Journal of Applied Bacteriology Symposium Supplement 1992, 73, 39-8s

Substrate capturing and growth in various ecosystems J.C. Gottschal Department of Microbiology, Kerklaan, Haren , The Netherlands

1. Introduction, 39s 2. Oligotrophy a n d ultramicrobacteria, 40s 2.1 Changes in cell size, 40s 2.2 Ultramicrobacteria in the environment, 40s

2.3 Ultramicrobacteria and oligotrophy, 42s 3. Conclusions, 45s 4. References, 46s

1. INTRODUCTION

properties of those bacteria which seem to prevail in extremely nutrient-deprived (‘oligotrophic’) environments. It is becoming increasingly accepted by microbiologists that in such environments the majority of micro-organisms are much smaller than those encountered in laboratory cultures, that these organisms may well be quantitatively very important for recycling organic matter, that a considerable fraction of these populations is unable to adapt to growth in the presence of carbon concentrations much higher than ambient (‘obligately oligotrophic’), and last, but not least, that hardly anything is known about their physiological properties (Oppenheimer 1952; Tabor et al. 1981; Morita 1985; Carlucci et al. 1986; Ishida et al. 1986, 1989; Hood & MacDonell 1987; Simon 1987; Cho & Azam 1988; Eguchi & Ishida 1990). The reason that we know so little about the real nature of these very small ubiquitous bacteria (often referred to as ‘ultramicrobacteria’) is mainly that considerable problems are involved in their isolation and growth in pure culture. To a large extent this is certainly caused by the difficulties encountered in growing them on solid media (very often visible colonies are not formed) and in obtaining appropriate cell densities (more than 106/ml). However, perhaps the most significant stumbling block remains the uncertainty about whether the cultures finally obtained represent those present as very small cells in the original sample. I n most cases the laboratorygrown strains no longer bear any morphological resemblance to the originals. Yet recent progress does shed some new light on the questions about the nature and the role of these tiny microbes. Although the emphasis of this paper will be on the bacteria in the ‘oligotrophic’ aquatic aerobic environment, soils also have to be considered, mostly as environments which are very poor in readily utilizable nutrients and are known to harbour various types of oligotrophic bacteria (Ohta & Hattori 1983; Hattori 1984; Williams 1985; Olsen & Bakken 1987; Morita 1988; Whang & Hattori 1988). In anaerobic habitats the situation may be completely different. T o date there are no published examples of

In order to avoid extinction, organisms have to produce progeny, which in general terms implies an increase in biomass. For micro-organisms this is usually reflected in an increase in the number of viable cells. Of course, this requires not only an adequate availability of carbon and other nutrients for biosynthesis of new cell material but also a sufficient supply of utilizable energy. Whereas phototrophic organisms can rely directly on light as a relatively abundant source of energy and thus in many (aquatic) habitats will be limited by other nutrients, heterotrophic microorganisms are dependent on the availability of organic compounds both for biosynthesis and for energy generation. Mainly on account of their own metabolic activity, however, microbes experience a severe lack of available carbon and energy sources in most natural environments. This is true for soils (Hattori 1984; Williams 1985), many freshwater habitats (Kuznetsov et af. 1979; Fry 1990) and, perhaps most significantly, marine waters (Williams 1971; Morita 1988). This important aspect of the ecology of micro-organisms has been known for several decades and consequently has resulted in the general acceptance of the need to study growth of bacteria not only in the presence of high concentrations of carbon and energy sources but especially under conditions of nutrient limitation. This has been done extensively by growing them under carbon and/or energy limitation in chemostat cultures. These studies have revealed a wealth of information on the response of bacteria (mainly single species) to conditions of severely restricted availability of growth substrates. Such studies have been reviewed extensively (see for example Jannasch & Mateles 1974; Veldkamp 1977; Tempest & Neijssel 1978; Gottschal & Dijkhuizen 1988). It must be emphasized that however important these studies are for our understanding of the physiology and the growth properties of bacteria there is growing concern about how much they tell us of the Correspondence to :Dr J . C . Gottschal, Department of Microbiology, Kerkluan 30, 9751 N N Haren, The Netherlands.

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anaerobic oligotrophic species ; neither does it seem very likely that there will be. Such habitats do not usually seem to be poor in organic carbon. Yet, growth in such environments is certainly not unrestricted. O n the contrary, in spite of the relative abundance of organic carbon, growth of specific organisms may be severely restricted by the limiting availability of electron acceptors. Because of the large number of different electron acceptors commonly found in most soils and sediment ecosystems (e.g. oxygen, oxidized nitrogenous compounds, iron, manganese, various sulphur compounds and carbon dioxide), a highly dynamic situation exists in which, depending on the input of organic matter, different energy-yielding redox reactions dominate. Of course this is the result of the sequential and sometimes simultaneous activities of many different microbial species, with those electron acceptors exhibiting the highest midpoint redox potentials taking precedence over those with more negative values (Zehnder & Stumm 1988). Progress in our knowledge of the use of various metals as electron acceptors and the recent demonstration of the co-existence of strictly anaerobic species with aerobic ones may have important consequences for the possible use of microorganisms in the treatment of contaminated habitats (Zehnder & Stumm 1988; Gerritse et al. 1990; Lovley & Lonergan 1990; Steinberg & Oremland 1990; Lovley et al. 1991; Oremland et al. 1991).

2. OLIGOTROPHY AND ULTRAMICROBACTERIA 2.1 Changes in cell size

Possibly the first observations on growth conditions resulting in small bacterial cells were reported as early as 1928 (see Morita 1985 and references therein). Very small cells with a diameter of less than 0.3 pm were obtained by continued cultivation of heterotrophic bacteria at low nutrient levels. Various terms have been introduced to describe bacteria of this and smaller sizes. T o avoid confusion I will follow the terminology proposed by Torrella & Morita (1981) who described them as ‘ultramicrobacteria’. Although changes in cell size in response to nutritional conditions have been known to occur for quite some time (see for example Schaechter et al. 1958), it has probably been the introduction of chemostat cultivation which has resulted in renewed interest in this phenomenon. For example, Tempest et al. (1967), in the course of a detailed study on the influence of very low growth rates on the physiology of Klebsiella (formerly Aerobacter) aerogenes, showed quite convincingly the gradual decrease in cell size when the dilution rate (D)was reduced from 1-0/h to O.l/h. As this reduction in size was most evident in length, near

coccoid cells were formed at the lower growth rates. Very similar results were obtained for two different bacteria grown under lactate limitation in continuous culture at dilution rates from 0.01/h to 0.46/h (Matin & Veldkamp 1978). Over this range of dilution rates the cell volumes changed approximately twofold from 0.34 to 0.69 pm3 for a Spirillum sp. and from 0-65 to 1.38 pm3 for a Pseudomonas sp. In batch culture the cell volume increased somewhat further, to 0.78 and 2.32 pm3 for Spirillum sp. (pmax = 0-35/h) and Pseudomonas sp. (pmax = 0-64/h), respectively. In fact, a decrease in cell size appears to be a general phenomenon in response to decreasing growth rates in chemostat cultures of both aerobic and anaerobic cultures (Gottschal, unpublished observations). As yet few studies have been designed to study this effect in detail. A recent example is the work of Moyer & Morita (1989) who observed a significant increase in cell volume of a marine Vibrio sp. ANT300 from 0.48 to 1.16 pm3 at dilution rates of 0.015-0.170/h, respectively, and 5.94 pm3 for log-phase cells in batch culture growing at a specific rate of 0*144/h. Nystrom & Kjelleberg (1989) also observed a strong correlation between the specific growth rate and the cell volume of another marine heterotrophic bacterium, Vzbrio sp. S14. With increasingly rich media the median cell volume increased from 0.7 to 2.6 pm3 with a concomitant increase of the specific growth rate from 0.28 to 1.5/h. These observations indicate a relationship between nutrient availability and the cell size of cultures grown in the laboratory. Of course, the most extreme condition of nutrient deprivation is true starvation, incubation of bacteria in the absence of utilizable energy sources. Possibly the first study in which changes in cell size were studied in response to starvation of a marine Vzbrio sp. (ANT300) was published in 1976 by Novitsky & Morita. A dramatic decrease in cell size was observed. It was shown most clearly by the ability of more than 50% of the viable cells to pass through filters with a pore size of 0.4 p m whereas at the onset of starvation only slightly more than 60% of the cells were filterable through filters with pore sizes of 3.0 pm and none passed through pores of 0.4 pm. T h e morphology of the cells had changed from curved rods to tiny coccoid cells. Since this pioneering study many others have concentrated on this starvation-induced miniaturization process which very often appears to be accompanied b j a significant increase in the number of cells (Novitsky & Morita 1978; Amy et al. 1983; Baker et al. 1983; Kjelleberg et al. 1983, 1987; Morita 1985; Hood et al. 1986). 2.2 Ultramicrobacteria in the environment

The importance of the above observations is much greater than a better understanding of the physiology of bacteria under starvation conditions alone. T h e conclusion that

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many ‘normal-sized’ bacteria, as we know them from laboratory cultures, change into very small cells has lent strong support to the idea that the filterable (0-45 pm pore sizes) cells found ubiquitously in almost any natural environment are also the result of a response to starvation conditions. Such cells were reported first by Oppenheimer (1952) in seawater and since then numerous studies have endorsed and extended these observations to freshwater environments and soils (Bae et al. 1972; Ishida & Kadota 1981; Tabor et al. 1981; Torrella & Morita 1981; MacDonnell & Hood 1982; Bakken & Olsen 1987; Hood & MacDonell 1987; Simon 1987; Cho & Azam 1988; Simon & Azam 1989). This new evidence of the existence of large numbers of very small bacteria in various environments has not only stressed their likely ecological significance but also suggested a possible alternative explanation for their existence in nature-namely that they could represent a group of micro-organisms specialized for growth in the presence of exceedingly low concentrations of carbon and energy substrates. In fact this had been implied by Torrella & Morita (1981) and MacDonell & Hood (1982) when they defined ultramicrobacteria as those cells which are less than 0.3 pm in diameter, which are (initially) not or hardly capable of growing in nutrient-rich media and which do not increase significantly in size in such media. This definition suggests that ultramicrobacteria would possess the important traits of oligotrophic bacteria. Unfortunately, a generally accepted and precise definition of oligotrophic bacteria does not appear to exist (Fry 1990). As a general rule, such organisms are supposed to be predominant in nutrient-poor environments, to be isolated on media ‘very low’ in nutrients and to grow relatively fast in such nutrient-poor media in comparison with non-oligotrophic (‘copiotrophic’) organisms (e.g. Kuznetsov et al. 1979; Poindexter 1981; Hattori 1984; Williams 1985; Fry 1990). This description is sufficiently vague as to be of little help in teaching us more about the properties of ultramicrobacteria. Therefore, it appears much more informative to attempt to answer the following questions concerning the properties of those ultramicrobacteria which have been studied in some detail. How abundant are ultramicrobacteria relative to the total number of bacteria present in an ecosystem? Are these small bacteria metabolically active and, if so, do they contribute significantly to the turnover of organic carbon in their natural environments ? T o what extent are ultramicrobacteria viable, in the sense that they can be shown to multiply when transferred to laboratory media supplied with appropriate nutrients and do they retain their morphological and physiological characteristics when grown under laboratory conditions? Do these small bacteria belong to known species that

have fragmented in response to the severe nutrientlimiting conditions in their environment or do they in fact represent an unknown class of bacteria? (5) Are ultramicrobacteria generally to be considered (facultatively/obligately) oligotrophic organisms ? Bae et al. (1972) used several direct microscopical methods and observed that in samples from six different soils taken at a depth of 6 cm below the surface vegetation all microbial cells were less than 0.9 pm in diameter and of these more than 70% were less than 0.3 pm, i.e. within the size range of ultramicrobacteria. O n the basis of direct microscopic counts Bakken & Olsen (1987) stated that the great majority of bacteria in soils consisted of cells that are very much smaller than those of most-described known species. I t is of interest that they noted further that only 0.2% of the smallest size fraction (diameter 0.6 p m did so. Unfortunately, only fullstrength nutrient medium was used. Of course it could have suppressed growth of any oligotrophic (micro)bacteria. Ultramicrobacteria are also commonly observed in freshwater ecosystems. Daley & Hobbie (1975) noted that in several ponds and water reservoirs a large fraction (up to 90%) of the bacteria seen with epifluorescence techniques were small cocci with a diameter of 0.2-0.8 pm. Ishida & Kadota (1981 and references therein) stated that the oligotrophic bacteria were distributed as the dominant organisms in the ‘oligotrophic’ and ‘mesotrophic’ areas of Lake Biwa. Subsequently they showed that these organisms had a cell width of no more than 0.14-0.17 pm and a length of 0.65-0.75 pm, thus suggesting that ultramicrobacteria dominated in this lake environment. Simon (1987) noted that in Lake Constance, Germany, between 40 and 95% of the bacterial cells fell within the size range of ultramicrobacteria. I n East Twin Lake-some 100 km west of Fairbanks, Alaska-O.4 pm filterable organisms with a mean cell volume of 0.016 pm3 dominated the bacterial population (Button & Robertson 1989). Thus, some evidence is available to suggest that extremely small organisms prevail both in soil and in freshwater environments. Yet, most observations on the occurrence of such microbes have been made in marine environments. Oppenheimer (1952) showed that, whereas freshwater samples could be sterilized by passing through filters with an effective pore size of 0.4 pm, marine samples contained cells which were not quantitatively retained by such filters. These observations were supported later by Anderson & Heffernan (1965) who showed that filterable (0.45 pm pore size) bacteria could be obtained from marine but not from several terrestrial samples. I t took approximately 10 years before Watson et al. (1977) showed that, in a large number of seawater samples, 2 7 5 % of the directly counted bac-

425 J . C . G O T T S C H A L

teria fell within the size range of ultramicrobacteria. This was confirmed by Tabor et al. (1981) who observed that in many samples from the deep sea a large fraction (up to approx. 80%) of the total colony-forming population consisted of 04.5 pm filterable bacteria. Among these they identified several strains of well-known species (e.g. Pseudomonas, Vihrio, Alcaligrnes etc.). From an estuarine environment-the Perdido Bay of the Gulf of M e x i c v MacDonell & Hood (1982) isolated 25 bacteria from surface water that had been filtered through 0.2 pm polycarbonate filters. Unfortunately, no information was obtained on the (relative) abundance of the ultramicrobacteria in the original samples. In a subsequent publication (Hood & MacDoneil 1987), very low numbers of such bacteria relative to the total number of colony-forming units on nutrientrich media werc reported (approx. O.loA~).This figure dropped to undetectable levels (less than 1 per lo6) in samples which were relatively heavily contaminated as a result of human impact. Information on the relative numbers of ultramicrobacteria was further provided by Maeda & Taga (1983) who reported that up to 90% of the bacteria observed were smaller than 0.8 p m in diameter (which strictly speaking does not make them ultramicrobacteria) in samples taken from three different bays in Japan. In samples obtained from the open sea they noted that approx. 75% of the total number of bacteria was of similar size. More recently, Cho & Azam (1988) studied the role of (small) bacteria in the cycling of organic carbon in the North Pacific gyre. Based on epifluorescence microscopy and on the results of C3H]thymidine incorporation experiments, they came to the conclusion that 98% of the bacterial population passed through 1 pm Nuclepore filters, that most of these cells were less than 0.1 pm3 in volume (thus for cocci less than 0.6 pm in diameter), that more than 95% of the bacteria were free-living and that nearly the entire carbon flux passed through these freeliving bacteria. This brings us to the important question of the activity and the possible role of these very small bacteria in the mineralization of organic matter in ‘oligotrophic’ ecosystems. 2.3 Ultramicrobacteria and oligotrophy

The study of the physiological properties of ultramicrobacteria is seriously hampered by the difficulties encountered in growing such organisms to sufficient density, if thcy can be grown at all under laboratory conditions. Generally, only a very small fraction of the total number of‘ bacteria observed microscopically and/or shown to be active in the field arc capable of’ forming visible colonies on solidified media o r growing to detectable density in liquid culture media. This appears to be true for ultrarnicrobacteria which constitute such an important portion of the

naturally-occurring bacteria (see above). I t seems very likely, therefore, that many of these bacteria have acquired properties highly adapted to growth in the presence of extremely low concentrations of nutrients (Ishida & Kadota 1981; Roszak & Colwell 1981; Tabor et al. 1981; Torrella & Morita 1981; MacDonell & Hood 1982; Carlucci et al. 1986; Whang & Hattori 1988; Ishida et al. 1986, 1989; Eguchi & Ishida 1990). In other words, it is quite reasonable to suppose that many of these organisms are either obligate or at least facultative oligotrophs. If this is the case it is extremely important to use nutrient concentrations which are orders of magnitude lower than those used in routine laboratory practice when attempting their isolation. If many of the very small bacteria also represent cells of well-known species, miniaturized in response to starvation conditions (Novitsky & Morita 1976; Morita 1985, 1988; Kjelleberg et al. 1987; Roszak & Colwell 1987), then it would seem at least prudent to grow them a t first in dilute nutrient solutions and to ‘gradually adapt’ them to higher nutrient concentrations (MacDonell & Hood 1982). Deciding which of these two possible explanations for the occurrence of ultramicrobacteria is most feasible constitutes a major challenge for microbial ecologists studying the role of bacteria in the field. The pioneering work done by Torrella & Morita (1981) illustrates clearly the complexity of the problem. Samples from seawater were concentrated on 0.2 pm polycarbonate membrane filters and transferred to small blocks of nutrient agar (depth 2 mm) mounted on microscope slides, incubated to allow growth to occur and viewed directly under a microscope to follow the development of microcolonies resulting from the growth of individual cells. Three distinct patterns were observed. (1)

Small cells increased in size and grew relatively quickly thus forming microbial colonies. (2) Small cells grew slowly without increasing in size thus developing into microcolonies which usually stopped growing after a few cell divisions. (3) Small cells, which made up the majority of those visible under the microscope as judged from the presented pictures, did not grow. The first response would appear typical for the type of bacteria usually isolated from marine environments and studied in laboratory cultures. T h e second was interpreted as indicating the presence of bacteria adapted to the very low nutrient concentrations encountered in seawater. The third showed that the chosen growth conditions were inadcquate for growth of most of the bacteria present in the sample or that the cells had lost the ability to divide all together, possibly by the filtration treatment. This third response, and to some extent the second as well, may also indicate that the cells are oligotrophic and require much

S U B S T R A T E C A P T U R I N G A N D GROWTH 43s

lower nutrient concentrations than those used in this study. Finally, but not very likely, the fact that concentration of the cells by filtration through 0.2 p m filters was used could mean that an important fraction of the real ultramicrobacteria had been missed in this study. Nevertheless, this study is the first example of ultramicrobacteria demonstrating that these cells may remain very small while growing actively. In a freshwater environment-Lake Biwa-Ishida & Kadota (1981) obtained evidence for the occurrence of very small bacteria and also presented evidence suggesting their oligotrophic nature. In the non-eutrophic areas of the lake oligotrophic bacteria (defined as cells capable of growth in the presence of < 1 mg C/1) were the dominant organisms. Several strains were isolated by repeated subculturing and dilution to extinction in liquid media containing 0.5 mg trypticase (BBL) and 0.05 mg yeast extract (Difco) per litre of aged lake water. As growth could not be followed by direct microscopy or turbidity in media with such very low carbon contents, a most probable number (MPN) method based on ['4C]glutamate incorporation activity was used. The numbers of viable cells were determined after incubation for 8 weeks at different concentrations of added nutrients. For at least five strains an optimum existed in the range of 5-500 mg C/1. At 5000 mg C/l no glutamate uptake was detected at all. Of particular interest is that scanning electron microscopy revealed that the size of cells of one of the strains was indeed very small (0.7 pm long and 0.15 pm wide) and remained unchanged during growth at nutrient concentrations of M O O mg C/l. This suggests that this strain is a typical example of an ultramicrobacterium with obvious oligotrophic properties, thus supporting the observations of Torrella & Morita (1981) discussed above. More recently, Ishida et al. (1986) extended their studies on the occurrence and activity of oligotrophic bacteria to the marine environment. Samples were taken from the Southern China Sea and the West Pacific Ocean south of Japan. Most probable number dilution series were prepared in which the incorporation of 14C-labelled substrates with a high specific radioactivity (present as a mixture of glutamate, glucose, protein hydrolysate and acetate) was measured in a medium of very low trypticase (0.5 mg/l) plus yeast extract (0.05 mg/l) and one that was 1000 times more concentrated. T h e MPN activity counts in the strongly diluted medium were always one to two orders of magnitude higher than those in the less diluted medium. These results were interpreted as indicating that 45-98% of the total metabolically active heterotrophs were obligate oligotrophs. T h e remaining bacteria were considered to be facultative oligotrophs capable of growth at both very low and relatively high nutrient concentrations. Unfortunately, the ratio of the total number of these heterotrophs (up to lo3104/ml) to the total number of bacteria counted directly

was only 0-02-0.08%. As no information was given on the size of the bacteria, it is difficult to relate these results to previous observations on ultramicrobacteria. Although the existence of a distinction between types of oligotrophic bacteria is generally assumed (eg. Fry 1990) the concept has been challenged by Martin & McLeod (1984) who demonstrated that the oligotrophic property of bacteria depended on the type of substrate used. Furthermore, in a 'minority report' of the Dahlem Conference (Berlin) on growth of bacteria in extreme environments, Koch (1979) expressed the view that Escherichia coli also should perhaps be considered an oligotroph as it is quite capable of growth at very low nutrient concentrations (for example in carbon-limited chemostats). Although this will most likely be true for carbon concentrations as low as 10 mg C/l and higher it is not necessarily the case (and so far it certainly has not been firmly established) for carbon concentrations in the range most often prevailing in seawater and oligotrophic lakes (i.e. < 1 mg of total organic C/l). Yet the distinction between obligately and facultatively oligotrophic organisms and attempts at a physiological characterization of ultramicrobacteria in this way is confused further by results from the interesting studies on bacteria isolated from the Perdido Bay estuary of the Mexican Gulf (MacDonell & Hood 1982; Hood & MacDonell 1987). Most of the isolates from the filtrate of 0.2 pm-filtered samples proved to be totally incapable of growth in various full-strength nutrient broths but did grow (and were isolated) on 10 times diluted broth. Moreover, two of these strains were tested on various concentrations of trypticase broth and exhibited an optimum in cell densities (after 36 h of incubation) at 200-300 mg C/l. No growth was observed at or above approx. 1000 mg C/l. Most noticeably, however, by employing heavy inocula on agar medium containing a nutrient broth of approx. 2000 mg C/1, cells of one strain proved capable of adapting to higher nutrient concentrations. After repeated transfers, every 48 h for 6 weeks, the ability to reach high cell densities within 36 h at 20&300 mg C/1 had been lost and optimum growth was obtained in trypticase broth of 1000 mg C/l and above. Concomitantly with this nutrient adaptation, the mean cell size increased to values normally encountered in laboratory cultures (0.51.0 by 24-3.0 pm). In combination with a limited survey of the response of a total of 27 similar strains to standard biochemical identification tests the authors concluded that the marine ultramicrobacteria belonged to identifiable taxa which had adapted to the very low nutrient levels experienced in the ecosystem. In the second paper on Gulf Coast estuarine organisms attention was focussed specifically on the adaptive response of these bacteria. Again a considerable number of bacteria were isolated and could be identified with known taxa, with Vibrio spp. representing the majority of identified organisms. Some of these Vibrio spp.

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were starved, in some cases for periods up to 3 years, and tested subsequently for the ability to grow at high and very low (2-5 mg C/1) nutrient levels. The most remarkable results were that some strains had produced very small, 0.2 pm filterable, cells and a considerable fraction of these cells had become obligately oligotrophic in the sense that growth occurred only in the presence of very low nutrient concentrations. The majority of the cells behaved, however, as facultative oligotrophs, growth occurred both on high and low nutrient media. If a strain of Vtbrro Juvralts was exposed for 60 d to very low nutrient concentrations (actual values not reported) in a continuous flow system with a calculated dilution rate of approx. 0.04/h very small cells (0.4 pm filterable) and some (1&15%) obligately oligotrophic cells were detected. It appears reasonable that, as suggested by the authors, at least a portion of the ultramicrobacteria represent bacterial forms which are part of a continuous spectrum of physiological types ranging from large, metabolically very active to extreme]! small, metabolically less active or even ‘dead’ cells with the actual position within this spectrum determined by the nutritional conditions of the ecosystem. Nevertheless, it must be emphasized that however true this may be for adaptability to nutrient levels above approx. 3-5 mg C/1 this appears much less certain for carbon concentrations well below 1 mg/l, for the simple reason that appropriate experiments including organisms adapted to growth under such conditions have not yet been done! This still allows for the possibility that a certain fraction of the ultramicrobacteria does not belong to known species and i s not capable of physiological adaptation to much higher nutrient levels (genetic adaptation can of course not be ruled out, as is the case for the adaptations described above). Such microbes could well possess specialized properties making them uniquely adapted to their nutrient-deprived way of life. Whatever the case, it seems beyond doubt that ultramicrobacteria must be capable of expressing highly efficient uptake mechanisms as a major component of their strategy to generate energ) and to grow in environments with extremely low nutrient concentrations. This is the traditionai view (Hirsch rt al. 1979) on the need of oligotrophic bacteria for low Michaelis-Menten half-saturation constants for substrate uptake. Numerous examples are available of copiotrophic and some oligotrophic bacteria with both low and high affinity uptake systems for various organic and/or inorganic substrates This topic has been discussed quite extensively in several publications (Tempest & Neijssel 1978; Akagi & Taga 1980; Bell 1984; Morita 1984; Kjelleberg & Hermansson 1987; Gottschal 1990). T h e general response observed so far appears to be a switch from low to much higher affinity systems in response to changes from relative nutrient abundance to more substrate-deprived conditions. Moreover, recent results from studies on argin-

ine and leucine uptake systems of marine heterotrophs demonstrated that even during starvation the high affinity systems remained fully operative or even increased several fold (Facquin & Oliver 1984; MHrden et al. 1987). I t has also been argued that changes in cell morphology especially those resulting in increased surface to volume ratios (S/V) may contribute to sequestering nutrients efficiently at very low concentrations (Matin & Veldkamp 1978 ; Poindexter 1981). A high S/V ratio has even been proposed as a very important characteristic of ‘model’ oligotrophs (Hirsch et al. 1979). It seems unlikely, however, that this factor alone should be of great consequence for most bacteria unless the change in size and/or morphology is very dramatic, for example as observed for prosthecate bacteria which form long, thin ‘stalks’ upon nutrient deprivation (Poindexter 1981). Thus a Spirtllum sp. studied in chemostats by Matin & Veldkamp (1978) at dilution rates from 0.35/h to 0.01/h changed its S/V ratio only from 6.3 to 9.3 , respectively. A somewhat larger difference was found by MacDonell & Hood (1987) adapting ultramicrobacterial spheres of 0.2 p m diameter (S/V = 31) to high nutrient conditions with cell sizes of up to 0.4 by 1.5 p m with a S/V ratio of 11. T h e marine Vibrio sp. ANT300, studied by Moyer & Morita (1989), appeared as a voluminous 6 p m 3 organism in batch culture and as a tiny almost spherical cell of 0.045 pm3 following growth at D = 0.015/h in the chemostat and subsequent starvation. Assuming cell sizes (which were not reported for these cells) based on these volumes (1.2 by 6.0 pm and 0.4 by 0.5 pm, respectively) S/V ratios of 3.8 for the batch culture cells and 14 for the starved cells can be estimated. Thus, even quite considerable changes in cell size do not appear to influence the S/V ratio more than 10-fold. However, the change in size does affect the volume of the cells very strongly: spherical cells of 0.2 pm exhibit a volume of only 0.004 pm3 which is 1500 times less than the larger ANT300 cells. An important consequence of this behaviour appears to be that the water content of the cells goes down considerably, at least by a factor of 2 (Simon & Azam 1989). T h e combination of these factors with the commonly observed derepression of many metabolic enzymes and substrate uptake systems in very slowly growing or starving cultures (see for references Gottschal 1990) may aid considerably in maintaining nutrient pool sizes in the mmol/l range in an environment in which nutrient concentrations are in the order of 10-9-10-12 mol/l. Obviously this requires a large uptake potential per cell with low half-saturation constants. In order to ensure the flux of substrate into the cell at ambient concentrations, low whole cell Michaelis-Menten constants for solute transport alone are not enough: V,,, values for the uptake process (and subsequent metabolism) should also be sufficient. T h e concept of specific affinity has been developed to give a realistic measure of nutrient

SUBSTRATE CAPTURING AND GROWTH 45s

uptake (and growth) of micro-organisms at ambient (low) nutrient concentrations (Button 1985; Gottschal 1985 ; Button & Robertson 1989). T h e specific affinity [a,(l/g cells/h)] represents the specific nutrient uptake rate per unit of substrate. At infinitely low substrate concentrations its value can be obtained from the specific substrate uptake rate vs the substrate concentration curve by taking the initial slope. This value is then designated a: which actually is nothing other than the maximum spec@ uptake activity (V,,,,,) divided by the half-saturation constant for uptake (KT).This concept of specific affinity may be particularly helpful in the study of oligotrophic organisms as it allows relatively straightforward and meaningful comparison of different micro-organisms with respect to their substrate sequestering potential. In other words, ‘model’ oligotrophs (Hirsch et al. 1979) must be expected to have high a: values, especially for those substrates which are crucial for growth and energy generation in their natural environment. Unfortunately, such values are usually not directly available from the literature. In some cases, however, they may be obtained by recalculation of data on specific uptake rates, half-saturation constants, nutrient concentrations and microbial biomass. A large number of such calculations for the uptake of many different carbon compounds by various organisms was compiled by Button (1985). What becomes immediately evident from inspection of these data is that the calculated a: values vary over three to four orders of magnitude. This is true not only for different organisms, but also for different substrates with one and the same bacterium. Furthermore, it appears that most of the reported values are less than 10 (very poor specific affinity) and only some are well above 100. It should be noted that most of the results were obtained with organisms isolated and cultivated in conventional, nutrient-rich media. Thus there was no selection for organisms with high affinity systems. No values of uptake measurements done in the field have been included. These would have been of particular interest in the light of the presumed highly adapted physiology of oligotrophic organisms in nutrient-poor environments. However, it is certainly remarkable that the highest reported a: value (1750 l/g cells/h; for uptake of arginine) cited in this review was found for Corynebacterium sp. strain 198, a marine strain, grown under arginine limitation in continuous culture. The same organism also showed high u: values for glucose uptake during glucose (a: = 388) and glucose+ arginine (a: = 625) limited growth. When Cytophaga johnsonae was grown under glucose limitation it was also found to have a high specific affinity for glucose, with a: values of 440 and 250 for dilution rates of 0.03/h and O.l5/h,respectively. Referring to unpublished results from his own laboratory, a very high value of 1064 was reported by Button for the uptake of toluene by Pseudomonas sp.

strain T2, a marine strain. In a more recent publication, however, values of 30&500 were the highest obtained for fully ‘induced’ cells (Robertson & Button 1987). However, much higher specific affinities appear to exist for some organisms. For example Aeromonas hydrophila, obtained from drinking water, was shown to be capable of growing on carbon compounds at concentrations of 10 pg C/1 for the individual compounds (van der Kooij & Hijnen 1988). Not too surprisingly this property is reflected in very high specific affinities: 140 000, 9000, 6500 and 55 000 for arginine, glucose, acetate and oleate, respectively, but some uncertainty remains as no direct data on biomass concentrations were given. Obviously, the strain of A. hydrophila possesses oligotrophic features suggesting that oligotrophic natural populations would have similarly high specific affinities. Unfortunately, no pure culture data are available to substantiate this, but Button & Robertson (1989) showed that 0.4 pm-filtered fractions of Alaskan lake water incubated with a I4C-amino acid mixture (2.5 nmol/l of total amino acids) had a: values of 1100-1300 l/g cells/h. Moreover, preliminary results from the laboratory of Button (Schut, unpublished results) indicated that a pure culture of a 0.4 p m filterable organism had an a: value of 4880 when tested with the same amino acid mixture. Very recently Schut, in my laboratory (unpublished), succeeded in isolating in pure culture several marine strains from Resurrection Bay (Alaska) which morphologically are to be considered ultramicrobacteria thus offering the unique possibility of studying the physiology of such organisms in greater detail. In a study of heterotrophic marine bacteria by Eguchi & Ishida (1990)it was concluded that in samples (0.5 m below the surface) from the south-eastern coastal area of Japan in the Pacific Ocean, more than 85% of the heterotrophic bacteria were obligately oligotrophic. Uptake experiments with these samples revealed very high a: values: 4800 and 98 000 for glycine and glutamate, respectively. Similar experiments with samples from more polluted near-shore sites with only 37% obligate oligotrophs (the remainder of the cells were copiotrophs and facultatively oligotrophic species) indicated a much lower specific affinity for glycine uptake: 75 l/g cells/h. Unfortunately these data must be considered as preliminary and relatively inaccurate due to uncertainty concerning the actual nutrient concentrations in the sample.

3. CONCLUSIONS However tempting it is to conclude that (obligate) oligotrophs express the highest specific affinities for scarcely available substrates, it is also quite obvious that facultative oligotrophs and even some copiotrophs may be equally effective in sequestering substrates under conditions of nutrient depletion. Results reported by the Japanese

46s J . C . G O T T S C H A L

research group mentioned above indicated that the facultatively oligotrophic strain KE-10, grown in a low carbon medium (0.5 mg organic matter,il), possessed an excessively high specific affinity for leucine, approximately 57 000 l/g cells/h (Ishida et a/. 1989). Similarly, starvation for 48 h resulted in a threefold increase (from 170 to 424 l/g cellsfh) in the specific affinity for leucine of the marine Vibrio sp. strain S14 (Marden et a / . 1987). These results again illustrate the enormous flexibility of many bacteria in response to the requirements for efficient substrate-capturing imposed on them by different environments. T h i s extraordinary adaptability is not in keeping with the view that in order to conquer extreme nutrient deprivation microorganisms need to become highll- specialized. I n many cases this appears to work out differently. For example, chemostat enrichments performed under conditions of oxygen limitation with various carbon and energy sources did not result in selection of microaerophilic bacteria, supposedly specialized in growth at very low oxygen tension. O n the contrary, heterotrophic aerobes, which also grew perfectly well at air saturation, became dominant in these cultures and, due to their extraordinarily high affinity for oxygen (see also Button 1985), allowed very oxygensensitive anaerobes (such as methanogens) to grow at high densities in the same culture (Gerritse et a / . 1990). Similarly, it appears that selective enrichments under conditions of multiple substrate limitation do not always result in the emergence of specialized organisms for each of these substrates, but very versatile and flexible organisms are selected which obtain their selective advantage from the very fact that they scavenge most of these substrates simuitaneously (e.g. Gottschal 1986).

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