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Home , Biogeochemical cycle, Microbial ecology, Microbial loop, Photoheterotroph

Journal of Experimental Marine and 366 (2008) 99–103

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Journal of Experimental and Ecology

journal homepage: www.elsevier.com/locate/jembe

The – 25 years later

Tom Fenchel

Marine Biological Laboratory, University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingør, Denmark article info abstract

Keywords: The background for and the subsequent impact on biological oceanography of the article Azam et al. [Azam, F., Functional types of microbes Fenchel, T., Field, J.G., Gray, J.S., Meyer-Reil, L.A., Thingstad, F. 1983. The ecological role of water-column Importance of names microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257-263.] is discussed. It is concluded that its influence in terms of Microbial loop stimulating further research and in changing the earlier paradigm of marine food chains was to a large Recent discoveries extent due to the fact that the term “microbial loop” occurred there for the first time. The progress in understanding microbial biota and microbial interaction during the last 25 years will also be reviewed. © 2008 Elsevier B.V. All rights reserved.

1. Introduction organisms was substantial, meaning that microbes play a major role in the transformation of matter and energy in the plankton. More so- The term “microbial loop” was originally coined by Azam et al. phisticated methods for estimating in situ bacterial growth rates had also (1983), a paper on which both John and I were co-authors. The term has been developed (Hagström et al., 1979; Fuhrman and Azam, 1982). since then been a staple in the vocabulary of biological oceanography. Altogether it was recognised that play a substantial role The paper summarised and connected a variety of discoveries made comparable to that of the primary producers in terms of element cycling during the preceding decade by several marine biologists. Primarily it in the (Kirchman et al., 1982; Williams, 1981). was shown that the classic view of the structure of marine plankton It remained a problem to understand the fate of the apparently communities as presented by e.g., Steele (1976) was incomplete and substantial bacterial production. The bacterial density in the water too simplified. column usually remains relatively constant around 106 cells ml-1,but While the existence of tiny , bacteria and heterotrophic other evidence indicated that bacteria were multiplying relatively fast in the marine water column had been recognised for a long time, with an average generation time of a day or less. Eventually it became most biological oceanographers believed that what really mattered in clear that bacterial density is mainly controlled by the grazing activity of the cycle of the water column was only the production of large small and that in particular representatives of various phytoplankters, especially and dinoflagellates that served as taxonomic groups of flagellates are important. Such organisms occur food for , mainly , that again served as food for at densities of around 103 cells per ml seawater. It could also be small fish. concluded that their functional response to bacterial density and It had actually been recognised for some time that tiny photosyn- their capacity to ingest bacteria showed that ambient concentrations thetic plankton organisms (measuring 2-20 μm) also play a significant of bacteria were sufficient to sustain populations of these micro- role as primary producers (see Platt and Li, 1986). Bacteria in the water phagotrophs and also that grazing could explain the relatively constant column had also been studied and quantified using plate counts for density of bacteria in the water column. Under some circumstances the several decades (e.g., Zobell, 1946). It had also been noted that micro- of bacteria and protozoa showed regular prey- scopic counts of bacteria exceeded plate counts by up to two orders of predator cycles that could be followed over longer time periods (Fenchel, magnitude, but it was generally believed that the bulk of microscopic 1982a,b,c). counts mainly included dead or metabolically inactive cells. Improve- The Azam et al. (1983) paper reviewed the available literature at the mentsintechniquesforcountingbacteria(Hobbie et al., 1977) combined time. It concluded that a substantial part of the with the use of 14C-labelled substrates such as glucose and amino acids was lost to the environment in the form of dissolved organic matter as showed that a substantial part of the bacterial communities estimated had been shown by Fogg (1983) among others, and that dissolved from microscopic counts was actually metabolically active (Wright and organic matter was utilised by bacteria that were again consumed by Hobbie, 1965, Hobbie et al., 1972; Meyer-Reil, 1978), and Pomeroy (1974) protozoa, and finally that the protozoa could then enter the had shown that the O2-uptake by the smallest size fraction of planktonic formed by larger creatures. Given a suitable graphical presentation, this new pathway in plankton dynamics forms a “loop” on the classic food chain (Fig. 1). The findings also showed that the characteristic E-mail address: [email protected]. time scale for the population dynamics and processes in the water

0022-0981/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2008.07.013 100 T. Fenchel / Journal of Experimental Marine Biology and Ecology 366 (2008) 99–103

Fig. 1. The microbial loop as described in Azam et al. (1983) (solid arrows) and with later additions (stippled arrows). DOC refers to dissolved organic matter.

column in much shorter than hitherto recognised – bacteria as well as celerates mineralisation and thus regenerated production in their predators typically have generation times of less than a day. limited systems. During the years following the Azam et al. (1983) paper there was a spate of studies on the microbial loop treating a diversity of aspects 3. New players in the microbial loop related to it; for a long period these dominated biological oceano- graphy and many new discoveries were made. In the present mini- It early became apparent that there were more functional types of review I will list some of the highlights of research in the microbial loop organisms involved in the microbial loop than just , since the publication of Azam et al. (1983). Excellent in-depth reviews heterotrophic bacteria, and phagotrophic protists. In particular, it was of most aspects can be found in Kirchman (2000). Here I will only found that unicellular photosynthetic such as the mention some important developments since 1983 and especially ubiquitous play a substantial role as primary producers highlight aspects that I believe will be important in future research and and the more recently discovered minute prochlorophytes (Prochlor- that open up new questions. Finally I will discuss why especially Azam ophyton) were shown to dominate in oligotrophic et al. (1983) had such a large impact. oceanic waters (Chisholm, 1988, 1992; Cambell and Vaulot, 1993). The bacterium Roseobacter and relatives are so-called aerobe 2. Carbon cycling – link or sink? ; they contain a like anaerobic phototrophic bacteria. Roseobacter cannot perform photosynthesis, A large part of the earlier effort was to document the quantitative role but it can generate ATP from light energy. Roseobacter often constitutes of the microbial loop and a variety of new methods were developed a large fraction of the bacterial plankton biota and it belongs to the mainly for estimating protozoan grazing rates and in situ growth rates relatively few quantitatively important plankton bacteria that are easy and growth efficiencies of bacteria (e.g., Kirchman et al., 1982; Fuhrman to isolate and grow on agar plates. The finding represents a novel and and Azam, 1982; Andersen and Fenchel, 1985; Sherr and Sherr, 1988; probably important energy input into the microbial loop (Kolber et al., Sherr et al., 1999; Caron and Goldman,1990; Del Giorgio and Cole, 1998) 2001). and these efforts also included modelling (Thingstad, 1992; Blackburn The most important new functional group is constituted by . It et al., 1996). It became clear that the relative role of the microbial loop was found that viral particles occur at densities in seawater of about was not invariant over time and space. Rather the microbial loop dom- 109 ml-1 and their turnover rate could be estimated from their survival inates oligotrophic waters whereas as the classical plankton food chain rate in water in the absence of host cells (e.g., Proctor and Fuhrman, predominates under circumstances when there is a fresh supply of 1990; Bratbak et al.,1992; Thingstad et al.,1993). Viral attacks also play a such as during the spring bloom in temperate waters role for eukaryotic . More recently, experimental studies have and in areas. This stands to reason in that for clarified a number of aspects concerning the role of . Almost all dissolved mineral nutrients favours small organisms and primary bacteria seem to have at least one host specific virus. Studies in the field production is then mainly based on mineral nutrients regenerated in and in laboratory systems have demonstrated the dynamics of virus- the water column (Chisholm, 1992; Kiørboe, 1993). host systems and the of resistance and how virus may induce Another question that arose was the so called “link or sink” problem, lysis and thus recycles some dissolved organic matter from bacteria that is, to what extent the microbial loop represents a loss of fixed (Suttle, 1994; Suttle and Chan, 1994; Middelboe, 2000; Middelboe and carbon to the system or whether it primarily channels fixed carbon to Riemann, 2002, Middelboe and Jørgensen, 2006; Middelboe et al., 2001). higher levels of the food chain. It seems that the consensus now would Mortality caused by viruses is of a similar magnitude as protozoan be that the microbial loop is primarily a sink (see discussion in grazing; however, the fact that virus – in contrast to protozoan grazing – Williams, 2000). This follows from the fact that the microbial loop is highly host-specific means that the effect is different. Rather than includes several trophic levels and so a large fraction of the organic affecting the total number of bacteria, viruses drive successional changes carbon is dissipated as CO2 along the process. The main effect of the of the bacterial biota and perhaps viruses sustain higher bacterial species microbial loop on element cycling in the water column is that it ac- diversity in plankton than would be the case in the absence of viruses. T. Fenchel / Journal of Experimental Marine Biology and Ecology 366 (2008) 99–103 101

This is because the most numerous types of bacteria are the most ability of local extinction is low. It has long been known that it is susceptible to viral attack. possible to isolate bacterial strains using enrichment cultures of Mixotrophy represent another complexity that was later added to bacterial strains from environments in which they cannot thrive the microbial loop. Mixotrophic protists are capable of both phagocy- such as thermophiles in cold seawater or obligate anaerobes in the tosis and phototrophy, a phenomenon that has proven to be widely aerobic water column (e.g., Isaksen et al., 1994). Also, isolated DNA distributed. There are different types of mixotrophy. Some protozoa, strands deriving from lysed cells may float around in the environment. in particular , feed on phototrophic algae, but retain their Therefore DNA-extraction and subsequent multiplying and sequencing in a functional state for several days and can utilise the RNA-genes may provide a rather misleading and inflated picture of the photosynthates – so-called retention (Stoecker et al., 1989; microbial diversity in terms of the number of organisms that actually Jones, 1994). Other species possess chloroplasts and are at the same play a role at a particular place and time. This problem gets worse with time phagotrophs – this is common among dinoflagellates and certain PCR and low copy number techniques. Similarly the extraction of heterokont flagellates; other forms again harbour phototrophic eukaryotic DNA also provides a picture of a large unexplored diversity symbionts, but in accordance with current understanding of the origin of microbial (e.g., López-Garcia et al., 2001). But this is of chloroplasts (e.g., Margulis, 1981) there is sometimes not a clear simply not supported by direct observations which indicate a finite distinction between possessing a chloroplast or a green symbiont – this number of generally known and named species with a cosmopolitan is in particular a common phenomenon among dinoflagellates. Such distribution (Fenchel and Finlay, 2006). Also, when “novel species” are may make an important contribution to energetics of the referred to, what is really meant is that there is no corresponding water column (e.g., Havskum and Hansen, 1997). sequence in a database – but most unicellular eukaryotes have been described based on morphological traits and may have been known for 4. The diversity of the microbial biota a century or more, but their rRNA-genes have never been sequenced. Among plankton bacteria there are metabolic specialists such as A major effect of the new understanding of plankton food chains was ammonia oxidisers and oxidisers of C-1 compounds. But there is also an increased interest in the organisms involved – their diversity and evidence to indicate that the majority of plankton bacteria are generalist their functional properties. And is probable that we now have a that can utilise a wide spectrum of low molecular weight relatively complete picture of unicellular eukaryotes in plankton. The organics in addition to species that can hydrolyse various polymers such reason is that – except, perhaps, in the case of the tiniest eukaryotes - as cellulose and chitin (Mou et al., 2008). such organisms can be identified microscopically and also it has proven The nature of the diversity of microbial plankton remains an easy to culture these forms in the laboratory, primarily because it is important topic to explorer further. But the central question is the relatively easy to pick individual cells. The interest in phagotrophic functional diversity rather than the diversity of rRNA-sequences. eukaryotes also led to an increased interest in the taxonomy and Sequencing RNA-genes is an insufficient tool beyond recording the functional biology of these organisms (e.g., Patterson and Larsen, 1991). major taxonomic groups of bacteria that may be present. Detection Regarding prokaryotes, things are more complicated. Phototrophic of the presence of gene families involved in metabolic traits may prokaryotes () have proven relatively easy to isolate into provide more information. But first of all further attempts to culture pure cultures, but with a few exceptions the majority of heterotrophic the organisms and to determine their phenoptypic properties will be bacteria have shown to be resistant to attempts to culture them. Only a necessary and methods such as may then establish the in situ minority of marine species form colonies on nutrient agar plates – the quantitative role of the different strains (Giovannoni and Stingl, 2007). classical way to obtain pure cultures. So the primary difficulty is the ability to pick individual cells. There may be several reasons why so few 5. The spatial heterogeneity of plankton planktonic bacteria are capable of forming colonies on agar plates. One is that many bacteria are obligatory that cannot thrive in The tendency of particulate organic and inorganic matter to traditional nutrient rich media (Pointdexter, 1981). It is also possible flocculate (so called ) has been known for some time and that using rich rich media induce rapid growth in the bacteria that in the associated microbial biota have drawn considerable interest. Such turn activate lysogenic viruses so that colony formation of the bacteria particles tend to sink and are the principal vehicle for transporting fails (Hagström et al., 2001). However, more recently there has been organic matter from the to the surface of sediments at success in culturing the ubiquitous so-called SAR 11 clade (Candidatus greater depths. One important question is how rapidly the particles Pelagibacter ubique) and future successes are likely (Giovannoni and mineralise during their passage from surface waters until they reach Stingl, 2007). Despite insight deriving from methods of molecular the sediments. genetics (see below) it will be important to develop methods for Marine snow particles are rapidly colonised by microbial commu- growing these organisms in the laboratory in order to unravel their nities, the composition of which differs somewhat from the biota in functional properties. free suspension. In particular bacteria of the Cytophaga type, which are DNA-extraction and sequencing RNA-genes has revealed an ap- known to hydrolyse various polymers such as cellulose, are common parently huge diversity of so called “unculturable” plankton bacteria on suspended particles (DeLong et al., 1993). Marine snow particles (Giovannoni et al.,1990; Bitshigi et al.,1991; Moon-van der Straay et al., also harbour various bacteria-feeding protozoa so that each 2001) and interesting discoveries such as that archaebacteria play an constitutes a small microbial . Solid surfaces in seawater are important role in marine plankton have followed (DeLong, 1992). rapidly colonised by bacteria; at first bacteria attach reversibly, but However, some evidence indicates that the numbers of metabolically eventually they may form colonies embedded in mucus resulting in a active and relatively numerous bacterial species are in fact relatively biofilm. Evidence indicates that a large fraction of the microbial activity more modest (Hagström et al., 2000, 2002; Giovannoni and Stingl, in the water column takes place on such suspended particles that 2007). Primarily, it is not known to what extent genetic distances may also serve as food for zooplankton such as copepods (Ploug and actually correlate with phenotypic differentiation and much genetic Grossart, 1999; Artolozaga et al., 2000; Kiørboe et al., 2002, 2003; variation is likely to be selectively neutral. Sequence differences may Grossart et al., 2003). also reflect minor functional differences such as adaptations to dif- In contrast to earlier belief, many or most plankton bacteria are ferent temperatures. Although a theoretical species concept for motile and show towards point source of organic matter prokaryotes does not exist, such genetic variation may be compared (Fenchel, 2001; Grossart et al., 2001). Such point sources may be living to “” within species. Also, due the huge absolute pop- algae that excrete dissolved organic matter or it may be cells that lyse ulation sizes the dispersal potential of bacteria is high and the prob- due to attack from viruses or from predators. Given that there are about 102 T. Fenchel / Journal of Experimental Marine Biology and Ecology 366 (2008) 99–103

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