Microb Ecol (1984) 10:95-98
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
The Maintenance of Bdellovibrio at Low Prey Density M a z a l Varon*, M i r i a m Fine, a n d A n a t Stein The Institute of Life Sciences, Division of Microbial and Molecular Ecology, The Hebrew University, Jerusalem, Israel
Abstract. A m a t h e m a t i c a l m o d e l for the interaction o f Bdellovibrio a n d its prey predicted that a relatively high p r e y density (7 x 105 cells m1-1) would be required for the e s t a b l i s h m e n t o f an equilibrium in a m i x e d p o p u l a t i o n [8]. T h e present report shows that Bdellovibrio can be m a i n tained in a c o n t i n u o u s culture w h e n the prey cell density is m u c h lower (2-5 x 104 cells m l - 1 ) , a n d closer to that o f naturally occurring bacterial p o p u l a t i o n s in sea waters.
Introduction Bdellovibrio is a b a c t e r i u m that depends for its growth on other bacteria which serve as its prey. In the absence o f potential prey, the Bdellovibrio starves. In
order to a v o i d starvation, it m u s t locate a suitable p r e y b a c t e r i u m within a limited period o f time. T h e location o f p r e y is a p p a r e n t l y a r a n d o m process a n d its p r o b a b i l i t y can be calculated on the basis o f k n o w n p a r a m e t e r s such as size, speed, etc. H o w e v e r , a chance collision is not sufficient to establish an irreversible a t t a c h m e n t a n d the frequency o f effective collisions, that is, those leading to i n a c t i v a t i o n o f the p r e y a n d growth o f the predator, m a y be relatively small. Based on d a t a o b t a i n e d with nongrowing populations, and using the L o t k a - V o l t e r r a differential equations, we h a v e calculated that a prey density o f 7 • 105 cells m l -j is required for the e s t a b l i s h m e n t o f an equilibrium in actively growing p o p u l a t i o n s o f prey a n d p r e d a t o r [8]. Such bacterial densities are significantly higher t h a n those generally reported for natural e n v i r o n m e n t s . We therefore a t t e m p t e d to find out e x p e r i m e n t a l l y whether, in spite o f the theroretical considerations, a Bdellovibrio p o p u l a t i o n could be m a i n t a i n e d at prey densities lower t h a n 7 • 105 cells m1-1.
Materials and Methods Bacterial Strains and Their Enumeration. BM4 and Photobacterium leiognathi E28 have been described before [8]. Both strains were streptomycin-resistant. Viable counts of Bdellovibrio were done on lawns of P. leiognathi spread over plates of MPY/10 medium. Viable counts of the photobacteria were done by the spread plate technique on MPY plates. Media. Medium MPY * Present address: Department of Microbiology, Tel Aviv University, Ramat Aviv, Israel.
96
M. Varon et al.
contained (g per liter) peptone (5), yeast extract (3), NaC1 (29.72), MgSO, 7H:O (6.16), MgC12 6H20 (5.0), CaC12 H20 (1.47), KC1 (0.75), pH 7.4. To obtain lower prey densities, the peptone and yeast extract were diluted 1:10 (MPY/10); 1:15, 1:100; 1:500 and 1:2,000. For simplification, the nutrient concentration in MPY medium was designated as z = 500 (relative units), and the nutrient concentrations in the dilute media were 50, 33.5, 5, 1, 0.25, accordingly. All media were supplemented with streptomycin (100 tzg/ml) and putrescine (1 x 10-3M). Streptomycin was used to reduce the chances of contamination;putrescine was required to insure a normal intraperiplasmic cycle at low cell densities (Varon et al., Archives of Microbiology, in press).
Chemostat A New Brunswick Bench Top Chemostat, Model C30 with a l-liter vessel was used in all the experiments. The dilution rate was D = 0.0017 min-t; agitation, 400 rev/min; aeration, 200 ml/ min; temperature, 26"C.
Experimental Procedure The organism whose growth was studied in these experiments was the Bdellovibrio and, since it cannot consume dissolved organic matter, the prey served as the limiting substrate. The level of prey in the growth vessel could be determined in either one of two ways: (1) fill the reservoir with a buffer suspension of pregrown photobacteria and let them flow into the growth vessel at different dilution rates; (2) let the photobacteria grow inside the growth vessel and determine their level by changing the dilution rate of the inflowing nutrients or keeping the dilution rate constant and changing the nutrient concentrationin the reservoir. The latter technique was chosen mainlybecause of the technical difficulty of accurately maintainingthe very low dilution rates required. Preliminary experiments were run in order to establish the nutrient concentrations which would give, under the above specific conditions, steady states at various levels in the range of 2 x 104 to 2 x 108 cells m1-1. In each experiment, the prey was inoculated first and allowed to grow and reach a steady state. Only then was the predator introduced. Samples were withdrawn once or twice daily and counted as described above.
Results and D i s c u s s i o n F i g u r e 1 s h o w s t h a t b d e l l o v i b r i o s are m a i n t a i n e d i n a 2 - m e m b e r e d c o n t i n u o u s c u l t u r e i f t h e p r e y d e n s i t y is i n t h e r a n g e o f 7 x 105-2 x 108 cells m1-1. S t r o n g o s c i l l a t i o n s i n t h e p o p u l a t i o n s o f p r e y a n d p r e d a t o r are o b s e r v e d w h e n the cell d e n s i t y is high. T h e o s c i l l a t i o n s b e c o m e s m a l l e r i n a m p l i t u d e w i t h d e s c r e a s i n g b a c t e r i a l d e n s i t y . A t a n e v e n l o w e r p r e y d e n s i t y , t h e p a t t e r n is different: i n 3 o u t o f 5 e x p e r i m e n t s , t h e b d e l l o v i b r i o s were c o m p l e t e l y w a s h e d o u t (Fig. 2, left). I n 2 o t h e r e x p e r i m e n t s , t h e d e c r e a s e i n t h e n u m b e r o f b d e l l o v i b r i o s s t o p p e d b e f o r e t h e y h a d b e e n c o m p l e t e l y w a s h e d o u t a n d t h e y a p p e a r e d to h a v e r e a c h e d a state o f e q u i l i b r i u m . T h e l e v e l a t w h i c h t h i s e q u i l i b r i u m w a s m a i n t a i n e d w a s 4 - 7 x 102 m1-1 i n o n e e x p e r i m e n t (Fig. 2, fight) a n d a b o v e 103 m l -~ i n a s e c o n d e x p e r i m e n t . T h e p r e y d e n s i t y at this stage w a s a l w a y s i n t h e r a n g e o f 2 - 5 x 104 cells m l - L T h e m a i n t e n a n c e o f Bdellovibrio i n t h e c o n t i n u o u s c u l t u r e o n p r e y b a c t e r i a at a d e n s i t y l o w e r t h a n 7 x 105 cells m l -~ s t a n d s i n c o n t r a d i c t i o n w i t h t h e p r e d i c t i o n m a d e a b o v e . H o w e v e r , this t h e o r e t i c a l v a l u e was o b t a i n e d a s s u m i n g
Bdellovibrio at Low Prey Density
97
iorl I
E
i
I
Z
g
1
2
I
10 6 I
Io 5
~
I
I
104 I
o 107
|
I
I0 ~
g
o.
-o
g 102
I
I
I
I 1
g
I i
~" to
o
o I0 ~
L
0
L
i
4
J
. . . . . .
0
,
4
8
Time
,
i
IZ (doys)
J
16
,
. . . .
0
,
4
8
,
9
i2
I
,
0
,
1
1
4
1
I
B
l
l
i
0
l
4
Time (doys)
Fig. 1. T w o - m e m b e r e d continuous cultures o f Bdellovibrio BM4 and P. leiognathi at different nutrient concentrations. Left, Z = 33.5; center, Z -- 5; right, Z = 1. G r o w t h conditions are described in the text. Fig. 2. T w o - m e m b e r e d continuous cultures ofBdellovibrio BM4 a n d P. leiognathi in m e d i u m o f Z = 0.25. Two experiments are s h o w n which represent the 2 different patterns that were observed in this m e d i u m .
random distribution of the bacteria. Ideally, this should be the case in our experiments, but uneven distribution of bacteria in the continuous culture apparatus is a well-known phenomenon: wall growth has often been demonstrated [3, 5, 6]. Various surfaces inside the growth vessel (plastic coated magnet, metallic temperature probe, etc.) could also adsorb organic substrates to various degrees and allow local development of adhering prey bacteria, thus forming local foci of bacterial growth. The Bdellovibrio may find its way to these same foci where the prey concentration is sufficiently high for interaction. If so, then artificial increase of surfaces in the growth vessel should allow the maintenance of the bdellovibros in the continuous culture, even at lower prey cell densities. Similar phenomena were observed in regard to the utilization of organic substrates by other bacteria [1, 2, 9]; Jannasch [2] described an aquatic bacterial isolate whose metabolic activity at low nutrient concentrations was considerably enhanced by the presence of inert particles. Heukelekian and Heller [1] have found that the inclusion of glass beads in the growth medium greatly enhanced the growth ofEscherichia coli. In both cases, the effect of the particles was only manifested at low substrate concentrations; the particles made no difference at high substrate concentration. The opposite effect, namely a reduced surface area available for bacterial adherence, could also be experimentally achieved. This has recently been done by Ratnam et al. [5]. These authors cultivated mixed cultures of the protozoan Tetrahymena pyriformis and the bacterium E. coli in chemostats previously treated with a silicone compound. They found that the silicone treatment
98
M. Varon et at.
reduced the density o f the attached bacteria by 2 orders o f magnitude or more, and that this significantly affected the predator-prey interaction. This was due to the fact that adhered bacteria are i m m u n e to the Tetrahymena attack while those in suspension are preyed upon. Since bdellovibrios do not appear to m a k e such a distinction between adhered or suspended prey, the o u t c o m e o f silicone treatment can be expected to be different for Bdellovibro; in this case, p r e v e n t i o n o f wall growth w o u l d not greatly affect the n u m b e r o f available prey, but w o u l d affect their distribution. I f local patches o f prey are the explanation for the existence o f Bdellovibrio at low prey density, their absence should invariably result in complete w a s h o u t o f predator at z = 0.25. In the natural e n v i r o n m e n t , as in the chemostat, local concentration o f heterotrophie bacteria on surfaces is an often observed p h e n o m e n o n [4]. The survival o f Bdellovibrio in seawater and other e n v i r o n m e n t s characterized by dilute bacterial populations would be difficult to u n d e r s t a n d on the basis o f average counts o f the bacterial populations. The highest estimates for bacterial n u m b e r s in euphoric offshore a n d oceanic waters are 0 . 5 - 1 0 x 105 ceils ml -t [7]. Obviously, n o t all o f t h e m can be regarded as a potential prey even for a b r o a d - s p e c t r u m Bdellovibrio species. M o r e o v e r , a large fraction o f the total n u m b e r is c o m p o s e d o f " m i n i b a c t e r i a " which probably would not be i n v a d e d by a bdellovibrio because o f size limitation. Thus, even cell densities as high as 10 • 10 s cells ml -I do not insure the existence o f Bdellovibrio. However, the adherence o f bacteria to surfaces, air-water, or water-water interfaces could lead to the f o r m a t i o n o f local concentrations a b o v e those required for m a i n taining a low but stable p o p u l a t i o n o f Bdellovibrio.
References 1. Heukelekian H, HeUerA (1940) Relation between food concentration and surface for bacterial growth. J Bacteriol 40:547-558 2, Jannasch HW (1979) Microbial ecology of aquatic low nutrient habitats. In: Shilo M (ed) Strategies of microbiai life in extreme environments. Verlag Chemic, Weinheim, pp 243-260 3. Kubitschek HE (1970) Introduction to research with continuous cultures. Prentice-Hall Inc, Englewood Cliffs, New Jersey 4. Marshall KC (1976) Interfaces in microbial ecology. Harvard University Press, Cambridge, London 5. Ratnam DA, Pavlou S, Fredrickson AG (1982) Effects of attachment of bacteria to ehemostat wails in a microbial predator-prey relationship. Biotechnol Bioeng 24:2675-2694 6. Van den Ende P (1973) Predator-prey interactions in continuous culture. Science 181:562-564 7. Van Es FB, Meyer-Reil L-A (1982) Biomass and metabolic activity of heterotrophic marine bacteria. In: Marshall KC (ed) Advances in Microbial Ecology, Vol 6, pp 111-170. Plenum Press, New York 8. Varon M, Zeig/er BP (1978) Bacteria/ predator-prey interactions at low prey density. App] Environ Microbiol 36:11-I 7 9. Zobell CE (1943) The effect of solid surface upon bacterial activity. J Bacteriol 46:39-56
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
The Maintenance of Bdellovibrio at Low Prey Density M a z a l Varon*, M i r i a m Fine, a n d A n a t Stein The Institute of Life Sciences, Division of Microbial and Molecular Ecology, The Hebrew University, Jerusalem, Israel
Abstract. A m a t h e m a t i c a l m o d e l for the interaction o f Bdellovibrio a n d its prey predicted that a relatively high p r e y density (7 x 105 cells m1-1) would be required for the e s t a b l i s h m e n t o f an equilibrium in a m i x e d p o p u l a t i o n [8]. T h e present report shows that Bdellovibrio can be m a i n tained in a c o n t i n u o u s culture w h e n the prey cell density is m u c h lower (2-5 x 104 cells m l - 1 ) , a n d closer to that o f naturally occurring bacterial p o p u l a t i o n s in sea waters.
Introduction Bdellovibrio is a b a c t e r i u m that depends for its growth on other bacteria which serve as its prey. In the absence o f potential prey, the Bdellovibrio starves. In
order to a v o i d starvation, it m u s t locate a suitable p r e y b a c t e r i u m within a limited period o f time. T h e location o f p r e y is a p p a r e n t l y a r a n d o m process a n d its p r o b a b i l i t y can be calculated on the basis o f k n o w n p a r a m e t e r s such as size, speed, etc. H o w e v e r , a chance collision is not sufficient to establish an irreversible a t t a c h m e n t a n d the frequency o f effective collisions, that is, those leading to i n a c t i v a t i o n o f the p r e y a n d growth o f the predator, m a y be relatively small. Based on d a t a o b t a i n e d with nongrowing populations, and using the L o t k a - V o l t e r r a differential equations, we h a v e calculated that a prey density o f 7 • 105 cells m l -j is required for the e s t a b l i s h m e n t o f an equilibrium in actively growing p o p u l a t i o n s o f prey a n d p r e d a t o r [8]. Such bacterial densities are significantly higher t h a n those generally reported for natural e n v i r o n m e n t s . We therefore a t t e m p t e d to find out e x p e r i m e n t a l l y whether, in spite o f the theroretical considerations, a Bdellovibrio p o p u l a t i o n could be m a i n t a i n e d at prey densities lower t h a n 7 • 105 cells m1-1.
Materials and Methods Bacterial Strains and Their Enumeration. BM4 and Photobacterium leiognathi E28 have been described before [8]. Both strains were streptomycin-resistant. Viable counts of Bdellovibrio were done on lawns of P. leiognathi spread over plates of MPY/10 medium. Viable counts of the photobacteria were done by the spread plate technique on MPY plates. Media. Medium MPY * Present address: Department of Microbiology, Tel Aviv University, Ramat Aviv, Israel.
96
M. Varon et al.
contained (g per liter) peptone (5), yeast extract (3), NaC1 (29.72), MgSO, 7H:O (6.16), MgC12 6H20 (5.0), CaC12 H20 (1.47), KC1 (0.75), pH 7.4. To obtain lower prey densities, the peptone and yeast extract were diluted 1:10 (MPY/10); 1:15, 1:100; 1:500 and 1:2,000. For simplification, the nutrient concentration in MPY medium was designated as z = 500 (relative units), and the nutrient concentrations in the dilute media were 50, 33.5, 5, 1, 0.25, accordingly. All media were supplemented with streptomycin (100 tzg/ml) and putrescine (1 x 10-3M). Streptomycin was used to reduce the chances of contamination;putrescine was required to insure a normal intraperiplasmic cycle at low cell densities (Varon et al., Archives of Microbiology, in press).
Chemostat A New Brunswick Bench Top Chemostat, Model C30 with a l-liter vessel was used in all the experiments. The dilution rate was D = 0.0017 min-t; agitation, 400 rev/min; aeration, 200 ml/ min; temperature, 26"C.
Experimental Procedure The organism whose growth was studied in these experiments was the Bdellovibrio and, since it cannot consume dissolved organic matter, the prey served as the limiting substrate. The level of prey in the growth vessel could be determined in either one of two ways: (1) fill the reservoir with a buffer suspension of pregrown photobacteria and let them flow into the growth vessel at different dilution rates; (2) let the photobacteria grow inside the growth vessel and determine their level by changing the dilution rate of the inflowing nutrients or keeping the dilution rate constant and changing the nutrient concentrationin the reservoir. The latter technique was chosen mainlybecause of the technical difficulty of accurately maintainingthe very low dilution rates required. Preliminary experiments were run in order to establish the nutrient concentrations which would give, under the above specific conditions, steady states at various levels in the range of 2 x 104 to 2 x 108 cells m1-1. In each experiment, the prey was inoculated first and allowed to grow and reach a steady state. Only then was the predator introduced. Samples were withdrawn once or twice daily and counted as described above.
Results and D i s c u s s i o n F i g u r e 1 s h o w s t h a t b d e l l o v i b r i o s are m a i n t a i n e d i n a 2 - m e m b e r e d c o n t i n u o u s c u l t u r e i f t h e p r e y d e n s i t y is i n t h e r a n g e o f 7 x 105-2 x 108 cells m1-1. S t r o n g o s c i l l a t i o n s i n t h e p o p u l a t i o n s o f p r e y a n d p r e d a t o r are o b s e r v e d w h e n the cell d e n s i t y is high. T h e o s c i l l a t i o n s b e c o m e s m a l l e r i n a m p l i t u d e w i t h d e s c r e a s i n g b a c t e r i a l d e n s i t y . A t a n e v e n l o w e r p r e y d e n s i t y , t h e p a t t e r n is different: i n 3 o u t o f 5 e x p e r i m e n t s , t h e b d e l l o v i b r i o s were c o m p l e t e l y w a s h e d o u t (Fig. 2, left). I n 2 o t h e r e x p e r i m e n t s , t h e d e c r e a s e i n t h e n u m b e r o f b d e l l o v i b r i o s s t o p p e d b e f o r e t h e y h a d b e e n c o m p l e t e l y w a s h e d o u t a n d t h e y a p p e a r e d to h a v e r e a c h e d a state o f e q u i l i b r i u m . T h e l e v e l a t w h i c h t h i s e q u i l i b r i u m w a s m a i n t a i n e d w a s 4 - 7 x 102 m1-1 i n o n e e x p e r i m e n t (Fig. 2, fight) a n d a b o v e 103 m l -~ i n a s e c o n d e x p e r i m e n t . T h e p r e y d e n s i t y at this stage w a s a l w a y s i n t h e r a n g e o f 2 - 5 x 104 cells m l - L T h e m a i n t e n a n c e o f Bdellovibrio i n t h e c o n t i n u o u s c u l t u r e o n p r e y b a c t e r i a at a d e n s i t y l o w e r t h a n 7 x 105 cells m l -~ s t a n d s i n c o n t r a d i c t i o n w i t h t h e p r e d i c t i o n m a d e a b o v e . H o w e v e r , this t h e o r e t i c a l v a l u e was o b t a i n e d a s s u m i n g
Bdellovibrio at Low Prey Density
97
iorl I
E
i
I
Z
g
1
2
I
10 6 I
Io 5
~
I
I
104 I
o 107
|
I
I0 ~
g
o.
-o
g 102
I
I
I
I 1
g
I i
~" to
o
o I0 ~
L
0
L
i
4
J
. . . . . .
0
,
4
8
Time
,
i
IZ (doys)
J
16
,
. . . .
0
,
4
8
,
9
i2
I
,
0
,
1
1
4
1
I
B
l
l
i
0
l
4
Time (doys)
Fig. 1. T w o - m e m b e r e d continuous cultures o f Bdellovibrio BM4 and P. leiognathi at different nutrient concentrations. Left, Z = 33.5; center, Z -- 5; right, Z = 1. G r o w t h conditions are described in the text. Fig. 2. T w o - m e m b e r e d continuous cultures ofBdellovibrio BM4 a n d P. leiognathi in m e d i u m o f Z = 0.25. Two experiments are s h o w n which represent the 2 different patterns that were observed in this m e d i u m .
random distribution of the bacteria. Ideally, this should be the case in our experiments, but uneven distribution of bacteria in the continuous culture apparatus is a well-known phenomenon: wall growth has often been demonstrated [3, 5, 6]. Various surfaces inside the growth vessel (plastic coated magnet, metallic temperature probe, etc.) could also adsorb organic substrates to various degrees and allow local development of adhering prey bacteria, thus forming local foci of bacterial growth. The Bdellovibrio may find its way to these same foci where the prey concentration is sufficiently high for interaction. If so, then artificial increase of surfaces in the growth vessel should allow the maintenance of the bdellovibros in the continuous culture, even at lower prey cell densities. Similar phenomena were observed in regard to the utilization of organic substrates by other bacteria [1, 2, 9]; Jannasch [2] described an aquatic bacterial isolate whose metabolic activity at low nutrient concentrations was considerably enhanced by the presence of inert particles. Heukelekian and Heller [1] have found that the inclusion of glass beads in the growth medium greatly enhanced the growth ofEscherichia coli. In both cases, the effect of the particles was only manifested at low substrate concentrations; the particles made no difference at high substrate concentration. The opposite effect, namely a reduced surface area available for bacterial adherence, could also be experimentally achieved. This has recently been done by Ratnam et al. [5]. These authors cultivated mixed cultures of the protozoan Tetrahymena pyriformis and the bacterium E. coli in chemostats previously treated with a silicone compound. They found that the silicone treatment
98
M. Varon et at.
reduced the density o f the attached bacteria by 2 orders o f magnitude or more, and that this significantly affected the predator-prey interaction. This was due to the fact that adhered bacteria are i m m u n e to the Tetrahymena attack while those in suspension are preyed upon. Since bdellovibrios do not appear to m a k e such a distinction between adhered or suspended prey, the o u t c o m e o f silicone treatment can be expected to be different for Bdellovibro; in this case, p r e v e n t i o n o f wall growth w o u l d not greatly affect the n u m b e r o f available prey, but w o u l d affect their distribution. I f local patches o f prey are the explanation for the existence o f Bdellovibrio at low prey density, their absence should invariably result in complete w a s h o u t o f predator at z = 0.25. In the natural e n v i r o n m e n t , as in the chemostat, local concentration o f heterotrophie bacteria on surfaces is an often observed p h e n o m e n o n [4]. The survival o f Bdellovibrio in seawater and other e n v i r o n m e n t s characterized by dilute bacterial populations would be difficult to u n d e r s t a n d on the basis o f average counts o f the bacterial populations. The highest estimates for bacterial n u m b e r s in euphoric offshore a n d oceanic waters are 0 . 5 - 1 0 x 105 ceils ml -t [7]. Obviously, n o t all o f t h e m can be regarded as a potential prey even for a b r o a d - s p e c t r u m Bdellovibrio species. M o r e o v e r , a large fraction o f the total n u m b e r is c o m p o s e d o f " m i n i b a c t e r i a " which probably would not be i n v a d e d by a bdellovibrio because o f size limitation. Thus, even cell densities as high as 10 • 10 s cells ml -I do not insure the existence o f Bdellovibrio. However, the adherence o f bacteria to surfaces, air-water, or water-water interfaces could lead to the f o r m a t i o n o f local concentrations a b o v e those required for m a i n taining a low but stable p o p u l a t i o n o f Bdellovibrio.
References 1. Heukelekian H, HeUerA (1940) Relation between food concentration and surface for bacterial growth. J Bacteriol 40:547-558 2, Jannasch HW (1979) Microbial ecology of aquatic low nutrient habitats. In: Shilo M (ed) Strategies of microbiai life in extreme environments. Verlag Chemic, Weinheim, pp 243-260 3. Kubitschek HE (1970) Introduction to research with continuous cultures. Prentice-Hall Inc, Englewood Cliffs, New Jersey 4. Marshall KC (1976) Interfaces in microbial ecology. Harvard University Press, Cambridge, London 5. Ratnam DA, Pavlou S, Fredrickson AG (1982) Effects of attachment of bacteria to ehemostat wails in a microbial predator-prey relationship. Biotechnol Bioeng 24:2675-2694 6. Van den Ende P (1973) Predator-prey interactions in continuous culture. Science 181:562-564 7. Van Es FB, Meyer-Reil L-A (1982) Biomass and metabolic activity of heterotrophic marine bacteria. In: Marshall KC (ed) Advances in Microbial Ecology, Vol 6, pp 111-170. Plenum Press, New York 8. Varon M, Zeig/er BP (1978) Bacteria/ predator-prey interactions at low prey density. App] Environ Microbiol 36:11-I 7 9. Zobell CE (1943) The effect of solid surface upon bacterial activity. J Bacteriol 46:39-56
