomized by the finding of new anaerobic marine bacteria which oxidize propane and n-butane via sulphate reduction (KNIEMEYER et al., 2007). DUBILIER (2007) ends her essay with the well-known eternal questions: ‘Who is there?’ and ‘What are they doing?’ It can be read as „more of the same“ which

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem 617 may, however, trigger another development, i.e., the advent of the computational period of microbial ecology. HUGENHOLTZ (2007) sees this as inevitable consequence of the increased power of sequencing and points out that the limiting step may be the storage capacity and the power of computer to make sense of the huge amount of data. OCHMAN (2007), on the other hand, expects major advances from the sequencing of single cells rather than whole communities. In contrast with these visions of an exploding number of sequences are the expectations of KUYPERS and JØRGENSEN (2007) who see the future of microbial ecology in a clever combination of those methods that are focused on “who is there” (FISH, fluorescent in situ hybridization) with those that answer the question “what are they doing” (SIMS, secondary ion mass spectroscopy) as shown by ORPHAN et al. (2001). The approach of single cell analysis is advocated also by DETHLEFSEN and RELMAN (2007) who envision a study of single bacteria in nanolitre or picolitre sized devices that includes their ageing, an endeavor which has hitherto been undertaken only for populations. Evolutionary adaptation studied at the level of single bacterial cells and small time steps is a new approach which brings microbial ecology and evolution close to those questions studied in animals and plants. Also STEPANAUSKAS and SIERACKI (2007) suggest to focus on single cell analysis to study, at the same time, phylogenetic and metabolic markers of uncultured bacteria. After the establishment of genomics, proteomics and metabolomics ROHWER (2007) expects the dawning of real-time microbial ecology, which may avoid the problems of current-day methods, i.e., the application of radiolabeled substrates to isolated portions of water. Also in this approach, however, computing time may become the limiting step. A data storm is anticipated also by STROUS (2007) who mentions climate change as one field in which microbial ecology has something substantial to say – an aspect not or rarely mentioned by other experts who are peering into their crystal balls. A massive plea for the science of the future, i.e., metagenomics, comes from a panel assembled around leading scientists and published by the National Academic Press in 2007 (http://www.nap.edu.catalog/11902.thml). There, “The dawning of a new microbial age” is conjured, since “in metagenomics, the power of genomic analysis is applied to entire communities of microbes, bypassing the need to isolate and culture individual bacterial community members. The new approach and its attendant technologies will bring to light the myriad capabilities of microbial communities that drive the planet’s energy and nutrient cycles, maintain the health of its inhabitants, and shape the evolution of life. Metagenomics will generate knowledge of microbial interactions so that they can be harnessed to improve human health, food security, and energy production.” It is common that new methods are hailed as the solution to old problems, but rarely have they been acclaimed as salvation for humanity (genetically modified organisms, stem cell research …). It seems that microbial ecologist have problems either to look beyond the immediate future of their science, or – if they do so – to keep in mind some modesty regarding economic, social and cultural constraints for their scientific visions. In addition, some of them have an impoverished vision of the past – it is not enough for a careful historical reconstruction of Aquatic Microbial Ecology to just mention, for instance, ANTONIE VON LEEUWENHOEK as one of the founders of single-cell microbial ecology (DETHLEFSEN and RELMAN, 2007). In the June 2007 issue of Oceanography, KIRCHMAN and PEDRÓS-ALIÓ argue that within the next 10 years all microbes of the sea will be cultured and sequenced, which produces more job opportunities for bioinformaticians. Basing their argument on the citation frequency of the famous paper of Sargasso microbial metagenomics in different journals, KIRCHMAN and PEDRÓS-ALIÓ predict that marine microbial ecology will become the lead discipline in the field of microbiology. They also assume that by 2017 strange microbes, such as Pyrococcus furiosus, will become new tabloid stars and will be better known among kids than Tyran- nosaurus rex. By that time, traditional methods in microbial ecology will have disappeared and a net of automatic monitoring stations will be available to follow the activity of microbes in the ocean. They also predict, that microbial ecology will become more important to fol-

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com 618 R. PSENNER et al. low the global carbon cycle than physical models. While some of the predictions may have been made with tongue in cheek, others sound more realistic or – to say the least – depict the last years of development with a certain marine and microbial bias. TIEDJIE (2000) sees the future of microbial ecology in microarray techniques to analyze community composition and expression. By 2019, he predicts the advent of a powerful interface between molecular tools and computational biology, but no new small subunit ribosomal RNA sequences and – regrettably – still no prokaryotic species definition.

5. Open Questions

Do the cited concepts (water desert – microcosm – ecosystem) apply to aquatic microbial ecology, i.e., does aquatic microbial ecology repeat the history of planktology? It does, in a sense, but there are some differences: Microbial ecology has progressed in reverse order, i.e., from fluxes and processes to species and populations. It has opened our eyes to the pivotal role of bacteria and bacterivores in the cycling of matter, but also to neglected areas of the biosphere, i.e., ice and the subsurface where > 99% of fresh water resides. Microbial ecologists are now attempting to close four gaps, i.e., single cell physiology under in situ conditions, phylogenetic taxonomy with environmental constraints, population ecology linked to element fluxes, and biodiversity in concert with biogeography. This may help us to answer the old questions of “what is there?” and “what is their metabolic potential”? What bacteria really do and how they interact with other species and with the environment, is another question, so far barely investigated, but it may become a major topic of microbial ecology in the near future. Our impression is that aquatic microbial ecology is characterized by a plurality not only of different research practices but also of theories and concepts. Thus one might conceptualize Aquatic Microbial Ecology as a discipline comprising different research styles that might even exist at the same time (pluralistic epistemology). First, there are a number of researchers who are seeking to detect as many different species, populations, ecotypes or genomes as possible (gene hunters, biodiversity aficionados, biogeographers). Second, there are those scientists focusing on certain well-defined groups to understand their role or behaviour in a particular habitat or ecosystem (physiologists and autecologists). Finally, there is a third group of researchers who are measuring and modeling the cycling of carbon, nitrogen or other elements by “bacteria” (biogeochemists).7 Will it be possible to reconcile those oppos- ing or unrelated trends? Is this necessary at all or don’t they complement each other rather well? If so, how can this be handled in a fruitful way? By investing, on the methodological level, in more computer power and machines? Shall we follow the suggestion to study single cells or rather to engage in large scale metagenomics? Or should we do both? If so, how could this be brought together productively? Just to fuse genomics, proteomics and metabolomics isn’t ecology – but then, what is ecology? Does ecology consist in relating all these “omics” to particular places, drawing comparisons between them and seeking a theory and practice of localities? Or should microbial ecologists care more about general ecological ideas, such as stability and diversity, or, for instance, the interaction between protists, bacteria, Archaea and viruses in different aquatic environments? And is it good to simply forget about old problems, such as viability, starvation, dormancy, activity and rates – just because new (molecular) methods are more sexy and trendy? It was almost 150 years after DARWIN that we realized that the first 3 billion years of evolution cannot be explained with the classical ingredients of evolutionary theory, i.e., spe-

7 These three groups might be eventually identified with the three research styles the basic concepts niche, microcosm and energy that, according to SCHWARZ (2003), simultaneously stabilize and advance aquatic ecology. However, this requires a more detailed study.

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.revhydro.com Aquatic Microbial Ecology: Water Desert, Microcosm, Ecosystem 619 cies and sexual reproduction (COHAN, 2002). Also the grand theories in ecology are based to a large extent on the classical species concept, so bacteria have played a minor role in the development of concepts in both ecology and evolution. We thus have to ask ourselves if microbial ecology may either be implemented in existing ecological theories or whether it is the source of new theories. This gives rise to the further question how these theories can be confirmed by experiments. One might argue that progress in the field of aquatic microbial ecology was hitherto driven by methods rather than by theories and concepts, epitomized in a report of the American Academy of Microbiology (STAHL and TIEDJE, 2002) which sum- marized the steps towards progress: “Develop new technologies ... for measuring the activity of microorganisms in the environment, .. approaches to cultivating currently uncultivable species, ... methods for rapid determination of key physiological traits and activities.” The report, however, states also that “... first we need to learn to ask questions before we can propose answers”.

6. Conclusions

In 2000 TIEDJIE, recalling 20 years of microbial ecology, argues that we should probably focus our analysis more on the relation between instruments, objects and methods rather than on the search for general theories and concepts. However, TIEDJIE found mainstream microbial ecologists more akin to astronomers, clearly a science of observation, and not so much to ecologists. We think that the variegated domain of ecology certainly encompasses features of an observational science while it is also a science where experiments are designed. Ecol- ogy seems to follow a “pluralistic epistemology”8 and thus includes both characteristics of experimental sciences, namely representing and intervening.9 Ecologists not only visually or mathematically reconstruct their objects – as astronomers do – but also manipulate and change these objects while exploring them. This is also what scientists do who are working in the field of ecological microbial ecology. So far, general ecology and aquatic microbial ecology seem to be structurally similar. But what if future progress in aquatic microbial ecology is neither driven by the application of general concepts and theories (as suggested by TIEDJE), nor by the application and adjustment of new methods and practices (as in the past), but by the necessity to tackle problems of global scale? Then the driving force – beside the intrinsic aim of disciplines to study the principles – will be the necessity to understand the role of microbes in the cycling of the elements on our globe. This might be called use-inspired basic research, a mode of research that embraces both the development of tools for technical manipulation and control and, at the same time, an understanding of the phenomenon focused on.10 As every scientific enterprise, be it applied or basic research, it is inspired by curiosity and might finally bring us back to the very beginnings of limnology. In 1896 FRANÇOIS-ALPHONSE FOREL expressed a thought that applies to past, recent and future scientific endeavors: “Quelle est la cause de l’attrait evident qu’exerce la limnologie sur les naturalistes contemporains ? C’est le charme de l‘inconnu.”11

8 The concept has been invented by PAUL FEYERABEND (1975). 9 This is the title of one of the most fruitful and comprehensible textbooks in the philosophy of science, written by the Canadian philosopher IAN HACKING and first published in 1983. 10 This is one of four research modes that DONALD STOKES (1997) identified to characterise the range of possibilities of doing research in a way that either seeks more control (pure applied research) or a better understanding (pure basic research) of the phenomena under consideration. 11 “What is the reason of the apparent appeal of limnology to contemporary naturalists? It’s the attrac- tion of the unknown.“

7. Acknowledgements

We are grateful to RUDI AMANN and JAKOB PERNTHALER for stimulating conversations. For linguistic improvement and discussion we would like to thank ALFRED NORDMANN. ALBIN ALFREIDER was sup- ported by the Austrian Science Fund, FWF (P17649).

8. References

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Manuscript received December 3rd, 2007; revised February 15th, 2008; accepted February 28th, 2008