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7 Measuring Heterotrophic Activity in Plankton
7 Measuring Heterotrophic Activity in Plankton JOHN E. HOBBIE Marine Biological Laboratory, Woods Hole, M A 02543, U S A
I. Introduction
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A. Heterotrophic activity . B. Oxygen measurements . . . C. Radioisotopemethods . . . . D. Improvements in radioisotope methods . . E. Ecological importance of heterotrophic activity measurements 11. Backgroundofmethods . . . . . . A. Theory of uptake measurements . B. Mathematical description . . . . c. v,,,, T , - ( K + S) . . . . . . D. Different types of heterotrophic activity measurements 111. Methods . . . . . . . . A. Standardmethod . * . . . . B. Kinetic analysis . . . C. Tracer-level additions . . . D. Addition of high level of substrate . . . E. Thymidine incorporation . . . F. Leucine incorporation . . . . References . . .
235 235 236 237 238 240 240 240 242 243 244 247 247 248 248 248 249 249 250
I. Introduction A.
Heterotrophic activity
In order to understand what microbes are actually doing in nature, as opposed to what microbes are capable of doing in the laboratory, ecologists must make measurements of rates of microbial processes in the real world. One obvious and important process is metabolism and growth METHODS IN MICROBIOLOGY VOLUME 22 ISBN 0-12-521522-3
Copyright @ 1990 by Academic Press Limited All rights of reproduction in any form reserved.
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of bacteria on organic compounds supplied from outside the cell. This heterotrophic growth decomposes most of the organic matter produced in the biosphere of land and water. In the water of lakes and oceans, measurements of bacterial heterotrophy are difficult because the great dilution of both substrates and organisms allows only low rates of activity, which laboratory techniques cannot measure, and because of the sensitivity of microbes to manipulation, which means that the rates are changed by, for example, incubations of 24 h or by filtration through a 10-pm pore size filter. For these reasons, techniques have been developed to measure low rates of heterotrophy either in undisturbed waters or in samples of lake or ocean water incubated briefly with radioisotopes. The techniques discussed here have given a tremendous amount of information about the role, rates of activity, and factors limiting the heterotrophic bacteria in the plankton. Only some of the methods, those using radioisotopes, are presented in detail here. These are all variations on the theme of the addition of extremely low concentrations of labelled organic substrates to water samples. Anyone working in this field should understand the background and theory of this basic theme and then choose the variation of the method that best suits the needs and circumstances of the investigation. Two kinds of measurements have been made, relative and absolute. Absolute measures give true rates of microbial metabolism and growth while relative measures give values which are positively correlated with the true rates but are not identical. For example, the incorporation of 14C glucose into bacteria is easily measured and the rate correlates well with the rate of growth of bacteria in laboratory cultures. Glucose, however, is just one substrate of many the bacteria are using and so glucose uptake is a relative measure. Absolute rates of growth can be derived from changes in bacterial numbers (if no predators are present) or from the incorporation of thymidine or specific amino acids into DNA or protein. B. Oxygen measurements
The first successful measurements of heterotrophic activity were of changes in the concentration of oxygen in a water mass. Oxygen, however, constantly exchanges with the atmosphere across the water surface so the water mass must be isolated from the surface for a long enough period for the changes to build up to a measurable level. A stratified lake, where deep-lying water is isolated from the surface water by a thermocline, is one situation where oxygen changes are often measured. This deep water is also in contact with the sediment, however, so the technique is useful only for a measure of all the processes occurring in the water mass plus some
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occurring in the sediment. These include animal respiration and microbial oxidation of sulphide and methane. Riley (1951) made better use of the concept in his examination of water masses at various depths of the Sargasso Sea, in the western North Atlantic Ocean. He assumed that the water mass was at one time at the surface of the ocean and that its oxygen content was at equilibrium with the atmosphere. Later, a few months to decades, the water mass has moved deeper in the ocean and is isolated from the surface waters by temperature and salinity stratification. Because the temperature of the water mass is unchanged over the months or years, the initial concentration of the oxygen may be calculated. When a water sample from the deep-lying water mass is collected and the oxygen measured, the rate of use of oxygen may be measured if the elapsed time is obtained from the physics of the ocean system. Riley measured rates of <0.01 to 1.0 ml O2 I-' year-' with this method. Better estimates have now been made by Jenkins (1977) who used tritium (3H) and its stable isotope daughter 3He as a clock to calculate the period of isolation of the water mass. This method is complicated and has not been widely used. In productive lakes and coastal oceanic waters, changes in oxygen may also be measured in short-term incubations in bottles. The measurements, which are made after a &24-h incubation of the water sample, will include the respiration of animals and algae unless the water is first filtered through a 1-pm mesh (e.g. Nytex). This filtration step may (Hopkinson ef al., 1989) or may not (Williams, 1981) increase the rate of metabolism. Hopkinson ef af. (1989) attributed the increase to the release of control of bacteria by the removal of predatory protozoans. With great care and high-precision techniques, the oxygen method may be extended to offshore waters (Griffith, 1988). C. Radioisotope methods
In the late 1950s radioisotopes became available and marine biologists soon began adding them to water samples to measure first algal photosynthesis (incubations in the light with Hi4CO;) and then bacterial heterotrophy (incubations in the dark with Hi4CO:). The photosynthesis measurements worked well because it was a true tracer experiment. That is, the HL4CO; added only a tiny amount to the rather large pool of HCO; (2 mM in sea water) and as a result the rate of incorporation did not increase because of higher amounts of substrate. The bacterial heterotrophy measurement assumed that the dark incorporation of Hi4CO; was all due to bacteria and that it represented the 6% of the total carbon in heterotrophic growth taken up as inorganic carbon (Sorokin,
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JOHN E. HOBBIE
1965). In reality, algae also take up some Hi4CO; in the dark and this and other sources of error make the method unworkable. . Next, ecologists began to add 14Corganic compounds to water samples in experiments analogous to the Hi4CO; photosynthesis measurement. Unlike the Hi4C03 measurements, the concentration of substrate (organic compound) could not be measured. Also, the uptake measurement was not a tracer-level experiment because the amount of organic compound added, for example ''C-glucose, was hundreds to thousands of times greater than the natural level present; in this situation the higher amounts of substrate increased the uptake rate. Parsons and Strickland (1962) were the first to apply kinetic analysis to measurements in the sea by measuring uptake and incorporation of glucose into microbes at a series of concentrations of added glucose. They found that uptake by the entire community of microbes in a sample of sea water could be described by the same Michaelis-Menten-type equation that describes uptake of a laboratory culture. A maximum velocity of uptake (V,,,) could be obtained which they called a relative heterotrophic potential. This heterotrophic potential has proven to be very useful as it correlates well with bacterial growth and activity. While it only measures one substrate of the hundreds present, the heterotrophic potential is a sensitive way of measuring when and where microbes are active, their relative rates of activity, and their response to such events as phytoplankton growth peaks and pollution. Wright and Hobbie (1966) examined this method in detail and found that the turnover time of the organic substrate could be calculated as well as a single value for the substrate concentration (S) plus the halfsaturation constant for uptake ( K ) . This turnover time tells how fast the natural level of substrate is being cycled by microbes; the (K S) gives the maximum value for the substrate concentration. The V,,, increases when microbial cells become better adapted to using a particular substrate, perhaps by an increase in the number of transport sites, and also increases when the number of cells increases. The V,,, range is four orders of magnitude from deep lakes to polluted ponds (Hobbie and Rublee, 1977).
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D. Improvements in radioisotope methods
It is obvious that some of the 14Csubstrate that is taken up is respired as 14C02during the experiment. This may be collected after the experiment on a piece of paper soaked with an organic base such as phenethylamine and the radioactivity measured by liquid scintillation counting (Hobbie and Crawford, 1969). One major problem with working with a plankton sample is that many
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different types of organisms are present. When radiolabelled organic compounds are added to a sample, bacteria, algae, and even the larvae of many marine animals will take up the organic compound. The solution is to add extremely low amounts of substrate in the experiment. At low substrate levels, bacteria are the only group adapted for uptake and as a consequence, they are responsible for almost all of the uptake of an organic compound. One way to reduce the amount of substrate is to use tritiated compounds (Azam and Holm-Hansen, 1973). This has the disadvantage that it is difficult to correct for respiration losses of the 3H as 3
~
~
0
.
If the experimental question can be answered by studies of a mixed culture of bacteria, then the sample is filtered through a 1.0-pm or 0.6-pm pore size filter (such as the Nuclepore type) and the bacteria in the filtrate are allowed to grow. Their activity may be compared over time or after different treatments by measuring the uptake of a radioactively labelled compound, such as ''C-glucose, added at quite high concentrations. The uptake gives the relative heterotrophic potential or the maximum velocity of uptake. Tranvik and Hofle (1987) used 80 pg glucose I-' to test the ability of cultures to use easily degradable substances. The advent of HPLC (high-performance liquid chromatography) has allowed measurement of the actual concentration of individual organic compounds in sea water that has not been modified in any way such as by desalting. These concentration data plus the turnover information allow calculation of the actual flux of different organic compounds through the bacteria (e.g. Fuhrman and Ferguson, 1986). As noted previously, the measurement of the flux of one o r ten compounds does still not give the actual heterotrophic activity or growth because hundreds of compounds may be used by the natural bacteria. Such a flux measurement is an ideal relative activity measurement but does require a high level of chemical equipment and skill. The most promising development for measuring absolute heterotrophic activity is two techniques for the direct measurement of bacterial growth. Both make use of vital components of bacteria that must be synthesized before division. The first of these techniques (Fuhrman and Azam, 1982) measures the rate of incorporation of 3H-thymidine, a precursor of DNA. Bacteria do incorporate externally supplied thymidine through a salvage pathway and the amount of thymidine incorporated may be translated into bacterial DNA and then into bacterial biomass. The rates of production measured in nature with this technique are reasonable and for the most part fit with ecological constraints such as the total amount of organic matter available. However, the technique as presently developed is not perfect and must be used with care. One problem is that sometimes there is
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an increase in DNA without any change in the number or biomass of the bacteria. Another problem is that it is not possible to determine the actual amount of DNA in the small bacteria that live in the plankton. Any attempt to calibrate using natural bacteria results in a rapid increase in the rate of growth and a potential switch to endogenous production of thymidine. The second promising technique uses the incorporation of 3H-leucine into bacterial protein as the basic measurement. Kirchman et al. (1985) pointed out that most of the heterotrophic bacteria take up 10 nM leucine and incorporate it directly into protein. Recent measurements by Simon and Azam (1989) confirm that leucine is a constant fraction of the total amino acids in bacterial protein and that protein is a constant per cent of bacterial dry weight and carbon. Thus, leucine incorporation may be translated into increase in carbon (growth). E. Ecological importance of heterotrophic activity measurements
Heterotrophic activity measurements have proven to be a valuable ecological tool. With the use of these techniques, it is now known when during the year the bacteria are active and that bacterial activity is positively correlated with algal photosynthesis in the plankton over a range of three orders of magnitude. Up to 60% of the carbon fixed in photosynthesis is broken down by bacteria in the water column. There are high rates of heterotrophic activity by bacteria in warm waters and low rates in cold waters. This low activity in cold waters may allow more of the algal carbon to reach the higher levels of the food chain and may in this way account for the high production of fish in colder waters (Hopkinson et al., 1989; Pomeroy and Deibel, 1986). 11. Background of methods
A. Theory of uptake measurements
The essential elements to understand are: (1) Bacteria in natural waters live in an environment containing only a few micrograms per litre (10100 nM) of each of the simple organic compounds used as substrates. They are well adapted to these concentrations and have transport systems with half-saturation constants that allow uptake at this low level. These transport systems are often very specific for individual substrates. The bacteria may take up many substrates simultaneously. (2) When radioactively labelled substrate is added to water samples at this concentration
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
24 1
or lower, then bacteria take up most of the substrate while the other organisms in the water (algae, animals) take up only a little substrate. Sometimes bacteria may have multiple uptake systems for the same substrate. (3) Uptake can be described by Michaelis-Menten kinetics; it is not necessary to measure the concentration of substrate in order to determine some parameters that are ecologically useful. These elements are explained by means of an imaginary experiment (Fig. 1) in which ''C-glucose is added to a sample of lake or ocean water, the water incubated for 1 h, the water filtered through a membrane filter, and the uptake of I4C into the particles measured with liquid scintillation counting. Actually, the uptake was measured at 10 different concentrations of added ''C-glucose (called A).
V
w
S
- A l g glucose /litre
Fig. 1. Uptake velocity ( v ) of added ''C-glucose ( A ) in the presence of a known amount of substrate (S) by a bacterial population (curve A) and by a second
population (curve B) (algal or bacterial population) and the uptake for the total plankton (curve C). Kinetics terms are defined in the text. The example is imaginary.
We will first assume that we have measured the natural substrate concentration (S) and that it is 2 pg glucose I-'. The S and the added A is taken up according to curve A by a population of bacteria. There is another uptake system present in the water sample (curve B) which might belong to bacteria of another type or to algae or might even be a different transport system of the A bacteria. The uptake of the entire plankton population is the sum of bacteria plus algae/bacteria (curve C). It is
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obvious that uptake due to bacteria must be measured at close to the natural substrate concentration ( S ) or else there will be interference from curve B. The four additions at 1, 2, 3 and 4 pg 1-’ would give the best estimate while the measurements at 6, 12, 18, and 24 pg 1-’ would give neither information about the bacteria nor information about what was actually happening at S (the imaginary vertical line where A = 0). The uptake of substrate by the bacteria (curve A) becomes saturated as A is The value increased until a maximum velocity of uptake is reached (Vmax). of the substrate concentration when the uptake rate is half the V,,, is K, the half-saturation constant.
B. Mathematical description We will now assume that we do not know the natural substrate concentration (the usual situation). As explained in Wright and Hobbie (1966), the velocity of uptake ( v ) at substrate (S A ) in Fig. 1 is
+
where f is the fraction of the isotope added that is taken up and t is the time of incubation. Equation 1 may also be rearranged as tlf = (S
+ A)/v(S+A)
(2)
Bacterial uptake (curve A) follows a saturation curve or the MichaelisMenten equation
a linear transformation is employed so that To better estimate Vmax, (S
+ A)/v(S+A)= ( K + S + A)/Vmax
(4)
Combining equations 2 and 4 gives tlf = ( K
+ S)/Vm,, + A/Vm,,
(5)
Accordingly, the data that produced curve C in Fig. 1 may be replotted according to equation 5 to produce Fig. 2. Note that this transformation is employed because it is not necessary to know the value of S and in nature this is usually extremely difficult to measure. The plot employs the two values that are known, A or the value of the substrate added in the experiment, and tlf which is the inverse of the fractional uptake per unit of time of the radiolabelled substrate.
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
'KtS'
243
A
Mg glucose/litre
Fig. 2. The data of Fig. 1 (curve C) transformed by equations 4 and 5. The Y axis is incubation time (h) divided by the fractional uptake of the radioisotope and the X axis is the added ''C-glucose in vg 1-'.
c.
vmax,
T , - (K
+ S)
It can be seen that curve C in Fig. 1 does, indeed, appear to be the result of the uptake by two populations. If we want to estimate the V,,, of the bacterial population (which is the inverse of the slope), then the portion of the curve closest to the point where A = 0 should be used. In this artificial example we know that V,,, is 1 (Fig. 1) so that the interference from the other population introduced an error. If the entire curve is used, the estimated V,,, is 2.3. If the four points closest to the Y axis are used, the estimated V,,, is close to 1. The example above illustrates the error possible when too high substrate concentrations are used in the measurement; but errors can also arise when too low concentrations are used. Fuhrman and Ferguson (1986) showed very well (Fig. 3) by the addition of extremely small quantities of substrate that there were two different uptake systems, one with a very low K and low V,,,, present in this sample of ocean water. They actually measured 4.5 nM serine in the water by HPLC which is close to the value of K + S from the extrapolation of the whole curve (they ignored the two points closest to the Y axis). My interpretation of the low K, low V,,, uptake curve is that it reveals some basic properties of the membrane transport system or of the concentrations in the space immediately next to this system.
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35r
30 -
2o
I
I
25
t/f (hr) 15 10 -
nM Added Serine Fig. 3. Uptake of 3H-serineby bacteria in a water sample from New York Bight on 9 February 1984. Data plotted according to equation 5. The figure is modified from Fig. 1 of Fuhrman and Ferguson (1986).
To avoid these errors, it is usually safe to measure uptake at 10-40 nM for sugars and 1-10 nM for amino acids. Another bit of information is the extrapolation of the curve to the X axis where the intercept gives - (K S). Thus, from equation 5 when t/f is 0, then A = -(K S). Neither quantity can be measured but from their sum we know a maximum value for the substrate (in Fig. 2 it was about 2 pg glucose I-', in Fig. 3 it is 1.4 nM if the two low points are ignored). Finally, we may also measure the turnover time of the substrate (T) as the extrapolation to the Y axis. From equation 5, the intercept on the Y axis is (K + S)/Vmax.When A = 0, then from equation 4
+
+
(K + S)/V,,,
=
( S ) / V ( ~or, T
(6)
because the natural concentration of substrate (S) divided by the velocity of uptake (v(~)) is the turnover time, T. Again, neither quantity can be easily measured. In Fig. 2, T is about 2 h and in Fig. 3 it is 23 h (again, ignoring two points closest to the Y axis).
D. Different types of heterotrophic activity measurements The five types of measurements described earlier may now be described in terms of their relation to the theory given above. First, are the
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measurements using kinetic analysis. It is important to use as low concentrations as possible of added substrate but sometimes there must be a trade-off. The microbial activity may be so low that more isotope must be added in order to get significant uptake after an incubation of 0.5-4 h. This means that the concentrations may be higher than optimal. So the rule is to add as low concentrations as possible while still obtaining enough radioisotope in the particles on the filter for good counting statistics. At a minimum 400 cpm are necessary; several thousand counts per minute are ideal. In very clear oceanic waters, the cpm may be increased by filtering more water through the filter. When the actual T and V,,, are needed, then 14C must be used and the respired I4CO2 collected and counted. When relative results are adequate and respiration can be ignored, then tritiated compounds may be used (these are available at much higher specific activity and so very low concentrations of added substrate are possible). The second type of measurement is the tracer level addition. In this type of experiment, a single concentration of substrate is used but the concentration is extremely low. In Figs 2 and 3 the single addition would be very close to the A = 0 point. If the addition is low enough, then a good estimate of the Y intercept may be obtained. For ''C-labelled compounds, different compounds are available at different specific activities and this may limit which may be used. In a recent catalog, ~-U-'~C-glucose was available at 230 mCi mmol-' while ~-3-l~C-serine was available at only 5060 mCi mmol-'. For 3H-labelled compounds the specific activity is much higher. For example, Azam and Holm-Hansen (1973) used glucose at 8600 mCi mmol-'. When the activity of bacteria alone is being measured, either in a laboratory culture or in a mixed culture of bacteria from nature grown on filtered sea or lake water, then a third type of measurement may be the easiest one to make. This is the determination of the V,,, of the culture through the addition of a single high level of substrate. In Fig. 1, curve A represents the uptake of bacteria; a high-level addition of greater than 5 pg glucose I-' would be adequate to estimate the V,,,. The advantages are that only one quick measurement must be made and high amounts of radioactivity are incorporated. The disadvantage is that only one type of information (V,,,) is obtained but for experimental manipulations this is often enough to determine an effect. The fourth type of measurement is the use of 'H-thymidine to measure the actual growth of bacteria (Fuhrman and Azam, 1982). The method has been thoroughly reviewed by Moriarty (1986) and will not be described in detail here. The addition of thymidine is made at 5-20 nM (0.9-3.8 pg I-') which are very low levels but are not tracer levels. Instead, they are the
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concentrations at which the de n o w synthesis of thymidine is completely inhibited. The transport mechanisms and uptake kinetics of thymidine are .similar to those of other organic compounds and at these concentrations it is the bacteria which take up most of the compound. In the growth measurement, it is the incorporation into DNA, not the transport into the cell, which is measured (the radioactivity remaining in the cells is measured after an extraction with cold TCA). When this is done, then any kinetic analysis, for example to give (K S), measures both the external concentration of thymidine and the amount of thymidine internally produced which diluted the isotope after it was transported into the cell. Finally, the amount incorporated is multiplied by a factor which is the number of bacteria per mole of DNA. Once the number of bacteria produced is known, the carbon and biomass may be calculated from appropriate factors. Overall, it appears that the results of bacterial growth analyses with thymidine give ecologically reasonable results. There are, however, enough inexplicable instances of high thymidine incorporation without bacterial growth, of problems with calibration (is there less DNA per cell in very small cells than in larger cells?), and of lack of thymidine uptake by certain types of bacteria that the method must be judged as promising but still imperfect in many ways. The fifth measurement is of the incorporation of leucine into bacterial protein. In the technique of Kirchman et al. (1985) as extended by Simon and Azam (1989), 3H-leucine is added to samples at a final concentration of 10 nM. This amount overwhelms the ambient leucine pool (about 1 nM) and maximizes leucine uptake. After incubation, the bacteria are extracted with hot TCA (to hydrolyse DNA and RNA) and then filtered onto 0.45pm pore size membrane filters for analysis of the incorporated tritium. The assumptions behind this method are that protein is a stable fraction of bacterial dry weight (63%), that leucine makes up a constant percentage of the total protein, that leucine at nM levels is taken up exclusively by marine bacteria, and that there is a consistent relationship between protein and cell volume for various sizes of bacteria. Thus, rates of production of bacterial protein may be translated directly into rates of production of bacterial carbon and biomass. With HPLC, Simon and Azam have successfully tested many of these assumptions on filtered samples that represent the bacteria between 0.2 and 0.6 pm. In most of the cases they examined, the leucine method gave comparable results to the thymidine method. This leucine method has fewer assumptions than the thymidine method for measuring bacterial heterotrophic activity and represents a very promising development.
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7 . MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
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A.
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Methods
Standard method
There is a standard method which is slightly modified for each of the various types of radioisotope experiments. However, once a method is chosen, tests must be made to decide upon incubation time and amount of labelled substrate to add. Samples are collected in acid-washed bottles of glass or plastic, held at the in situ temperature in the dark, and the radioisotope experiment begun as soon as possible. This holding period should be no longer than 30 min. Samples may be incubated in any volume of flask but 10-20 ml of sample are convenient to filter through a 25-mm diameter filter so that large test tubes may be used. If the respired I4CO2 is to be collected, then a 25-ml Erlenmeyer flask is suitable. Replicate or triplicate samples along with killed controls are necessary. In clean ocean water, special techniques will often prevent contamination by trace metals (such as Cu) and increase the rate of bacterial activity. Ferguson and Sunda (1984) achieved this by using a 30-1 Teflon-lined water sampler suspended on a plastic-coated hydrowire and closed with a Teflon messenger. They also carefully cleaned their glassware and rinsed with specially prepared distilled water. Isotope is diluted in distilled water with special care to avoid contamination from organic compounds and trace metals (Ferguson and Sunda, 1984). The isotope is delivered to the sample with automatic micropipettes so that, for example, 25, 50, 75, and 100 pl will produce the desired concentrations in 10 o r 20 ml of sample. The incubation is carried out in the dark. The time should be long enough to produce good uptake by the bacteria yet short enough that there is no significant growth or adaptation during the experiment. The ideal time must be chosen in a test so that the amount of uptake increases linearly over time. Incubation is ended by the addition of buffered formalin (0.4% final concentration, pH 8). For a control, the isotope is also added to a formalin-killed sample. When respired 14C02is to be measured, the incubation is carried out in a 25-ml Erlenmeyer flask (Hobbie and Crawford, 1969). After addition of the isotope, the flask is immediately sealed with a rubber serum stopper that has a plastic cup suspended from it. The cup contains a 25 x 51 mm piece of accordion-folded chromatographic paper (Whatman No. 1). After incubation, 0.2 ml of a 2 N H2S04 solution is injected through the septum to stop the uptake. Next, still working through the septum, 0.2 ml of an organic base (phenethylamine) is slowly added to the folded paper and the
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flask is then shaken for an hour at room temperature so that the I4CO2is absorbed. After this, the paper is placed in a scintillation vial with a scintillation cocktail and counted. Under these conditions, tests with a ''C02 standard showed that only 82% of the I4C was counted even after a counting correction is made with an internal standard. This loss might be due to absorption of the light inside the filter paper. Thus, the final counts were multiplied by 1.23. The samples are filtered through a membrane filter and rinsed with at least 5 ml of filtered sea water. The 25-mm diameter 0.45-pm pore size cellulose filters of Millipore or Sartorius work well and capture all of the bacteria. Filters are placed in standard counting vials and 1 ml ethyl acetate is added to dissolve the filter. The samples are counted with liquid scintillation and standard scintillation cocktails.
B.
Kinetic analysis
The method is that of Wright and Hobbie (1966) and Fuhrman and Ferguson (1986). The sample (10-50 ml) is incubated in flasks or in rinsed polyethylene bags (Whirlpak) with or without light. The final concentration of the added 3H- or 14C-labelledsubstrate is 10-100 nM or approximately 1-10 pg 1-'. Sugars, acetate, and amino acids may be used. The respired 14C02may be collected into an organic base or the respired 3H20 calculated from the loss from solution during freeze-drying.
C. Tracer-level additions The method is that of Azam and Holm-Hansen (1973). For analysis of glucose uptake in sea water they added 0.01 pg gIuc0se-6-~Hto 100 ml of sample (to give 0.5 nM) and incubated for 0.5-4 h. The loss of labelled intracellular pool material was minimized by not killing the sample with formalin before washing the filter with 20 ml of iced and filtered sea water.
D. Addition of high level of substrate The only change used in this method is that a single concentration of labelled substrate is used. Tranvik and Hofle (1987) used 80 pg glucose I-' in short-term measurements of the adaptation of microbes in a lakewater culture to taking up sugar.
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E. Thymidine incorporation The method is that of Fuhrman and Azam (1982) and Simon and Azam (1989). Methyl-l-[3H]-thymidine (70 Ci mmol-') is added to 10-ml samples to give a final concentration of 5 nM. Formalin-treated controls are run in parallel. The incubation times are 1 5 4 5 min depending upon the temperature (incubations are carried out in the dark at the in situ temperature). After stopping the incubation with formalin, samples are filtered onto membrane filters, extracted with ice-cold 5% TCA and radioassayed with liquid scintillation. The thymidine incorporation rates are converted to cell multiplication rates by multiplying by the factor of 1.18 x 10" cells produced per mol of thymidine incorporated. Cell multiplication rates are converted to carbon production by using the cell carbon values for the given cell size (Table I).
TABLE I Cell volumes, protein composition (in femtograms or lo-'' g), dry weight, and carbon content of average marine bacteria in the size range 0.026-0.4 pm3 (adapted from Simon and Azarn, 1989) Volume CLm3
fg
0.026 0.036 0.050 0.070 0.100 0.200 0.400
12.1 14.7 17.7 21.6 26.7 40.3 60.6
Protein
YOVOI.
%dw
Dry weight fg
fg
Carbon %dw
46.5 40.8 35.4 30.9 26.7 20.2 15.2
61.4 62.5 63.0 62.8 62.8 63.5 63.3
19.7 23.5 28.1 34.4 42.5 63.5 95.8
10.4 12.6 15.2 18.7 23.3 35.0 53.3
52.0 53.6 54.2 54.3 54.7 55.1 55.7
F. Leucine incorporation
In this method (Simon and Azam, 1989), [3,4,5-3H]-1-leucine (140 Ci mmol-') is added to triplicate 10 ml samples to produce a concentration of 10 nM. Formalin-treated controls are run in parallel. Sample incubations (20-40 min) are ended by the addition of formalin and then samples are extracted with 5% TCA at 95-100°C for 30 min to hydrolyse RNA and DNA. Longer extraction times lead to significant hydrolysis of protein. The extracted samples are cooled and filtered. One necessary correction is for the isotope dilution within the cell. Simon and Azam (1989) suggest that a factor of two is appropriate in many situations but the pool specific activity may also be measured with HPLC.
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JOHN E. HOBBIE
The conversion of 3H-leucine incorporated to carbon or biomass produced by the bacteria is made by the following formula (Simon and Azam, 1989): BPP = (mol 3H-leucine incorporated)(100/7.3)(131.2)(2)(0.86)
(7) where BPP is bacterial protein production (in g C), 10017.3 is 100 divided by the mol % of leucine in protein, 131.2is the formula weight of leucine, 2 is the intracellular isotope dilution of labelled leucine, and 0.86 converts protein to carbon. If the carbon per cell is known (from Table I), then BPP can also be converted into bacterial cell production. If the isotope dilution is not known, then using 1 instead of 2 in formula 7 gives a minimum estimate of BPP. Azam (personal communication) states that even if the isotope dilution is known from HPLC measurements, the absolute value for growth is still elusive because the dilution gives the maximum estimate of BPP. We still do not know how much of the isotope dilution is due to de novo synthesis by the active cells and how much is due to unlabelled leucine pools of the inactive cells. In coastal waters the range from minimum to maximum is about two-fold. References Azam, F. and Holm-Hansen, 0. (1973). Mar. Biol. 23, 191-196. Ferguson, R. L. and Sunda, W. G . (1984). Limnol. Oceanogr. 29, 258-274. Fuhrman, J. A. and Azam, F. (1982). Mar. Biol. 66, 109-120. Fuhrman, J. A. and Ferguson, R . L. (1986). Mar. Ecol. Prog. Ser. 33, 237-242. Griffith, P. C. (1988). Limnol. Oceanogr. 33, 632-638. Hobbie, J. E. and Crawford, C. C. (1969). Limnol. Oceanogr. 14, 528-532. Hobbie, J. E. and Rublee, P. (1977). In “Aquatic Microbial Communities” (J. Cairns Jr, Ed.), pp. 4 4 4 7 6 , Garland Publishing Co., New York and London. Hopkinson, C. S. Jr, Sherr, B. and Wiebe, W. J . (1989). Mar. Ecol. Prog. Ser. 51, 155-166. Jenkins, W. J. (1977). Science 196, 291-292. Kirchman, D. L., K’Ness, E. and Hodson, R. (1985). Appl. Environ. Microbiol. 49, 599-607. Moriarty, D. W. (1986). Adv. Microbiol. Ecol. 9, 245-292. Parsons, T. R. and Strickland, J . D. H. (1962). Deep-sea Res. 8, 211-222. Pomeroy, L. and Deibel, 0. (1986). Science 233, 359-361. Riley, G. A . (1951). Bull. Bingham Oceanogr. Coll. 13, 1-126. Simon, M. and Azam, F. (1989). Mar. Ecol. Prog. Ser. 51, 201-213. Sorokin, Yu. I. (1965). Mem. 1st. Ital. Idrobiol. 18, 187-205. Tranvik, L. J. and Hofle, M. G. (1987). Appl. Environ. Microbiol. 53, 482-488. Williams, P. J. LeB. (1981). Oceanologica Acfa 4, 359-364. Wright, R. T. and Hobbie, J. E. (1966). Ecology 47, 447-464.
I. Introduction
.
.
.
.
* .
. .
A. Heterotrophic activity . B. Oxygen measurements . . . C. Radioisotopemethods . . . . D. Improvements in radioisotope methods . . E. Ecological importance of heterotrophic activity measurements 11. Backgroundofmethods . . . . . . A. Theory of uptake measurements . B. Mathematical description . . . . c. v,,,, T , - ( K + S) . . . . . . D. Different types of heterotrophic activity measurements 111. Methods . . . . . . . . A. Standardmethod . * . . . . B. Kinetic analysis . . . C. Tracer-level additions . . . D. Addition of high level of substrate . . . E. Thymidine incorporation . . . F. Leucine incorporation . . . . References . . .
235 235 236 237 238 240 240 240 242 243 244 247 247 248 248 248 249 249 250
I. Introduction A.
Heterotrophic activity
In order to understand what microbes are actually doing in nature, as opposed to what microbes are capable of doing in the laboratory, ecologists must make measurements of rates of microbial processes in the real world. One obvious and important process is metabolism and growth METHODS IN MICROBIOLOGY VOLUME 22 ISBN 0-12-521522-3
Copyright @ 1990 by Academic Press Limited All rights of reproduction in any form reserved.
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of bacteria on organic compounds supplied from outside the cell. This heterotrophic growth decomposes most of the organic matter produced in the biosphere of land and water. In the water of lakes and oceans, measurements of bacterial heterotrophy are difficult because the great dilution of both substrates and organisms allows only low rates of activity, which laboratory techniques cannot measure, and because of the sensitivity of microbes to manipulation, which means that the rates are changed by, for example, incubations of 24 h or by filtration through a 10-pm pore size filter. For these reasons, techniques have been developed to measure low rates of heterotrophy either in undisturbed waters or in samples of lake or ocean water incubated briefly with radioisotopes. The techniques discussed here have given a tremendous amount of information about the role, rates of activity, and factors limiting the heterotrophic bacteria in the plankton. Only some of the methods, those using radioisotopes, are presented in detail here. These are all variations on the theme of the addition of extremely low concentrations of labelled organic substrates to water samples. Anyone working in this field should understand the background and theory of this basic theme and then choose the variation of the method that best suits the needs and circumstances of the investigation. Two kinds of measurements have been made, relative and absolute. Absolute measures give true rates of microbial metabolism and growth while relative measures give values which are positively correlated with the true rates but are not identical. For example, the incorporation of 14C glucose into bacteria is easily measured and the rate correlates well with the rate of growth of bacteria in laboratory cultures. Glucose, however, is just one substrate of many the bacteria are using and so glucose uptake is a relative measure. Absolute rates of growth can be derived from changes in bacterial numbers (if no predators are present) or from the incorporation of thymidine or specific amino acids into DNA or protein. B. Oxygen measurements
The first successful measurements of heterotrophic activity were of changes in the concentration of oxygen in a water mass. Oxygen, however, constantly exchanges with the atmosphere across the water surface so the water mass must be isolated from the surface for a long enough period for the changes to build up to a measurable level. A stratified lake, where deep-lying water is isolated from the surface water by a thermocline, is one situation where oxygen changes are often measured. This deep water is also in contact with the sediment, however, so the technique is useful only for a measure of all the processes occurring in the water mass plus some
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occurring in the sediment. These include animal respiration and microbial oxidation of sulphide and methane. Riley (1951) made better use of the concept in his examination of water masses at various depths of the Sargasso Sea, in the western North Atlantic Ocean. He assumed that the water mass was at one time at the surface of the ocean and that its oxygen content was at equilibrium with the atmosphere. Later, a few months to decades, the water mass has moved deeper in the ocean and is isolated from the surface waters by temperature and salinity stratification. Because the temperature of the water mass is unchanged over the months or years, the initial concentration of the oxygen may be calculated. When a water sample from the deep-lying water mass is collected and the oxygen measured, the rate of use of oxygen may be measured if the elapsed time is obtained from the physics of the ocean system. Riley measured rates of <0.01 to 1.0 ml O2 I-' year-' with this method. Better estimates have now been made by Jenkins (1977) who used tritium (3H) and its stable isotope daughter 3He as a clock to calculate the period of isolation of the water mass. This method is complicated and has not been widely used. In productive lakes and coastal oceanic waters, changes in oxygen may also be measured in short-term incubations in bottles. The measurements, which are made after a &24-h incubation of the water sample, will include the respiration of animals and algae unless the water is first filtered through a 1-pm mesh (e.g. Nytex). This filtration step may (Hopkinson ef al., 1989) or may not (Williams, 1981) increase the rate of metabolism. Hopkinson ef af. (1989) attributed the increase to the release of control of bacteria by the removal of predatory protozoans. With great care and high-precision techniques, the oxygen method may be extended to offshore waters (Griffith, 1988). C. Radioisotope methods
In the late 1950s radioisotopes became available and marine biologists soon began adding them to water samples to measure first algal photosynthesis (incubations in the light with Hi4CO;) and then bacterial heterotrophy (incubations in the dark with Hi4CO:). The photosynthesis measurements worked well because it was a true tracer experiment. That is, the HL4CO; added only a tiny amount to the rather large pool of HCO; (2 mM in sea water) and as a result the rate of incorporation did not increase because of higher amounts of substrate. The bacterial heterotrophy measurement assumed that the dark incorporation of Hi4CO; was all due to bacteria and that it represented the 6% of the total carbon in heterotrophic growth taken up as inorganic carbon (Sorokin,
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1965). In reality, algae also take up some Hi4CO; in the dark and this and other sources of error make the method unworkable. . Next, ecologists began to add 14Corganic compounds to water samples in experiments analogous to the Hi4CO; photosynthesis measurement. Unlike the Hi4C03 measurements, the concentration of substrate (organic compound) could not be measured. Also, the uptake measurement was not a tracer-level experiment because the amount of organic compound added, for example ''C-glucose, was hundreds to thousands of times greater than the natural level present; in this situation the higher amounts of substrate increased the uptake rate. Parsons and Strickland (1962) were the first to apply kinetic analysis to measurements in the sea by measuring uptake and incorporation of glucose into microbes at a series of concentrations of added glucose. They found that uptake by the entire community of microbes in a sample of sea water could be described by the same Michaelis-Menten-type equation that describes uptake of a laboratory culture. A maximum velocity of uptake (V,,,) could be obtained which they called a relative heterotrophic potential. This heterotrophic potential has proven to be very useful as it correlates well with bacterial growth and activity. While it only measures one substrate of the hundreds present, the heterotrophic potential is a sensitive way of measuring when and where microbes are active, their relative rates of activity, and their response to such events as phytoplankton growth peaks and pollution. Wright and Hobbie (1966) examined this method in detail and found that the turnover time of the organic substrate could be calculated as well as a single value for the substrate concentration (S) plus the halfsaturation constant for uptake ( K ) . This turnover time tells how fast the natural level of substrate is being cycled by microbes; the (K S) gives the maximum value for the substrate concentration. The V,,, increases when microbial cells become better adapted to using a particular substrate, perhaps by an increase in the number of transport sites, and also increases when the number of cells increases. The V,,, range is four orders of magnitude from deep lakes to polluted ponds (Hobbie and Rublee, 1977).
+
D. Improvements in radioisotope methods
It is obvious that some of the 14Csubstrate that is taken up is respired as 14C02during the experiment. This may be collected after the experiment on a piece of paper soaked with an organic base such as phenethylamine and the radioactivity measured by liquid scintillation counting (Hobbie and Crawford, 1969). One major problem with working with a plankton sample is that many
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
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different types of organisms are present. When radiolabelled organic compounds are added to a sample, bacteria, algae, and even the larvae of many marine animals will take up the organic compound. The solution is to add extremely low amounts of substrate in the experiment. At low substrate levels, bacteria are the only group adapted for uptake and as a consequence, they are responsible for almost all of the uptake of an organic compound. One way to reduce the amount of substrate is to use tritiated compounds (Azam and Holm-Hansen, 1973). This has the disadvantage that it is difficult to correct for respiration losses of the 3H as 3
~
~
0
.
If the experimental question can be answered by studies of a mixed culture of bacteria, then the sample is filtered through a 1.0-pm or 0.6-pm pore size filter (such as the Nuclepore type) and the bacteria in the filtrate are allowed to grow. Their activity may be compared over time or after different treatments by measuring the uptake of a radioactively labelled compound, such as ''C-glucose, added at quite high concentrations. The uptake gives the relative heterotrophic potential or the maximum velocity of uptake. Tranvik and Hofle (1987) used 80 pg glucose I-' to test the ability of cultures to use easily degradable substances. The advent of HPLC (high-performance liquid chromatography) has allowed measurement of the actual concentration of individual organic compounds in sea water that has not been modified in any way such as by desalting. These concentration data plus the turnover information allow calculation of the actual flux of different organic compounds through the bacteria (e.g. Fuhrman and Ferguson, 1986). As noted previously, the measurement of the flux of one o r ten compounds does still not give the actual heterotrophic activity or growth because hundreds of compounds may be used by the natural bacteria. Such a flux measurement is an ideal relative activity measurement but does require a high level of chemical equipment and skill. The most promising development for measuring absolute heterotrophic activity is two techniques for the direct measurement of bacterial growth. Both make use of vital components of bacteria that must be synthesized before division. The first of these techniques (Fuhrman and Azam, 1982) measures the rate of incorporation of 3H-thymidine, a precursor of DNA. Bacteria do incorporate externally supplied thymidine through a salvage pathway and the amount of thymidine incorporated may be translated into bacterial DNA and then into bacterial biomass. The rates of production measured in nature with this technique are reasonable and for the most part fit with ecological constraints such as the total amount of organic matter available. However, the technique as presently developed is not perfect and must be used with care. One problem is that sometimes there is
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an increase in DNA without any change in the number or biomass of the bacteria. Another problem is that it is not possible to determine the actual amount of DNA in the small bacteria that live in the plankton. Any attempt to calibrate using natural bacteria results in a rapid increase in the rate of growth and a potential switch to endogenous production of thymidine. The second promising technique uses the incorporation of 3H-leucine into bacterial protein as the basic measurement. Kirchman et al. (1985) pointed out that most of the heterotrophic bacteria take up 10 nM leucine and incorporate it directly into protein. Recent measurements by Simon and Azam (1989) confirm that leucine is a constant fraction of the total amino acids in bacterial protein and that protein is a constant per cent of bacterial dry weight and carbon. Thus, leucine incorporation may be translated into increase in carbon (growth). E. Ecological importance of heterotrophic activity measurements
Heterotrophic activity measurements have proven to be a valuable ecological tool. With the use of these techniques, it is now known when during the year the bacteria are active and that bacterial activity is positively correlated with algal photosynthesis in the plankton over a range of three orders of magnitude. Up to 60% of the carbon fixed in photosynthesis is broken down by bacteria in the water column. There are high rates of heterotrophic activity by bacteria in warm waters and low rates in cold waters. This low activity in cold waters may allow more of the algal carbon to reach the higher levels of the food chain and may in this way account for the high production of fish in colder waters (Hopkinson et al., 1989; Pomeroy and Deibel, 1986). 11. Background of methods
A. Theory of uptake measurements
The essential elements to understand are: (1) Bacteria in natural waters live in an environment containing only a few micrograms per litre (10100 nM) of each of the simple organic compounds used as substrates. They are well adapted to these concentrations and have transport systems with half-saturation constants that allow uptake at this low level. These transport systems are often very specific for individual substrates. The bacteria may take up many substrates simultaneously. (2) When radioactively labelled substrate is added to water samples at this concentration
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
24 1
or lower, then bacteria take up most of the substrate while the other organisms in the water (algae, animals) take up only a little substrate. Sometimes bacteria may have multiple uptake systems for the same substrate. (3) Uptake can be described by Michaelis-Menten kinetics; it is not necessary to measure the concentration of substrate in order to determine some parameters that are ecologically useful. These elements are explained by means of an imaginary experiment (Fig. 1) in which ''C-glucose is added to a sample of lake or ocean water, the water incubated for 1 h, the water filtered through a membrane filter, and the uptake of I4C into the particles measured with liquid scintillation counting. Actually, the uptake was measured at 10 different concentrations of added ''C-glucose (called A).
V
w
S
- A l g glucose /litre
Fig. 1. Uptake velocity ( v ) of added ''C-glucose ( A ) in the presence of a known amount of substrate (S) by a bacterial population (curve A) and by a second
population (curve B) (algal or bacterial population) and the uptake for the total plankton (curve C). Kinetics terms are defined in the text. The example is imaginary.
We will first assume that we have measured the natural substrate concentration (S) and that it is 2 pg glucose I-'. The S and the added A is taken up according to curve A by a population of bacteria. There is another uptake system present in the water sample (curve B) which might belong to bacteria of another type or to algae or might even be a different transport system of the A bacteria. The uptake of the entire plankton population is the sum of bacteria plus algae/bacteria (curve C). It is
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obvious that uptake due to bacteria must be measured at close to the natural substrate concentration ( S ) or else there will be interference from curve B. The four additions at 1, 2, 3 and 4 pg 1-’ would give the best estimate while the measurements at 6, 12, 18, and 24 pg 1-’ would give neither information about the bacteria nor information about what was actually happening at S (the imaginary vertical line where A = 0). The uptake of substrate by the bacteria (curve A) becomes saturated as A is The value increased until a maximum velocity of uptake is reached (Vmax). of the substrate concentration when the uptake rate is half the V,,, is K, the half-saturation constant.
B. Mathematical description We will now assume that we do not know the natural substrate concentration (the usual situation). As explained in Wright and Hobbie (1966), the velocity of uptake ( v ) at substrate (S A ) in Fig. 1 is
+
where f is the fraction of the isotope added that is taken up and t is the time of incubation. Equation 1 may also be rearranged as tlf = (S
+ A)/v(S+A)
(2)
Bacterial uptake (curve A) follows a saturation curve or the MichaelisMenten equation
a linear transformation is employed so that To better estimate Vmax, (S
+ A)/v(S+A)= ( K + S + A)/Vmax
(4)
Combining equations 2 and 4 gives tlf = ( K
+ S)/Vm,, + A/Vm,,
(5)
Accordingly, the data that produced curve C in Fig. 1 may be replotted according to equation 5 to produce Fig. 2. Note that this transformation is employed because it is not necessary to know the value of S and in nature this is usually extremely difficult to measure. The plot employs the two values that are known, A or the value of the substrate added in the experiment, and tlf which is the inverse of the fractional uptake per unit of time of the radiolabelled substrate.
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
'KtS'
243
A
Mg glucose/litre
Fig. 2. The data of Fig. 1 (curve C) transformed by equations 4 and 5. The Y axis is incubation time (h) divided by the fractional uptake of the radioisotope and the X axis is the added ''C-glucose in vg 1-'.
c.
vmax,
T , - (K
+ S)
It can be seen that curve C in Fig. 1 does, indeed, appear to be the result of the uptake by two populations. If we want to estimate the V,,, of the bacterial population (which is the inverse of the slope), then the portion of the curve closest to the point where A = 0 should be used. In this artificial example we know that V,,, is 1 (Fig. 1) so that the interference from the other population introduced an error. If the entire curve is used, the estimated V,,, is 2.3. If the four points closest to the Y axis are used, the estimated V,,, is close to 1. The example above illustrates the error possible when too high substrate concentrations are used in the measurement; but errors can also arise when too low concentrations are used. Fuhrman and Ferguson (1986) showed very well (Fig. 3) by the addition of extremely small quantities of substrate that there were two different uptake systems, one with a very low K and low V,,,, present in this sample of ocean water. They actually measured 4.5 nM serine in the water by HPLC which is close to the value of K + S from the extrapolation of the whole curve (they ignored the two points closest to the Y axis). My interpretation of the low K, low V,,, uptake curve is that it reveals some basic properties of the membrane transport system or of the concentrations in the space immediately next to this system.
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35r
30 -
2o
I
I
25
t/f (hr) 15 10 -
nM Added Serine Fig. 3. Uptake of 3H-serineby bacteria in a water sample from New York Bight on 9 February 1984. Data plotted according to equation 5. The figure is modified from Fig. 1 of Fuhrman and Ferguson (1986).
To avoid these errors, it is usually safe to measure uptake at 10-40 nM for sugars and 1-10 nM for amino acids. Another bit of information is the extrapolation of the curve to the X axis where the intercept gives - (K S). Thus, from equation 5 when t/f is 0, then A = -(K S). Neither quantity can be measured but from their sum we know a maximum value for the substrate (in Fig. 2 it was about 2 pg glucose I-', in Fig. 3 it is 1.4 nM if the two low points are ignored). Finally, we may also measure the turnover time of the substrate (T) as the extrapolation to the Y axis. From equation 5, the intercept on the Y axis is (K + S)/Vmax.When A = 0, then from equation 4
+
+
(K + S)/V,,,
=
( S ) / V ( ~or, T
(6)
because the natural concentration of substrate (S) divided by the velocity of uptake (v(~)) is the turnover time, T. Again, neither quantity can be easily measured. In Fig. 2, T is about 2 h and in Fig. 3 it is 23 h (again, ignoring two points closest to the Y axis).
D. Different types of heterotrophic activity measurements The five types of measurements described earlier may now be described in terms of their relation to the theory given above. First, are the
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measurements using kinetic analysis. It is important to use as low concentrations as possible of added substrate but sometimes there must be a trade-off. The microbial activity may be so low that more isotope must be added in order to get significant uptake after an incubation of 0.5-4 h. This means that the concentrations may be higher than optimal. So the rule is to add as low concentrations as possible while still obtaining enough radioisotope in the particles on the filter for good counting statistics. At a minimum 400 cpm are necessary; several thousand counts per minute are ideal. In very clear oceanic waters, the cpm may be increased by filtering more water through the filter. When the actual T and V,,, are needed, then 14C must be used and the respired I4CO2 collected and counted. When relative results are adequate and respiration can be ignored, then tritiated compounds may be used (these are available at much higher specific activity and so very low concentrations of added substrate are possible). The second type of measurement is the tracer level addition. In this type of experiment, a single concentration of substrate is used but the concentration is extremely low. In Figs 2 and 3 the single addition would be very close to the A = 0 point. If the addition is low enough, then a good estimate of the Y intercept may be obtained. For ''C-labelled compounds, different compounds are available at different specific activities and this may limit which may be used. In a recent catalog, ~-U-'~C-glucose was available at 230 mCi mmol-' while ~-3-l~C-serine was available at only 5060 mCi mmol-'. For 3H-labelled compounds the specific activity is much higher. For example, Azam and Holm-Hansen (1973) used glucose at 8600 mCi mmol-'. When the activity of bacteria alone is being measured, either in a laboratory culture or in a mixed culture of bacteria from nature grown on filtered sea or lake water, then a third type of measurement may be the easiest one to make. This is the determination of the V,,, of the culture through the addition of a single high level of substrate. In Fig. 1, curve A represents the uptake of bacteria; a high-level addition of greater than 5 pg glucose I-' would be adequate to estimate the V,,,. The advantages are that only one quick measurement must be made and high amounts of radioactivity are incorporated. The disadvantage is that only one type of information (V,,,) is obtained but for experimental manipulations this is often enough to determine an effect. The fourth type of measurement is the use of 'H-thymidine to measure the actual growth of bacteria (Fuhrman and Azam, 1982). The method has been thoroughly reviewed by Moriarty (1986) and will not be described in detail here. The addition of thymidine is made at 5-20 nM (0.9-3.8 pg I-') which are very low levels but are not tracer levels. Instead, they are the
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JOHN E. HOBBIE
concentrations at which the de n o w synthesis of thymidine is completely inhibited. The transport mechanisms and uptake kinetics of thymidine are .similar to those of other organic compounds and at these concentrations it is the bacteria which take up most of the compound. In the growth measurement, it is the incorporation into DNA, not the transport into the cell, which is measured (the radioactivity remaining in the cells is measured after an extraction with cold TCA). When this is done, then any kinetic analysis, for example to give (K S), measures both the external concentration of thymidine and the amount of thymidine internally produced which diluted the isotope after it was transported into the cell. Finally, the amount incorporated is multiplied by a factor which is the number of bacteria per mole of DNA. Once the number of bacteria produced is known, the carbon and biomass may be calculated from appropriate factors. Overall, it appears that the results of bacterial growth analyses with thymidine give ecologically reasonable results. There are, however, enough inexplicable instances of high thymidine incorporation without bacterial growth, of problems with calibration (is there less DNA per cell in very small cells than in larger cells?), and of lack of thymidine uptake by certain types of bacteria that the method must be judged as promising but still imperfect in many ways. The fifth measurement is of the incorporation of leucine into bacterial protein. In the technique of Kirchman et al. (1985) as extended by Simon and Azam (1989), 3H-leucine is added to samples at a final concentration of 10 nM. This amount overwhelms the ambient leucine pool (about 1 nM) and maximizes leucine uptake. After incubation, the bacteria are extracted with hot TCA (to hydrolyse DNA and RNA) and then filtered onto 0.45pm pore size membrane filters for analysis of the incorporated tritium. The assumptions behind this method are that protein is a stable fraction of bacterial dry weight (63%), that leucine makes up a constant percentage of the total protein, that leucine at nM levels is taken up exclusively by marine bacteria, and that there is a consistent relationship between protein and cell volume for various sizes of bacteria. Thus, rates of production of bacterial protein may be translated directly into rates of production of bacterial carbon and biomass. With HPLC, Simon and Azam have successfully tested many of these assumptions on filtered samples that represent the bacteria between 0.2 and 0.6 pm. In most of the cases they examined, the leucine method gave comparable results to the thymidine method. This leucine method has fewer assumptions than the thymidine method for measuring bacterial heterotrophic activity and represents a very promising development.
+
7 . MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
111.
A.
247
Methods
Standard method
There is a standard method which is slightly modified for each of the various types of radioisotope experiments. However, once a method is chosen, tests must be made to decide upon incubation time and amount of labelled substrate to add. Samples are collected in acid-washed bottles of glass or plastic, held at the in situ temperature in the dark, and the radioisotope experiment begun as soon as possible. This holding period should be no longer than 30 min. Samples may be incubated in any volume of flask but 10-20 ml of sample are convenient to filter through a 25-mm diameter filter so that large test tubes may be used. If the respired I4CO2 is to be collected, then a 25-ml Erlenmeyer flask is suitable. Replicate or triplicate samples along with killed controls are necessary. In clean ocean water, special techniques will often prevent contamination by trace metals (such as Cu) and increase the rate of bacterial activity. Ferguson and Sunda (1984) achieved this by using a 30-1 Teflon-lined water sampler suspended on a plastic-coated hydrowire and closed with a Teflon messenger. They also carefully cleaned their glassware and rinsed with specially prepared distilled water. Isotope is diluted in distilled water with special care to avoid contamination from organic compounds and trace metals (Ferguson and Sunda, 1984). The isotope is delivered to the sample with automatic micropipettes so that, for example, 25, 50, 75, and 100 pl will produce the desired concentrations in 10 o r 20 ml of sample. The incubation is carried out in the dark. The time should be long enough to produce good uptake by the bacteria yet short enough that there is no significant growth or adaptation during the experiment. The ideal time must be chosen in a test so that the amount of uptake increases linearly over time. Incubation is ended by the addition of buffered formalin (0.4% final concentration, pH 8). For a control, the isotope is also added to a formalin-killed sample. When respired 14C02is to be measured, the incubation is carried out in a 25-ml Erlenmeyer flask (Hobbie and Crawford, 1969). After addition of the isotope, the flask is immediately sealed with a rubber serum stopper that has a plastic cup suspended from it. The cup contains a 25 x 51 mm piece of accordion-folded chromatographic paper (Whatman No. 1). After incubation, 0.2 ml of a 2 N H2S04 solution is injected through the septum to stop the uptake. Next, still working through the septum, 0.2 ml of an organic base (phenethylamine) is slowly added to the folded paper and the
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flask is then shaken for an hour at room temperature so that the I4CO2is absorbed. After this, the paper is placed in a scintillation vial with a scintillation cocktail and counted. Under these conditions, tests with a ''C02 standard showed that only 82% of the I4C was counted even after a counting correction is made with an internal standard. This loss might be due to absorption of the light inside the filter paper. Thus, the final counts were multiplied by 1.23. The samples are filtered through a membrane filter and rinsed with at least 5 ml of filtered sea water. The 25-mm diameter 0.45-pm pore size cellulose filters of Millipore or Sartorius work well and capture all of the bacteria. Filters are placed in standard counting vials and 1 ml ethyl acetate is added to dissolve the filter. The samples are counted with liquid scintillation and standard scintillation cocktails.
B.
Kinetic analysis
The method is that of Wright and Hobbie (1966) and Fuhrman and Ferguson (1986). The sample (10-50 ml) is incubated in flasks or in rinsed polyethylene bags (Whirlpak) with or without light. The final concentration of the added 3H- or 14C-labelledsubstrate is 10-100 nM or approximately 1-10 pg 1-'. Sugars, acetate, and amino acids may be used. The respired 14C02may be collected into an organic base or the respired 3H20 calculated from the loss from solution during freeze-drying.
C. Tracer-level additions The method is that of Azam and Holm-Hansen (1973). For analysis of glucose uptake in sea water they added 0.01 pg gIuc0se-6-~Hto 100 ml of sample (to give 0.5 nM) and incubated for 0.5-4 h. The loss of labelled intracellular pool material was minimized by not killing the sample with formalin before washing the filter with 20 ml of iced and filtered sea water.
D. Addition of high level of substrate The only change used in this method is that a single concentration of labelled substrate is used. Tranvik and Hofle (1987) used 80 pg glucose I-' in short-term measurements of the adaptation of microbes in a lakewater culture to taking up sugar.
7. MEASURING HETEROTROPHIC ACTIVITY IN PLANKTON
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E. Thymidine incorporation The method is that of Fuhrman and Azam (1982) and Simon and Azam (1989). Methyl-l-[3H]-thymidine (70 Ci mmol-') is added to 10-ml samples to give a final concentration of 5 nM. Formalin-treated controls are run in parallel. The incubation times are 1 5 4 5 min depending upon the temperature (incubations are carried out in the dark at the in situ temperature). After stopping the incubation with formalin, samples are filtered onto membrane filters, extracted with ice-cold 5% TCA and radioassayed with liquid scintillation. The thymidine incorporation rates are converted to cell multiplication rates by multiplying by the factor of 1.18 x 10" cells produced per mol of thymidine incorporated. Cell multiplication rates are converted to carbon production by using the cell carbon values for the given cell size (Table I).
TABLE I Cell volumes, protein composition (in femtograms or lo-'' g), dry weight, and carbon content of average marine bacteria in the size range 0.026-0.4 pm3 (adapted from Simon and Azarn, 1989) Volume CLm3
fg
0.026 0.036 0.050 0.070 0.100 0.200 0.400
12.1 14.7 17.7 21.6 26.7 40.3 60.6
Protein
YOVOI.
%dw
Dry weight fg
fg
Carbon %dw
46.5 40.8 35.4 30.9 26.7 20.2 15.2
61.4 62.5 63.0 62.8 62.8 63.5 63.3
19.7 23.5 28.1 34.4 42.5 63.5 95.8
10.4 12.6 15.2 18.7 23.3 35.0 53.3
52.0 53.6 54.2 54.3 54.7 55.1 55.7
F. Leucine incorporation
In this method (Simon and Azam, 1989), [3,4,5-3H]-1-leucine (140 Ci mmol-') is added to triplicate 10 ml samples to produce a concentration of 10 nM. Formalin-treated controls are run in parallel. Sample incubations (20-40 min) are ended by the addition of formalin and then samples are extracted with 5% TCA at 95-100°C for 30 min to hydrolyse RNA and DNA. Longer extraction times lead to significant hydrolysis of protein. The extracted samples are cooled and filtered. One necessary correction is for the isotope dilution within the cell. Simon and Azam (1989) suggest that a factor of two is appropriate in many situations but the pool specific activity may also be measured with HPLC.
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JOHN E. HOBBIE
The conversion of 3H-leucine incorporated to carbon or biomass produced by the bacteria is made by the following formula (Simon and Azam, 1989): BPP = (mol 3H-leucine incorporated)(100/7.3)(131.2)(2)(0.86)
(7) where BPP is bacterial protein production (in g C), 10017.3 is 100 divided by the mol % of leucine in protein, 131.2is the formula weight of leucine, 2 is the intracellular isotope dilution of labelled leucine, and 0.86 converts protein to carbon. If the carbon per cell is known (from Table I), then BPP can also be converted into bacterial cell production. If the isotope dilution is not known, then using 1 instead of 2 in formula 7 gives a minimum estimate of BPP. Azam (personal communication) states that even if the isotope dilution is known from HPLC measurements, the absolute value for growth is still elusive because the dilution gives the maximum estimate of BPP. We still do not know how much of the isotope dilution is due to de novo synthesis by the active cells and how much is due to unlabelled leucine pools of the inactive cells. In coastal waters the range from minimum to maximum is about two-fold. References Azam, F. and Holm-Hansen, 0. (1973). Mar. Biol. 23, 191-196. Ferguson, R. L. and Sunda, W. G . (1984). Limnol. Oceanogr. 29, 258-274. Fuhrman, J. A. and Azam, F. (1982). Mar. Biol. 66, 109-120. Fuhrman, J. A. and Ferguson, R . L. (1986). Mar. Ecol. Prog. Ser. 33, 237-242. Griffith, P. C. (1988). Limnol. Oceanogr. 33, 632-638. Hobbie, J. E. and Crawford, C. C. (1969). Limnol. Oceanogr. 14, 528-532. Hobbie, J. E. and Rublee, P. (1977). In “Aquatic Microbial Communities” (J. Cairns Jr, Ed.), pp. 4 4 4 7 6 , Garland Publishing Co., New York and London. Hopkinson, C. S. Jr, Sherr, B. and Wiebe, W. J . (1989). Mar. Ecol. Prog. Ser. 51, 155-166. Jenkins, W. J. (1977). Science 196, 291-292. Kirchman, D. L., K’Ness, E. and Hodson, R. (1985). Appl. Environ. Microbiol. 49, 599-607. Moriarty, D. W. (1986). Adv. Microbiol. Ecol. 9, 245-292. Parsons, T. R. and Strickland, J . D. H. (1962). Deep-sea Res. 8, 211-222. Pomeroy, L. and Deibel, 0. (1986). Science 233, 359-361. Riley, G. A . (1951). Bull. Bingham Oceanogr. Coll. 13, 1-126. Simon, M. and Azam, F. (1989). Mar. Ecol. Prog. Ser. 51, 201-213. Sorokin, Yu. I. (1965). Mem. 1st. Ital. Idrobiol. 18, 187-205. Tranvik, L. J. and Hofle, M. G. (1987). Appl. Environ. Microbiol. 53, 482-488. Williams, P. J. LeB. (1981). Oceanologica Acfa 4, 359-364. Wright, R. T. and Hobbie, J. E. (1966). Ecology 47, 447-464.