Influence of temperature and substrate shocks on survival and succinic dehydrogenase activity of heterotrophic freshwater bacteria

Woter Re..curc It Vol. 13. PP- 11.1'4 to I 15.1. C Pergamon Press Lid 197'4. Printed in Great Brtt.ttn

(Mg/.3-135-1. 7t/ 12()1-11.195112.00 0

INFLUENCE OF TEMPERATURE AND SUBSTRATE SHOCKS ON SURVIVAL AND SUCCINIC D E H Y D R O G E N A S E ACTIVITY OF H E T E R O T R O P H I C FRESHWATER BACTERIA* W. REICHARDT Dept. of Microbiology, University of Maryland, College Park, MD 20742. U.S.A.

(Receiced 2 April 1979) Abstract--The influence of temperature shocks on aquatic bacteria was studied in laboratory experiments with regard to population shifts in lake water communities of aerobic heterotrophic bacteria including coliforms, as well as in synthetic mixed cultures of psychrotolerant aquatic isolates. In addition, succinic dehydrogenase activity was used as a parameter of bioactivity. Each experiment was designed to study concomitant effects of elevated levels of organic substrates. Simultaneous additions of complex nutrients such as 100-300 mg I- t of meat peptone to lake water caused a synergistic action of substrate and temperatureon population dynamics by depressing population densities at temperatures below 200C. In contrast, siimulation was observed at higher temperatures particularly for the coliform group. In mixed cultures consisting of Cytophaga. Chromobucterium and Arthrobacter as important members of the heterotrophic microflora of the investigated lake water. substrate accelerated death was induced by temperature shocks beyond the maximum growth temperatures. The succinic dehydrogenase activity of a psychrotolerant strain of Cytophaoa failed to show more sensitive responses to temperature shocks than did viable counts.

INTRODUCTION

To some extent, consequences of heated water discharges for the aquatic microflora may be inferred from pure culture experiments. But it appears questionable, whether experimental data collected from a few microbial cultures will ever be sufficient to cover most of the stimulation or inhibition of microbial activities which occur as direct or indirect consequences of thermal additions to the aquatic environment. As a first approach, information ought to be gathered from investigations in situ or using laboratory microcosms (Allen & Brock, 1968). At this stage, even such heterogeneous groups of organisms as zymogenic heterotrophic bacteria which are defined exclusively by their ability to form colonies on defined agar media may provide useful information (Guthrie et al., 1974; Guerrero et al., 1975; Verstraete et al., 1975). To establish causalities, synthetic mixed culture,s and pure cultures become necessary to obtain more d~tailed information. At first view, published statements on the effects of temperature shocks on aquatic bacteria appear contradictory, if the availability of nutrients is also taken into account (George & Gaudy, 1973; Klein & Wu, 1974; Guthrie et aL, 1974). Hence, this study deals with the combined influence of thermal and organic Ioadings on heterotrophic bacterial communi* This work has been financially supported by the Deutsche Forschungsgeraeinschaft as part of a grant (Re 271/7).

ties in water samples from regional lakes, which at temperatures around 5°C, are mostly dominated by facultatively psychrophilic bacteria (Reichardt, 1974). To bridge the gap between information on mixed cultures and pure cultures, a synthetic mixed culture consisting of three psychrotolerant bacterial strains was studied. In addition to viable counts, succinic deh~;drogenase activity was used as an indication of bioactivity. MATERIALS AND METHODS

Heating experiments were performed in magnetically stirred vessels of 1.5 1 capacity containing 800 ml of lake water (from Lake Constance litoral) or culture suspensions in mineral media (for preparation see: Reichardt, 1975). The most important experimental parameters have been illustrated in Fig. 1. At the basic temperature (To) sterilized solutions of meat peptone (Merck) as a complex substrate (S+), or glucose (in culture experiments) were added to the culture vessel before the heating period began. During heating periods of 30-60 rain increases of temperature (AT) were 5-35°C above ambient. While the time lag between the end of the exposure time (Atr) and incubation in the assay could be kept shorter than 30 rain, there were inevitable differences between the final temperature of exposition ('/'1) and the maximum temperature attained during the incubation (T2). Viable counts of aerobic heterotrophic bacteria were determined on a caseine peptone starch glycerol agar (Collins 8~ Willoughby, 1963) after 14 days of

1149

I 150

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°C

Final temperature, rOnge : 5"- 35"C

T~

I

-~ T2

ZXT I n c u botion of samples

AtT2

Basic tem~lmture,

At 'r

range: 5"- 25"C

Ranges :

Zncubotion tempera ture

Substmte adaptation Fxposure Transition period to increased period temperature O- 20 h 30 -60 rain 4~.30 rain

Fig. I. Diagram illustrating the most important parameters in experiments to study responses of heterotrophic bacteria in lake water samples to sudden increases of temperature. Symbols S" indicate the addition of dissolved organic substrates as compared with untreated controls (hatched parts, also in the following diagrams). incubation at 1 8 C (water samples) or 4 days of incubation at 20°C (cultures). Serial dilutions of the samples for spread plate inoculation were prepared

500 fl_lTo to'c

I

T2- 22"C

S=OJgt i Meat peptone At,= .~h At T-

h

400

in 0.005 M phosphate buffer, pH 7.0, with 10mg I - ' of Tween 80 kept at 20 C. Colonies of coliform bacteria were counted after one day of incubation at 37 C on commercial Endoagar (Oxoid) using 1-10ml-samples for filtration through 0.2 F~m Sartorius membrane filters. Synthetic mixed cultures were obtained from exponentially growing cultures of Cytophag, johnsonae, Chromohacterium liridum and Arthrohucter sp. with growth temperature maxima of 31.5, 31.5 and 34.5'C respectively. Culture suspensions were centrifuged, resuspended in a mineral medium (see above)

x

T

To- B'C

300

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S = O.Ig t 4 Meat peptone

A t T w Ih

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E

i

o

300

T Als= Oh

200 u

o

I

~. 2oo 8

I00

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20 35 T., "C ~o

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25

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Fig. 2. Lake water samples adapted to 10°C. Effects on population densities (viable counts) of increasing the water temperature for A t r = I h up to A T = 25°C above ambient. Trials with additional organic substrate (5) are indicated by white, controls by hatched columns. Sul~rate was added At, = 12 h prior to the temperature shift up. Standard deviations for n = 10 replicates,

TI ,

18

28

°C 10

20

AT, "C

Fig. 3. Lake water samples adapted to To = 8'C. Effects on population densities (viable counts) of increasing the water temperature for Atr = 1 h up to A T = 20°C above ambient. Additional substrate added immediately [At, = 0 h) before the shifts of temperature (white columns). Controls without additional substrate are indicated by hatched columns. Standard deviations for n = 10 replicates.

Heterotrophic freshwater bacteria

ll51

Heterotrophs isolated from populations acclimati:ed to TO - 7 " C =(

[]

T • 22°C

25"C

peptone

S = O . I q L-0 M e o t

1(30

Both nutrient addition and temperature shock significantly reduced viable counts (Fig. 5). When these stresses were applied together no significant cumulative effect was observed.

A t r ,= O . S h

E

A t r - Oh

_= o

Synthetic mixed cultures acclimati:ed to 20~'C

.u

o

50

o <.

15

22

~, "c Fig. 4. Lake water samples adapted to 7~C. Similar experiment as in Fig. 3, except for the shorter time of heating, ArT = 0.5 h. and, after adjustment to an optical density of 057s,m (photometer Eppendorff), mixed DI ~m = 0.010 together using equal volumes. Experiments were run at final dilutions equivalent to Oo = 10 -5. Activities of succinic dehydrogenase were determined in cell extracts in Triton X-100 using an INTPMS-method (Nachlas, 1960; Reichardt, 1979). Concentrations of protein were determined according to Lowry's method (Herbert et al., 1971).

Synthetic mixed cultures consisting of three aquatic isolates furnished evidence of the combined action of elevated temperatures and elevated levels of substrate on viable counts (Fig. 7). A temperature shift for 1 h from 20°C to 3YC, i.e. beyond the maximum temperature for growth of the strains employed, caused significant depressions only in the presence of 10 mM 1-~ of glucose. Though temperature maxima for Cytophaga and Chromobacterium were identical (31.YC), both these populations showed different responses.

Succinic dehydrogenase actit:ity of C. johnsonsae acclimatized to 5~'C and 20°C, respectirely In order to find a parameter of bioactivity which showed possibly higher sensitivity to temperature

K

%- ~s'c T ~ ' C rl

S=O.Ig

L"i

M ~ t peptooe

700

A t I - Oh

600

x 500

R ESU LTS

Heterotrophs isolated from populations acclimatized to 7°-10°C Temperature shock up to 35°C for 1 h duration had no significant numerical influence on recoveries from these populations (Figs. 2 and 3). 12 h after the addition of nutrient (0.1 g I- t of peptone), increased viable counts were obtained which were unaffected by subsequent temperature shock (Fig. 2). When temperature shock was applied at the time of nutrient addition, an increase of 10°C for 1 h significantly reduced recoveries, whilst an increase of 20°C had no effect (Fig. 3). If the increase of 10°C was limited to .0.5 h, then no decrease in counts was observed (Fig. 4).

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=_ 400

8 ¢3

o 300

o

200

II

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!

i!

Coliforms isolated from populations acclimatized to lO°C The coliform group showed different responses to temperature shock (Fig. 6) when compared with the general heterotrophic populations. Increases in temperature up to 35°C increased counts. However addition of 0.3 g I- t peptone reduced recoveries observo:l after temperature shock up to 35°C. Temperature shock alone at 40°C reduced counts whereas the highest recoveries were o b t a i n e d in the presence of peptone with this regime.

25

30

35

To, °¢ 0 ~T,

5

aO "C

Fi¢ 5. Lake water samples adapted to To -- 25:C. Effects on population densities of increases of water tem~-ratur¢ for Air = I h up to AT = IO°C above ambient. Additional substrate (S) added immediately before the shifts of temperature (white columns). Controls without additional substrate are indicated by hatched columns. Standard deviations for h --- I0 replicates.

1152

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T2= (22"C ~ ) 3 7 " C

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A t T = Ih

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_J-I- 7 FF-

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o

lit

IIItt

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1500

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20

25

30

35

a0

20

25

30

I00

T,, "c 0

I0

I,~

AT, *C

Fig. 6. Lake water samples adapted to To = 10°C. Effects on coliform counts on membrane filters of different increases of water temperature for ArT = 1 h up to AT = 30~C above ambient. Substrate additions (white columns) immediately before heating (At, = 0 h). Controls without additional substrate are hatched. shocks than did viable counts, succinic dehydrogenase activity in Cytophaga johnsonae was determined (Figs. 8 and 9). However, upon temperature shocks from 5°C up to 40°C activity proved to be fairly stable, the level being exclusively dependent on the level of substrate in the culture medium (Fig. 8). Similar results were obtained in experiments starting from a basic temperature of 20°C to which the culture had been adapted (Fig. 9). In this case, an accelerated loss of activity was observed above the maximum temperature for growth (at 40°C only) which was more pronounced in starved cultures than in those which were saturated with glucose.

2 0 - 3S'C

[ - ~u¢ose]

[*10 mM L-t g l u ¢ ~ ] % Zncr~

of

20 - 30"C

ZO- 30"C I0

20 - 35"C

'- -I0

DISCUSSION As the counting procedure used for coliforms does not allow to record the recovery of sublethally damaged cells, a distinction between inactivation and elimination of coliforms is not possible. On the other hand, prolonged incubation periods for viable counts of non-coliform bacteria permit to presume optimal recovery. Though total heterotrophic bacteria have been studied as only one complex, internal shifts in populations in response to stress become evident when additional data from a few selected groups are considered. They show that reduced recovery in the case of warming from 8°C to 18°C (Fig. 3) coincided with a striking decrease of Cytophagacolonies. This re~p~se can be explained by previous findings s h o ~ that aquatic isolates of Cytophaoa were in most cases facultatively psychrophilic (Reichardt, I974). Since preliminary estimates of other populations such as

At - Ih rm Clln)mW~erium livlclum Arthmbacter SO,

Fig. 7. Synthetic mixed cultures consisting of three Inychrotolerant strains of aquatic bacteria. Elects of teml~ature shifts (Atr = 1 h; Tffi 5° or 10° above ambient (as indicated by arrows) at mbmaximum and supranmximum tzmt~ratur¢~ on i ~ t s of r i f l e counts eatpt~mled as the l:~¢¢ntage of population d~-'~ies at ba~¢ temperature (20"C). Comparison of starved cuhur~ suspended in mineral medium (left) and cultures ~turated with 10 mM Iof glucose (right).

Heterotrophic freshwater bacteria

1153

Cytopha9¢l john~w~nae (C 21 ) I

40

30

*C

20

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r

O

,,

I

•,i;

¸¸

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•

i

,

,!

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I

2 3 4 SDH-units (x IOZ)/min-mg protein

Fig. 8. Cytophagajohnsonae, strain C21. Specific succinate dehydrogenase activities following temperature shifts (Atr= 1 h) from To = 5°C up to Tt = 40°C. Comparison of cultures exponentially growing on 10 mM I- t of glucose (white columns) and starved culture suspensions in mineral medium (hatched columns). gelatine liquefying or nitrate reducing bacteria showed no decline after temperature shifts up to 30°C, these might partly compensate the loss of facultatively psychrophilic bacteria at elevated temperatures. Heterotrophic microcosms developed at temperatures below 25°C have been reported to show temperature optima exceeding 25°C and thus to be not optimally adapted (Allen & Brock, 1968). This temperature limit is roughly identical with maximum water temperatures of most natural water bodies which reecive thermal effluents. A comparison of experiments started at 8°C and 25°C, respectively (Figs. 3 and 5), has shown that increases of viable counts, which were probably the result of shifts between different populations after the temperature shock, did not exceed the 25 ° limit. Hence, it may be concluded that those systems derived from lake water were dominated by heterotrophic bacteria which

could not be stimulated by temperatures of 30°C or higher. As shown by the coliforms which represent a small group of mesophilic bacteria, sudden shifts up of temperature could immediately load to higher population densities. This type of response has already boon known from pure culture experiments within the linear range of the Arrhenius function for growth rates (Ng et al., 1962). Effects of temperature shock must not be secluded from the concomitant availability of nutrients. Within a moderate range, Guthrie e t a l . (1974) consider thermal additions as a potential method for use in restoration of natural bacterial populations to sewage polluted aquatic environments. This point of view, however, must not b¢ generalized, since it originates mainly in a biased understanding of the most hoterogenous group of 'chromogenic' bacteria. Whereas the findings of Guthrie etal. (1974) suggest that those

Cytophoga johnsonae (C 21) i

!

45

40

°c !

30

I

I

I

Fig. 9. Same experiment as in Fig. 8, except for To -= 20°C ai~d Tl = 45°C.

t 154

W. REICH~,RDr

bacteria play a considerable part in self-purification processes preferrably at elevated temperatures, most predominant members of the "chromogenic" group in our lakes such as Cytophagas turned out to be sensitive to elevated temperatures (Reichardt, 19741. Whereas activated sludge bacteria grown in a chemostat were reportedly less sensitive to thermal stress at low growth rates and low nutrient concentrations (George & Gaudy, 1973), according to Klein & Wu (1974) starvation for nutrients could increase the susceptibility of heterotrophic bacteria to transient warming stress. Apart from a single response of the coliforms (see below), this study is in agreement with the results obtained by George & Gaudy (1973). Furthermore, as Klein & Wu's (1974) conclusions were drawn from experiments with resting cells of E. coli, the type of stress exerted is not readily comparable with that imposed on starved, but growing cells. Only when considering any changes of the availability of nutrients, i.e., elimination of essential substrates as well as their addition, as nutritiot:al stress in general, similar conclusions might be reached from both points of view. The results presented here, provide information on natural heterotrophic lake water communities which could be considered as starved for nutrients. They showed a substrate-induced partial reduction of viable counts of heterotrophic bacteria which could be 'reproduced' by shocking a synthetic mixed culture consisting of three psychrotolerant bacteria with thermal additions which exceeded, their maximum growth temperatures. Although it is nearly impossible to detect physiological phenomena such as 'substrate accelerated death' (Postgate & Hunter, 1964) in natural heterotrophic communities, obviously an analogous response had occurred on simultaneous shocks by elevated levels of temperature and substrate in mixed cultures. Whilst at moderate shifts up of temperature (up to 35°C) additional substrates imposed a stress on the coliforms, the reverse was found at 4WC. In agreement with Klein & Wu's (1974) statement a decline of coliform counts following maximal stress was prevented by additional nutrients. This finding corresponds also to those of Verstraete et al. (1975) observation that fecal bacteria are favored at high temperature and nutrient levels.

Finally, activlt 3 of succimc dehydrogenase as a parameter of bioacti~ity appeared less sensitive to thermal shock than viable counts which provided a more complex parameter. Correlations between viable counts and biochemical activity are therefore expected to change in situations where stress has been applied.

REFERENCES Allen S. D. & Brock T. D. (1968) The adaptation of heterotrophic microcosms to different temperatures. Ecoloyy

49. 343-346. George T. K. & Gaudy A. F. Jr. [19731 Transient response of continuously cultured heterogeneous populations to changes in temperature. Appl. MicrohioL 26, 796-803. Guerrero R., Roda F.. Abella C. & Torella F. (1975) Optimal growth temperatures and media parameters of bacterial communities from lakes of different trophic states. Verh. int. Verein. theor, angew. LimnoL 19, 2620-2626. Guthrie R. K.. Cherry D. S. & Ferebee R. N. 11974) A comparison of thermal loading effects on bacterial populations in polluted and non-polluted aquatic systems. Water Re.s. 8, 143-148. Herbert D., Phipps P. J. & Strange R. E. 119711 Chemical analysis of microbial cells. In Methods in Microhiolooy, edited by Norris J. R . & Ribbons D. W., Vol. 5B, 209-344. Klein D. & Wu S. (19741 Stress: a factor to be considered in heterotrophic microorganism enumeration from aquatic environments. Appl. Microhiol. 27, 429-431. Nachlas M. M., Margulies S. I. & Seligman A. M. 11960) A colorimetric method for the estimation of succinic dehydrogenase activity. J. biol. Chem. 235, 499-503. Ng H.. lngraham J. L. & Marr A. G. (1962) Damage and derepression in Escherichia coil resulting from growth at low temperatures. J. Bact. 84, 331-339. Postgate J. R. & Hunter J. R. (19641 Accelerated death of Aerobacter aerogenes starved in the presence of growth limiting substrates. J. yen. Mk'robiol. 34, 459473. Reichardt W. [1974) Zur Okophysiologie einiger Gew~isserbakterien aus der Flacobacterium-Cytophaga.Gruppe. Zbl. Bukt. 1. Abt. Oriy. A 227, 85-93. Reichardt W. (1975i Bacterial decomposition of different polysaccharides in a eutrophic lake. Verh. int. Verein. theor, angew. Limnol. 19, 2636-2642. Reichardt W. (1979) Significance of different methods to determine dehydrogenase (ETS)-activities in aquatic environments. Arch. Hydrobiol. Beih. Ergebnisse Limnol. 12, 105-114. Verstraete W., Voets J. P. & Vanstaen H. (1975) Shifts in microbial groups of river water upon passage through cooling systems. Encir. Pollut. 8, 275-281.