Three different methods for determining bacterial growth rate in rivers are described. Two of the methods are for bacteria in suspension: a recirculating experimental channel method and a radioactive tracer technique using super(35)SO sub(4). The third method is for bacteria attached to surfaces and specifically considers the surface of the common duckweed Lemna minor)

Baker, J.H.

English

Aquatic Commons

REPORT OF THE DIRECTOR 57 
GROWTH RATES OF BACTERIA IN RIVERS 
J. H. BAKER 
Ecosystems in general and rivers especially are not fixed in a particular 
state, but are continuously changing; sometimes rapidly, at other times 
imperceptibly, but never remaining exactly the same. Therefore, if we are 
to understand an ecosystem, we must observe and measure the way in which 
it is changing. One of the most important changes in the biota is brought 
about by growth, and this is just as relevant to the bacteria as it is to the more 
obvious components of river systems such as the fish. However, unlike fish, 
bacterial cells do not increase in size throughout their lives; hence we are 
concerned here with the growth of the population and not of in-
dividuals. Bacterial populations in rivers, as in most other environments, 
are numerically large; e.g. in the River Frome, Dorset, there are 
approximately 50000 bacteria per ml (Baker & Farr 1977), but because 
bacteria are so small (of the order of 10-6 m long) their total biomass is also 
small. 
In theory the determination of population growth is simply a matter of 
counting the entire population at time A and again at time B. The growth, 
which can be positive or negative, is then the difference between the two 
counts and can be called a growth rate when expressed per unit time. The 
difficulty of estimating growth arises because changes in the populations 
between time A and time B may arise from factors other than growth; for 
example grazing, wash out, recruitment from outside, etc. The problem is 
to isolate growth from all these other changes. Nevertheless it is relatively 
easy to count the numbers of bacteria per unit volume of samples of river 
water and observe how these change with time. There are wide fluctua-
tions from week to week, but over relatively long periods (years) the average 
population in the River Frome remains more or less constant (Baker & Farr 
1977). In other words the net change over a year in the numbers of 
bacteria suspended in the river is zero, but in the shorter term (hours or 
days) positive growth rates can be determined. Three different approaches 
will now be described. 
In order to limit the changes in the bacterial population to those due to 
growth, one can isolate a portion of water, e.g. in a flask, and determine the 
population changes within it. However, over a period of days, the condi-
tions such as temperature, aeration and nutrient concentration within the 
flask will differ markedly from those in the river, and the bacterial popula-
tion will change accordingly. Thus the growth rates of the bacterial pop-
ulation in the flask bear little or no resemblance to those occurring at the 
same time in the river. The desirability of isolating river water from 
extraneous influences, while retaining its essential characteristic of con-
tinuous flow, is common to many problems of lotic ecology, and hence a large 
Rep. Freshwat. biol.Ass. 49 (1981). 
58 FORTY-NINTH ANNUAL REPORT 
recirculating experimental channel has been built (illustrated in 43rd 
Annual Report). It consists of a race-track-shaped tank made of glass-
reinforced plastic, 53 m in circumference and with a trapezoidal cross-
section. It is filled to a depth of 70 cm with water which is circulated by an 
Archimedean screw pump at a velocity of approximately 0.25 m s_1, similar 
to the current velocity of the R. Frome. Borehole water is used, because it 
forms 74% of the discharge of the R. Frome (Paolillo 1969) and is virtually 
free of all living organisms (bacterial concentration < 5 ml -1). 
The circulating channel thus provided conditions much closer to those of 
the river than a simple flask, but it was still different in two obvious ways: (i) 
there was no sediment initially present; (ii) the channel was kept covered to 
prevent aerial contamination by birds and to inhibit the growth of 
algae. Under these conditions the bacterial population was periodically 
sampled and its size estimated by the viable or plate count technique using 
casein/peptone/starch agar (Jones, 1977). The number of bacteria rose 
steadily for about ten days and similar results were obtained when the 
experiment was repeated, as shown in Fig. 1. The lines are computed 
regressions based on the assumption that the bacteria grew logarithmic-
ally. This is a reasonable assumption, given that the most common form of 
bacterial multiplication is by binary fission, and it results in a good fit of the 
lines to the data. Thus the correlation coefficients were 0.96 and 
0.99. The slopes of the lines are directly related to the growth rates of the 
bacterial populations and an analysis of covariance shows that the two slopes 
are not significantly different from each other. Growth rates of bacteria 
are commonly expressed as the time taken for the population to double, and 
the computed common slope for these experiments gives a doubling time of 
42 h. 
REPORT OF THE DIRECTOR 59 
After ten days the bacterial population stopped increasing and oscillated 
around abundances typical of those in rivers (Ladle et al. 1977). The 
recirculating channel thus resembled a giant 'batch culture' of a natural 
bacterial population with bore-hole water as the medium. Nevertheless, as 
mentioned earlier, the recirculating channel differed greatly from a natural 
river in certain respects, and there was clearly a need to devise a different 
method which was applicable to natural as well as experimental 
situations. This second method approaches the determination of growth 
rates not by counting the number of bacteria at different times, but by deter-
mining the rate at which a metabolite is taken up by the population as a 
whole. It is therefore sometimes called a process-orientated method as 
opposed to organism-orientated methods. 
In the past the process which has often been examined is the incorporation 
of dissolved organic carbon compounds (DOC) into the bacterial 
biomass. Relatively simple organic compounds are usually used, such as 
acetate or glucose, labelled with radioactive carbon (14C). Stated simply, 
the theory assumes that the rate of uptake of the radioactive compound is 
proportional to the activity of the population before the compound was 
added, provided that the incubation time is of the order of hours. Two of 
the drawbacks to the 14C method are that (a) the radioactive material is 
probably quite different to the natural carbon substrate and (b) the addition 
of radioactive DOC may substantially affect the total concentration of avail-
able DOC. Hence this method might lead to erroneous results. 
One possible way to overcome these drawbacks is to use radioactive sul-
phur, in the form of sulphate (35S04), instead of 14C (Campbell & Baker 
1978a). Sulphur compounds (as in the R. Frome), is obtained from sul-
phate. Furthermore, sulphate is an essential element and, in the absence of 
reduced sulphur is naturally present in excess and the addition of radioactive 
sulphate is most unlikely to affect the growth rate of the bacteria. The 
method consists of adding a known quantity of 35S04 to a fixed volume of 
river water and incubating it in the dark at river temperature for a few 
hours. The river water is then passed through a fine filter to catch the 
bacteria. The bacteria are washed and the amount of radioactivity they 
have accumulated during the incubation period is determined. 
Some problems associated with this technique have been discussed by 
Campbell & Baker (1978a, b), but essentially it has to be assumed that the 
quantity of sulphate incorporated into the bacterial cells has a fixed 
relationship with the amount of carbon incorporated, and hence is directly 
proportional to the bacterial growth rate. The sulphate uptake rate varies 
during the year, but a value of 5 ng S l_1h_1 seems about average outside the 
periods of high algal activity. In order to turn this figure into a bacterial 
doubling time, we need to know the size of the bacterial population 
(1.5 x 105 ml-1) and the dry weight of an average bacterium 
(2 x 10-13g). This last figure is subject to considerable error. However if FIG. 1. Semi-logarithmic regression of bacterial numbers against time in the Waterston re-circulating channel. Each point is the mean of approximately ten determinations. 
60 FORTY-NINTH ANNUAL REPORT 
we make these assumptions then the bacterial doubling time, as calculated 
from the sulphate uptake rate, is 42 h (Hossell & Baker, 1979a). The fact 
that it is exactly the same as the figure obtained from the recirculating chan-
nel is purely fortuitous, but the similarity is encouraging. 
Both of the above methods are concerned with bacteria in suspension, but 
in a river many bacteria are attached to surfaces such as plant leaves and 
neither method can be easily adapted to this situation. Any surface in a 
river is continually bombarded by bacteria carried by the current. Some of 
these bacteria stick to the surface while many 'bounce' off again. Four 
possible fates await those organisms which attach themselves: they might 
multiply and form a colony; they might be eaten by a grazer; they might 
detach themselves again; or, lastly, they might remain there apparently 
doing nothing. These four possibilities were considered by Hossell & Baker 
(1979a) with respect to the surface of the common duckweed Lemna minor. 
Duckweed is a small, floating, disc-shaped plant a few millimetres in 
diameter. It is commonly found in still water; in rivers it occurs amongst 
the emergent vegetation and in the slow eddies, particularly during the sum-
mer. Bacteria on the surface of duckweed can be observed directly by 
staining them with phenolic aniline blue (Hossell & Baker 1979b). Thus 
the numbers of bacteria per unit area of duckweed surface can easily be 
counted. Reproduction in the duckweed is mainly asexual and new plants 
appear every few days. These new plants have no bacteria on them initially 
and, by counting the bacteria on plants of known ages, the gradual coloniza-
tion of the plant can be quantified. The results of such an experiment are 
illustrated in Fig. 2. 
It would be possible to use linear regression analysis directly on the data in 
Fig. 2, as was used in Fig. 1, but the resulting rate of increase would not be 
equivalent to the in situ growth rate of the bacteria which, as stated above, is 
the sum of four components. First let us deal with the two increasing com-
ponents, namely attachment and growth. Once more we can assume that 
growth is logarithmic and we can further reasonably assume that an 
approximately constant number of bacteria attach per unit time, i.e. the 
attachment rate is arithmetic in character. From these two assumptions it 
is possible to write a theoretical equation describing the mathematical form 
which an increase in number due to these two components alone would have 
(Hossell & Baker 1979a): 
Nt=(Nt-1e r)+A 
where r is the growth rate of the bacteria, Nt is the number of bacteria cm -2 
on plants aged t days and A is the attachment rate. Non-linear regression 
analysis was employed to obtain the best value of A for an arbitrary value of 
r and the experimentally determined values of Nt and Nt_1 The line of best 
fit (shown in Fig. 2) results in an attachment rate of 5.7 x 105 cm -2 day -1 
and an apparent doubling time of 164 h. 
This apparent doubling time does not take into account either detachment 
or grazing, both of which would reduce it. Detachment rates have been 
determined in the laboratory, but not in the river. In the laboratory the 
assumption was made that bacterial growth in one hour on a duckweed sur-
face, or in a beaker of river water, was negligible. Then the number of 
bacteria detaching from a known surface area of duckweed was determined 
by removing the plants after one hour from a beaker of previously sterile 
river water. The plants were placed in a similar beaker of sterile river 
water and the bacteria in the first beaker were counted. This process was 
repeated many times, and for the first ten hours the arithmetic detachment 
rate was approximately constant. When this detachment rate is added to 
the apparent rate of increase, a corrected doubling time of 61 h results 
(Hossell & Baker 1979a). No suitable method has so far been found to 
estimate the losses due to predation and so this corrected doubling time is 
still an underestimate. 
Bacterial growth rates are directly proportional to the prevailing 
temperature and thus in a river they would be expected to be lower in winter 
than in summer. The water temperatures during the recirculating channel 
experiments and 35S-uptake investigations were approximately the same. 
On the other hand the temperature of the duckweed plants on the surface of 
relatively still water during summer is likely to have been substantially 
higher. Nevertheless, bacterial growth rate due to the higher temperature 
is likely to be increased by, at most, a factor of two. 
At the present time there is a considerable interest in the productivity of 
ecosystems. The prediction of bacterial production rates in the R. Frome 
REPORT OF THE DIRECTOR 61 
FIG. 2. Mean population density of bacteria on duckweed plants of known age with the com-
puted curvilinear regression. Each point is the average of twenty counts. (After Hossell & 
Baker, 1979a: Freshwat. Biol. 9, 319-27). 
62 FORTY-NINTH ANNUAL REPORT 
from data so few as are presented here must be largely speculative. 
However, bacterial secondary production in the water column has been 
estimated from the 35S experiments, and Campbell& Baker (1978a) suggest 
a range of 6-13 mg C m - 3 d_1. 
The three methods described above for determining bacterial growth rates 
in rivers are open to quite serious criticisms, as are the methods used by other 
workers. Because of their fundamental importance to microbial ecology, 
however, it is well worth trying to obtain estimates of such growth rates, 
even though the methods are known to be inadequate. It should be noted 
that all three estimates are close together and hence despite their weaknesses 
they lend strength to each other. 
I thank the many members of the FBA River Laboratory staff and visitors 
who have helped in this work, particularly Dr P. G. C. Campbell, Mrs J. C. 
Hansford (nee Hossell) and Mr D. R. Orr. 
REFERENCES 
Baker, J. H. & Farr, I. S. (1977). Origins, characterization and dynamics of 
suspended bacteria in two chalk streams. Arch. Hydrobiol. 80, 308-26. 
Campbell, P. G. C. & Baker, J. H. (1978a). Estimation of bacterial production 
in fresh waters by the simultaneous measurement of 35S-sulphate and D-3H 
glucose uptake in the dark. Can. J. Microbiol. 24, 939-46. 
Campbell, P. G. C. & Baker, J. H. (1978b). Measurement of sulphate uptake in 
the dark by suspended micro-organisms: application to running waters. 
Verh. int. Vermin. theor. angew. Limnol. 20, 1423-8. 
Hossell, J. C. & Baker, J. H. (1979a). Estimation of the growth rates of epi-
phytic bacteria and Lemna minor in a river. Freshwat. Biol. 9, 319-27. 
Hossell, J. C. & Baker, J. H. (1979b). A note on the enumeration of epiphytic 
bacteria by microscopic methods with particular reference to two freshwater 
plants. J. appl. Bad. 46, 87-92. 
Jones, J. G. (1977). Variation in bacterial populations in time and space. Rep. 
Freshwat. biol. Ass. 45, 55-61. 
Ladle, M., Baker, J. H., Casey, H. & Farr, I. S. (1977). Preliminary results 
from a recirculating experimental system: observations in interactions between 
chalk stream water and inorganic sediment. In: Interactions between sedi-
ments and fresh water (ed. H. L. Golterman), 252-7. The Hague. Junk. 
Paolillo, S. A. G. (1969). Hydrogeology of the River Frome catchment. 
Memorie Note 1st. Geol. appl., Napoli, 11, 1-69. 


Growth rates of bacteria in rivers

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Growth rates of bacteria in rivers

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