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Bacterial survival in soil: Effect of clays and protozoa
Soil Biol. Biochem. Vol. 25, No. 5, pp. 525-531, 1993 Printedin Great Britain.All rightsreserved
0038-0717/93 $6.00 + 0.00 Copyright0 1993 PergamonPressLtd
REVIEW BACTERIAL
SURVIVAL AND
IN SOIL: EFFECT PROTOZOA
OF CLAYS
LAURA S. ENGLAND, HUNG LEE and JACK T. TREVORS*
Department
of Environmental
Biology,
University
(Accepted
of Guelph,
30 November
Guelph,
Ontario,
Canada
NIG 2W1
1992)
Summary-There are numerous interacting biological, physical and chemical factors that affect survival of both indigenous and introduced bacteria in soil. Three important factors arc clay type, clay content and predation by soil protozoans. These factors are important when considering release of non-engineered and genetically engineered microorganisms (GEMS) into soil. In this review we examine the influence of clays and predatory activity of protozoans on bacteria1 survival. These aspects are related because protozoa may ingest clay particles as well as bacteria. In addition, clays may protect bacteria from predation by protozoans by increasing the number of protective microhabitats available to bacteria in soil.
INTRODUCTION
Selected factors
content and predation by protozoans. The effects of nutrient availability, temperature, pH, microbial interactions, redox potential, salinity, radiation, light, antibiotics, capsules, slime layers and toxic compounds have been reviewed by Babich and Stotzky (1985), Clark (1965), Duxbury (1981), Gadd and Griffiths (1978), Henis (1987), Roszak and Colwell (1987) Shales et al. (1989) and Smit et a/. (1992).
that influence bacterial survival
Interest in using microorganisms for controlling plant diseases (Fuxa, 1991) enhancing productivity of agricultural crops (Keeler and Turner, 1991; Pimentel, 1985), mineral leaching (Brierley, 1985; Hughes and Poole, 1989), emulsifying (Berg et al., 1990; Jain et al., 1992; Van Dyke et al., 1991) and degrading toxic chemicals (Alexander, 1981; Briglia et al., 1990; Horvath, 1972; Jain et al., 1992; Leahy and Colwell, 1990; Schwien and Schmidt, 1982; Seech et al., 1991) has led to increased research into factors that affect bacterial survival, activity, dispersal and gene transfer in soil (Levin and Strauss, 1991; Trevors, 1991; Trevors and van Elsas, 1989). Both naturally occurring and genetically engineered microorganisms (GEMS) may be released deliberately or accidentally into soil and aquatic environments. It is therefore necessary to understand survival and activity of these organisms in the environment. The study of bacterial survival in soil is a challenge to researchers due to complex physical, chemical and biological interactions that occur in soil (Paul and Clark, 1989). A recognized problem is bacteriostasis, defined as a physiological resting state of bacteria characterized by inactivity (Brown, 1973; Ho and Ko, 1982; Ko and Chow, 1977). Another problem is the viable but non-culturable cells phenomenon (Roszak and Colwell, 1987). Bacteria may be surviving and growing in their environment but can not be recovered and cultured by conventional plating methods (Stotzky, 1989). Bacterial survival in soil can be influenced by a number of factors (Table 1) and also by clay type and
*Author
Selected clay characteristics Clays are silicate minerals belonging to the phyllosilicates, which have a layered structure. Two main types may be distinguished: the 2: 1 type (e.g. illite, bentonite, montmorillonite and vermiculite, the latter three with a strong swelling capacity after wetting) consisting of two layers of Si04 tetrahedrons and an octahedral layer of, e.g. AlO, (OH),; the 1: 1 type (e.g. kaolinite, a non-expansible clay) with one SiO, tetrahedral layer and one octahedral layer of, e.g. AlOz (OH), (Borchardt, 1979; Dixon, 1979; Fanning and Keramidas, 1979; Harter, 1979). Some commonly studied clays with regard to their influence on microorganisms include bentonite, montmorillonite, kaolinite, illite and vermiculite. The cation exchange capacity (CEC) of expansible clays is greater than non-expansible clays due to exchangeable cations on internal as well as external surfaces of the clay particles. For example the CEC of montmorillonite is 8&100me lOOg_‘, of illite 15-40 me 100 gg’, while that of kaolinite is 3-5 me lOOg-’ (Buckman and Brady, 1964). The type and amount of clay present in a soil can affect the soil matrix, and therefore, bacterial survival and activity. Clay particles generally impart a fine texture and a heavy nature to soil. The fine texture produces a large amount of total pore space but pore sizes are small. Clay particles generally have a ce-
for correspondence. 525
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LAURA S. ENGLAND et al.
menting effect on soil aggregation and clays of smaller particle sizes were found to form more stable aggregates than clays of larger particle sizes (Mazurak, 1950). Mazurak also suggested clay particle size has a role in the permeability of aggregates to water and gases, and pore size is controlled by clay particle size. The ranges of particle sizes of montmorillonite, illite and kaolinite are 0.01-1.0, 0.1-2.0 and 0.1-5.0 pm, respectively (Buckman and Brady, 1964). The roles of clays and protozoans on bacterial survival have not been fully investigated in a wide range of soils, even though they influence microbial populations (Heijnen et al., 1992; Kuikman, 1990; Stotzky and Rem, 1966). Information on claymicrobe interactions has come from laboratory and field studies (Heijnen et al., 1988; Marshall, 1968; Marshall and Roberts, 1963; Stotzky, 1966; Stotzky and Rem, 1966). We review the influence of clays and predation by protozoa on bacterial survival in soil. EFFECT OF CLAYS ON BACTERIAL
SURVIVAL
Some excellent studies by Marshall and colleagues provided evidence that bacterial survival is influenced
by clays. Marshall and Roberts (1963) examined the influence of fine particle materials (fine silica, montmorillonite and fly ash consisting mainly of burnt clay) on survival of Rhizobium trifolii in sandy soil. Both fly ash and montmorillonite increased Rhizobium survival as determined by 73 and 54%, respectively, nodulation of clover compared to 21% in the silica-amended and 28% in non-amended soil. The nature of this increased nodulation was not described, although it may be due to one or more of the microbe-clay interactions reported by Marshall (1968). When coated with montmorillonite, the electrophoretic mobility of Rhizobium cells was similar to montmorillonite. Secondly, an “edge-to-face” association between clay platelets and bacterial cell surfaces may account for the large amount of montmorillonite absorbed per unit area. The amount of montmorillonite absorbed per unit area was higher with cells possessing surface carboxyl groups. Thirdly, a montmorillonite envelope which surrounded bacterial cells may confer a protective effect from desiccation, high temperatures, X-rays and metabolically induced pH changes (Marshall, 1968). Stotzky and Rem (1966) reported that montmoril-
Table I. Some factors that affect bacterial survival in soil Factors Surfaces
Moisture
Soil texture
Plants (rhizosphere)
Bacterial movement
lnoculum density
comments Review of influence of surfaces on microbial activity Discussion of terrestrial subsurface with respect to microbial ecology Review of microbial adhesion to clays Discussion of interfaces and interfacial interactions with respect to microbial ecology Rhizobium sp. exhibit better survival with lower initial moisture content Rewetting dried soil increased bacterial population Water potential influenced survival of four rhizobial strains Short review on water activity in the soil and its effect on bacterial survival Rhizobium sp. final cell numbers were higher m silt loam than in loamy sand Recombinant PseudomonasPuorescens survived better in a loam than in a loamy sand f?. subrilis cell numbers declined rapidly in loamy sand and silt loam, PseudomonasPuoresrens cell numbers declined slowly in both soils but survival was better in silt loam than loamy sand Soil texture affected survival of rhizobial strains, better survival in sandy loams. silt loams and sandy clay loams Bacteria-plant species specificity, probable cause of rhizosphere effect: root exudates and growth factors synthesized by other bacteria, generalization that rhizosphere effect increases with plant age Bacteria-plant species specificity, amount of bacterial growth possibly related to quantity of substrates released Review-bacterial species present/absent in the rhizosphere, factors affecting bacterial survival in the rhizosphere Recombinant Pseudomonas sp., limited lateral migration, vertical movement to a depth of 30 cm (after 5 weeks) (possible association with roots of inoculated plants) Transport of bacteria with water through soil, six genera of bacteria tested, concluded cell size plus other unknown factors affect transport Recombinant Pseudomonas sp. downward transport through soil dependent on flow rate and number of times water was added Addition of water found to affect bacterial distribution, Pseudomonas strain inoculated on wheat roots was recovered 3.5 cm below inoculation site (after 24 h) when water was added to soil surface, 0.5 cm without water Rhizobium japonicum and Pseudomonas purida, both strains were not found in nonsterile soil below 2.7 cm in absence of a transporting agent or in the presence of plant roots, earthworm and combinations of a transporting agent and earthworm or plant roots enhanced transport Rhizobium sp., as inoculum density increased final population sizes increased in both a silt loam and a loamy sand
Sources Van Loosdrecht et al. (1990) Ghiorse and Wilson (1988) Stotzky (1985) Marshall (1976) Postma ef al. (1989) Lund and Goksoyr (1980) Mahler and Wollum (1981) Morita (1992) Postma et al. (1990) Van Elsas ef al. (1989) Van Elsas ef al. (1986)
Mahler and Wollum (1981) Curl and Truelove (1986) Bennett and Lynch (1981) Katznelson
(I 965)
Kluepfel er al. (1991)
Gannon er al. (1991) Trevors er al. (1990) Parke et cl. (1986)
Madsen and Alexander ( 1982)
Postma et al. (1990)
Bacterial survival in soil lonite stimulated bacterial respiration by maintaining the pH at a value suitable for sustained growth. Due to the higher CEC of montmorillonite, more H + ions can be exchanged with adsorbed basic cations. The low CEC of kaolinite could not provide this buffering capacity. They also attributed this respiratory stimulation in part to mineral nutrients provided by clays. They suggested both montmorillonite and kaolinite were equally effective in providing minerals for bacteria. Stotzky (1985) later suggested that exchangeable inorganic nutrients adsorbed on clays may be available to microbes. It is not known how bacterial survival or activity in soil may be affected due to greater nutrient availability from clays exhibiting higher CEC values. Roper and Marshall (1974) presented evidence that Escherichia coli was protected from phage lysis by sediments, montmorillonite or organic matter at salinity values below or above the required point for desorption and dispersal. At low electrolyte concentrations, this protection was provided by an envelope of colloidal materials sorbed to cells. At high electrolyte concentrations, protection resulted from both the colloidal envelope around cells and sorption of cells and phages to solid particles. The electrolyte (salt) concentration was varied by using different concentrations of sea water. Stutz et al. (1989) demonstrated that survival of Pseudomonas fluorescens in vermiculite was better than in montmorillonite, which was better than in illite. For example, after 6 weeks, survival in pure vermiculite was 2 x lo5 cfu gg’ whereas in pure montmorillonite, survival was 2 x lo3 cfu g-‘. No cfu were detected in pure illite. The experiment was conducted at 20°C and 70% relative humidity. It is not understood how different clay types influenced bacterial survival. This may be related to particle size-surface area, moisture retention, cation exchange capacity (CEC) or nutrient status. In recent years, the effect of adding bentonite to a loamy sand inoculated with R. Ieguminosarum biovar trifofii was examined by Heijnen et al. (1988). It was observed that cell numbers in non-sterile soil at 15°C and 18% moisture decreased from 10’ to 3 x lo5 cfu g-r dry soil after 60 days. In soil amended with 10% (w/w) bentonite, cell numbers remained at 10’ cfu g-r during the experiment. When inoculated into sterile soil, Rhizobium cell numbers increased from 10’ to 3 x lo* cfu g-’ dry soil during the first week, and remained unchanged for the remainder of the 60 day incubation. A similar trend was observed with the same microorganism in sterile soil amended with bentonite. These results showed the protective effect of bentonite depended on whether the soil was sterile or not. A decrease in introduced Rhizobium cells in non-sterile soil may have been due to competition by indigenous organisms or predation primarily by protozoa. These interactions would not be present in sterile soil. A Tn5 mutant of R. leguminosarum biovar trifolii survived better in a non-sterile
527
loamy sand agricultural soil amended with 10% (w/w) bentonite or 10% (w/w) kaolinite compared to unamended soil (Heijnen and van Veen, 1991). Bacteria were introduced into soils at log 7.4 to 7.7 cfu gg’ dry soil, and the indigenous clay content of unamended loamy sand soil was 3% (van Elsas et al., 1986). After 57 days, the log cfu gg’ soil were 8.05, 6.41 and 5.49 for soil amended with bentonite, kaolinite and unamended soil, respectively. Immunofluorescence counts for the same treatments were 8.09, 6.42 and 5.62, respectively, showing an excellent correlation between two different enumeration methods. One suggestion to explain increased cell survival in clay amended soils was that clays shielded microorganisms from predation by protozoans by forming protective microhabitats (Heijnen et al., 1988; Heijnen and van Veen, 1991). It is noteworthy that introduced bacteria survived better in soil amended with bentonite than with kaolinite. It is possible that addition of clay to soil creates new microhabitats from those initially present, especially if the soil had a low clay content (e.g. loamy sand soil). Heijnen and van Veen (1991) suggested the number of protective pore spaces increased with addition of bentonite. These authors used moisture retention curves to show increased pore spaces in the following order: loamy sand < loamy sand + 10% kaolinite < loamy sand + 5% bentonite < loamy sand + 10% bentonite. PROTOZOAN-BACTERIAL
INTERACTIONS
Predation was recognized as a significant factor affecting bacterial survival in soil more than 60 years ago (Cutler, 1927, cited in Fenchel, 1987). Of soil organisms that feed on bacteria, protozoa appear to be the group that has the most significant effect (Acea et al., 1988; Acea and Alexander, 1988; Danso et al., 1975; Heijnen et al., 1988). The possibility that slime molds, bacteriophages, Bdellovibrios and myxobacteria might be partially responsible for decline of bacteria in soil has not been shown to be the case (Acea et al., 1988). Bacteria are a major food source for protozoans such as flagellates, naked amoebae and some ciliates (Fenchel, 1987). Some amoebae exhibit definite bacterial preferences, for example, between Gram-positive and Gram-negative and between old and young cultures (Oehler, 1919, cited in Sandon, 1927). Within the free living phagotrophic protozoan group there are three feeding habits: (a) filter feeding, (b) raptorial feeding and (c) diffusion feeding. Filter feeding is the filtering of suspended food particles (e.g. bacteria) by some flagellates and ciliates. A filter-feeding ciliate such as Paramecium caudatum can ingest about 400 particles s-r (Fenchel, 1980). Raptorial feeding involves the individual ingestion of bacteria, both suspended and surface-bound, by small flagellates and amoebae. Diffusion feeding by some amoeboid protozoa, such as foraminifera and
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LAURA S. ENGLAND et al.
small heliozoans, involves extension of the pseudopodium to engulf the prey, followed by food vacuole formation (Fenchel, 1987). The number of protozoans in soil has been estimated at between 1000 and 500,000 organisms per g of moist soil (Sleigh, 1973). This agrees with the estimate of Campbell (1983) that protozoan numbers range from log 4 gg ’ soil or 38 g m-3 in the top 15 cm of an agricultural soil. Protozoans are important in carbon and nitrogen cycling in soil. The presence of protozoans in soil has been shown to increase the turnover and mineralization of carbon (Kuikman et al., 1990a) and nitrogen (Kuikman et al., 1990a, b; Kuikman and van Veen, 1989). Kuikman et al. (1990a) have concluded from experimental observations that protozoa increase the turnover rate of carbon and nitrogen by (a) consuming bacteria and releasing nitrogen as a byproduct and (b) increasing activity of bacterial populations and this in turn increases the amount of carbon metabolized by bacteria. A number of studies have been performed to assess if protozoans have an effect on introduced bacteria. When bacteria were added to sterile soil no decline in numbers occurred (Danso et al., 1975; Heijnen et al., 1988). If protozoans were also introduced into sterile soil, bacterial numbers declined (Heijnen et al., 1988). Generally, results have shown that when bacteria are added to non-sterile soils, bacterial populations decline (Chao and Alexander, 1981; Danso et al., 1975; Heijnen et al., 1988), while protozoan numbers increase (Chao and Alexander, 1981; Danso et al., 1975). The addition of 1% glucose to sterile soil, with added protozoa, delayed decline of Agrobacterium, Pseudomonas and Corynebacterium spp for approximately 2 days, while the protozoan population grew for the first 4-6 days then declined (Acea and Alexander, 1988). Danso et al. (1975) studied Rhizobium cells and protozoa in nonsterile soil. Rhizobial cell numbers declined, but were not eliminated, whereas protozoan numbers increased. The inability of protozoans to remove all prey was not attributed to another predator attacking the protozoa. In salt solution, protozoa were also incapable of eliminating the bacterial prey (Danso and Alexander, 1975). In sterile soil inoculated with both bacteria and protozoa, protozoa also did not eliminate all the bacteria (Danso et al., 1975). It was also noted that protective microhabitats were not the reason for bacterial survival because results were the same in sterile pond water as in soil (Danso et aI., 1975). It was suggested the inability of the predators to eliminate their prey was a result of protozoan numbers reaching a density where the energy used in finding the prey equalled that from feeding. Amoebae populations in both field and pot experiments were found to be present in large enough numbers and fluctuated in a manner that indicated their involvement in regulating bacterial numbers
(Clarholm, 1981). Flagellates and ciliates did not have a similar role. In the field study, the presence of amoebae resulted in a 60% decrease in bacterial populations. For example, during a rainfall, amoebae density was lo5 gg’ dry soil weight. The large rainfall caused a IO-fold increase in bacterial biomass after 2 days. Four days later, the amoebae population increased 20-fold while bacterial biomass declined. In pot studies not containing plants, amoebae numbers increased 6-fold over the initial value of 104g-’ dry soil weight. However, in pot studies containing wheat seedlings, this increase was 30-fold (Clarholm, 198 1). These populations were transient as 2 days later, amoebae numbers decreased to 3 times the initial value in planted soil and 2 times in unplanted pots. Bacteria are not evenly distributed throughout soil. Instead cells are found in small colonies (usually less than 10 cells) (Campbell, 1983) that cover about 0.1% of the available surface area (Wood, 1988). This suggests that protozoa must move through soil to seek bacteria and to avoid unfavourable conditions. Cultivation of soils will assist in bringing bacterial and protozoan populations into contact with each other (Wood, 1988). Vargas and Hattori (1986) reported that cell numbers of Aerobacter aerogenes in the presence of Colpodu sp. decreased from lo* to about lo4 cells gg’ soil in the outer zone of soil aggregates (1-2 mm) in 12 days. By contrast, 10’ cells gg’ soil remained in the inner aggregate zone after the same period of exposure. This suggests susceptibility of bacterial cells to predation is influenced by their spatial location in soil. Protozoa do not always respond to bacteria added to soil. Casida (1989) did not observe an increase in protozoan numbers on addition of Arthrobacter globiformis and Bacillus thuringiensis cells to soil. However, B. mycoides spores added to soil caused a marked increase in numbers of selected protozoans. It was not known if this response was associated with spore germination. The results suggest some preferential feeding. Even though E. coli is not a common soil microorganism, the protozoans increased in response to its addition in a manner similar to that when B. mycoides was added. Heijnen et al. (199 1) observed that Bodo sultans fed almost solely on R. leguminosarum biovar trifolii despite the presence of other bacterial species present in demineralized water. It should be noted that prior to inoculation into water, B. sultans was maintained exclusively on R. leguminosarum biovar trifolii. It is not known if this maintenance regime influenced subsequent feeding behaviour of protozoans during the experiment. In their study, addition of protozoans to 1O’cfu ml-’ rhizobial cells in distilled water resulted in a decline in cell numbers by 2-3 log units over 3540 days. Addition of 2.5% (w/v) bentonite led to little change in Rhizobium cell numbers in the presence of B. sultans. The protozoan population was not significantly affected by bentonite for the first 7
Bacterial survival in soil days, whereas after 15 days, protozoa were undetectable. It is noteworthy that clay particles were found to interfere with microscopic detection of this organism. They also reported that bentonite did not release substances toxic to protozoans (Heijnen et al., 1991). Free-living phagotrophic protozoa may not be able to distinguish between clay particles and bacteria, and ingest both. Fenchel (1980) observed that filter feeding ciliates ingested latex beads (dia studied 0.09-5.7 pm) as easily as bacteria. The optimal size ingested by 14 different ciliates ranged from 0.3 to 1 pm dia. Interestingly, this size range is very similar to the size of some bacterial cells. For example, Rhizobium sp. are rod shaped ranging in size from 0.5-0.9 by 1.2-3.0pm in length (Holt et al., 1977). Dimensions of the genus Pseudomonas are straight or curved rods of 0.5-1.0 by 1.54.0pm (Holt et al., 1977). The ranges of particle size of montmorillonite, illite and kaolinite are 0.01-1.0, 0.1-2.0 and 0.1-5.0 pm, respectively (Buckman and Brady, 1964). It is noteworthy that the l-5-pm clay sizes are in the same magnitude as some common soil bacteria. Fenchel (1980) suggested size selection may be a function of the size of the mouth apparatus. Selective feeding behaviour by protozoans has been documented (Sandon, 1927). If selective feeding behaviour by indigenous protozoans is common, it may be possible to select bacterial strains that are not ingested, or ingested at a low frequency, for use in soil. Bacterial cells adsorbed to clay particles exhibit an increase in mass and volume. This can affect the feeding behaviour of predatory protozoans because the combined clay-cell complex may be too large to ingest. Predation as a factor affecting bacterial survival in soil must be examined critically if introduced bacteria are to survive and perform the function they were introduced to do. Ramadan et al. (1990) suggested that bacterial populations added as inocula for biodegradation may fail to survive because of nutrient deficiencies or grazing by protozoans. CONCLUDING
REMARKS
Clays and protozoa play important roles in determining microbial survival and activity in soil. Clays may protect bacterial cells from predation by creating protective pore spaces. Also, protozoa may ingest clay particles indiscriminately, thereby sparing bacterial cells. This may reduce their food intake and decrease their reproductive potential. In recent years, information on clay-bacteria-protozoan interactions has been obtained using sterile and non-sterile soils amended with this mixture. Although this approach provides useful information, the laboratory conditions do not reflect those found in the field. There is a lack of information on whether the protective effects of added clays is also seen in soil with a high initial clay content. More studies are needed to determine if soil protozoa exhibit selective
529
feeding behaviour with respect to indigenous or introduced bacterial strains. An interesting study would be to determine if a protozoan reared on a bacterial strain it is not known to feed on will exhibit preferential feeding of this strain when provided with its preferred bacterial prey. It is not understood how indigenous protozoa compete with introduced protozoans. In environmental applications, bacterial cells will be released into different soil types under a variety of conditions. A range of soil type, bacterial or protozoan numbers and species has not been studied in detail, and hence requires further investigation. For specific applications such as bioremediation of contaminated soils, it would be useful to know if indigenous protozoa may affect the survival and degradative activity of introduced cells. A knowledge of these factors will assist researchers when formulating and releasing microbial cells for introduction into soils (Trevors et al., 1993) which may contain active protozoan populations. Acknowledgements-Our research on bacterial survival was supported by a grant from the Ontario Ministry of the Environment. L. S. England was the holder of a Postgraduate scholarship award from the Natural Sciences and Engineering Research
Council
of Canada.
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Henis Y. (1987) Survival and Dormancy of Microorganisms. Wiley, New York. Ho W. C. and Ko W. H. (1982) Characteristics of soil microbiostasis. Soil Biology & Biochemistry 14, 5899593. Holt J. G. (1977) The Shorter Bergey’s Manual of Dererminative Bacteriology, 8th edn. The Williams & Wilkins, Baltimore. Horvath R. S. (1972) Microbial co-metabolism and the degradation of organic compounds in nature. Bacteriology Reviews 36, 146155. Hughes M. N. and Poole R. K. (1989) Metals and Microorganisms. Chapman and Hall, New York. Jain D. K.. Lee H. and Trevors J. T. (1992), Effect of addition of Pseudomonas aeruginosa UG2 inocula or biosurfactants on biodegradation of selected hydrocarbons in soil. Journal of Industrial Microbiology. 10, 87-93. Katznelson H. (1965) Nature and importance of the rhizosphere. In Ecology of soil-borne plant parhogens: Prelude to bioloeical conrrol (K. F. Baker and W. C. Snvder. Eds), pp. 187-209. University of California Press, Berkeley, Los Angeles. Keeler K. H. and Turner C. E. (1991) Management of transgenic plants in the environment. In Risk Assessmen/ _ in Genetic Engineering: Environmental Release of Organ isms (M. Levin and H. Strauss, Eds), pp. 189-218. McGraw-Hill, New York. Kluepfel D. A., Kline E. L., Skipper H. D., Hughes T. A., Gooden D. T.. Drahos D. J.. Barrv G. F., Hemmine B. C. and Brandt E. J. (1991) The release of tracking of genetically engineered bacteria in the environment. Phytopathology 81, 348-352. Ko W. H. and Chow F. K. (1977) Characteristics of bacteriostasis in natural soils. Journal of General Microbiology 102, 295-298. Kuikman P. J. (1990) Mineralization of nitrogen by protozoan activity in soil. Ph.D. thesis, Agricultural University of Wageningen, The Netherlands. Kuikman P. J. and van Veen J. A. (1989) The impact of protozoa on the availability of bacterial nitrogen to vlants. Biology & Fertility of Soils 8, 13-18. Kuikman P. J.,Jansen A. G., van Veen J. A. and Zehnder A. J. B. (1990a) Protozoan predation and the turnover of soil organic carbon and nitrogen in the presence of plants. Biology and Fertility of Soils 10, 22-28. Kuikman P. J., van Elsas J. D., Jansen A. G., Burgers S. L. G. E. and van Veen J. A. (1990b) Population dynamics and activity of bacteria and protozoa in relation to their spatial distribution in soil. Soil Bio/og_y & Biochemistry 22, 1063-1073. Leahy J. G. and Colwell R. R. (1990) Microbial degradation of hydrocarbons in the environment. Microbiology Reviews 54, 305-315. Levin M. A. and Strauss H. S. (Eds) (1991) Risk As.~e.wnen/ in Genetic Engineering. McGraw-Hill, New York. Lund V. and Goksoyr J. (1980) Effects of water fluctuations on microbial mass and activity in soil. Microbial Ecoiog) 6, 115-123. Madsen E. L. and Alexander M. (1982) Transport of Rhizobium and Pseudomonas through soil. Soil Science Societ_v of America Journal 46, 557-560. Mahler R. L. and Wollum A. G. (1981) The influence ofsoil water potential and soil texture in the survival of Rhizobium japonicum and Rhizobium leguminosarum isolates in the soil. Soil Science Society of America Journal 45, 761-766. Marshall K. C. (1968) Interaction between colloidal montmorillonite and cells of Rhizobium species with different ionogenic surfaces. Biochimica et Biophysics Acta 1%. 1799186. Marshall K. C. (1976) Interfaces in Microbiul Ecology. Harvard University Press, Cambridge. Marshall K. C. and Roberts F. J. (1963) Influence of fine
Bacterial
survival
particle materials on survival of Rhizobium frifolii in sandy soil. Nufure 198, 410-411. Mazurak A. P. (1950) Aggregation of clay separates from bentonite, kaolin, and a hydrous-mica soil. Soil Science Society of America Proceedings 15, 18-24. Morita R. Y. (1992) Survival and recovery of microorganisms from environmental samples. In Microbial Ecology: Principles, Methods, and Applications (M. A. Levin, R. J. Seidler and M. Rogul, Eds), pp. 435453. McGraw-Hill, New York. Parke J. L., Moen R., Rovira A. D. and Bowen G. D. (1986) Soil water flow affects the rhizosphere distribution of a seed-borne biological control agent, PseudomonasJluorestens. Soil Biology & Biochemistry 18, 5833588. Paul E. A. and Clark F. E. (1989) Soil Microbiology and Biochemistry. Academic Press, New York. Pimentel D. (1985) Using genetic engineering for biological control: reducing ecological risks. In Engineered Organbms in the Environment: Sciemific Issues (H. 0. Halvorson, D. Pramer and M. Rogul, Eds), pp. 1299140. American Society for Microbiology, Washington. Postma J., Hok-A-Hin C. H. and Oude Voshaar J. H. (1990) Influence of the inoculum density on the growth and survival of Rhizobium leguminosarum biovar trlfolii introduced into sterile and non-sterile loamy sand and silt loam. FEMS Microbiology Ecology 13, 49-58. Postma J., van Veen J. A. and Walter S. (1989) Influence of different initial soil moisture contents on the distribution and population dynamics of introduced Rhizobium leguminosarum biovar trifolii. Soil Biology & Biochemistry 21, 437442. Ramadan M. A., El-Tayeb 0. M. and Alexander M. (1990) Inoculum size as a factor limiting success of inoculation for biodegradation. Applied and Environmental Microbiology 56, 139221396:Rooer M. M. and Marshall K. C. (1974) Modification of the \ interaction between Escherichiu coli and bacteriophage in saline sediment. Microbial Ecology 1, l-13. Roszak D. B. and Colwell R. R. (1987) Survival strategies of bacteria in the natural environment. Mirobiology Reviews 51, 365-379. Sandon H. (1927) The Composition and Distribution of the Protozoan Fauna of the Soil. Oliver and Boyd, Edinburgh. Schwien U. and Schmidt E. (1982) Improved degradation of monochlorophenols by a constructed strain. Applied Environmental Microbiology 44, 33-39. Seech A. G., Trevors J. T. and Bulman T. L. (1991) Biodegradation of pentachlorophenol in soil: the response to physical, chemical, and biological treatments. Canadian Journal of Microbiology 37, 440444. Shales S., Thake B. A., Frankland B., Khan D. H., Hutchinson J. D. and Mason C. F. (1989) Biological and ecological effects of oils. In The Fate and Effects of Oil in Freshwater (J. Green and M. W. Trett, Eds), pp. 1733195. Elsevier Applied Science, London. Sleigh M. A. (1973) The Biology of Protozoa. Edward Arnold, London. I
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Smit E., van Elsas J. D. and van Veen J. A. (1992) Risks associated with the application of genetically modified microorganisms in terrestrial ecosystems. FEMS Microbiology Reviews. 88, 263-278. Stotzky G. (1966) Influence of clay minerals on microorganisms. II. Effect of various clay species, homoionic clays, and other particles on bacteria. Candiun Journal of Microbiology 12, 831-848. Stotzky G. (1985). Mechanisms of adhesion of clays, with reference to soil systems. In Bacterial Adhesion: Mechanisms and Physiological SigniJicance (D. C. Savage and M. Fletcher, Eds), pp. 195-253. Plenum Press, New York. Stotzky G. (1989) Gene transfer among bacteria in soil. In Gene Transfer in the Environment (S. B. Levy and R. V. Miller, Eds), pp. 165-222. McGraw-Hill, New York. Stotzky G. and Rem L. T. (1966) Influence of clay minerals on microorganisms. I. Montmorillonite and kaolinite on bacteria. Canadian Journal of Microbiology 12, 547-563. Stutz E., Kahr G. and Defago G. (1989) Clays involved in suppression of tobacco black root rot by a strain of Psuedomonas fluorescens. Soil Biology & Biochemistry 21, 361-366. Trevors J. T. (1991) Respiratory activity of alginate-encapsulated Pseudomonusjkorescens cells introduced into soil. Applied Microbiology & Biotechnology 35, 416419. Trevors J. T. and van Elsas J. D. (1989) A review of selected methods in environmental microbial genetics. Canadian Journal of Microbiology 35, 895-902. Trevors J. T., van Elsas J. D., Lee H. and van Overbeek L. S. (1992) Encapsulation of bacterial cells for release into soil. Microbial Releases 1, 6169. Trevors J. T., van Elsas J. D., van Overbeek L. S. and Starodub M.-E. (1990) Transport of a genetically engineered Pseudomonas fiuorescens strain through a soil microcosm. Applied and Environmental Microbiology 56, 401408. Van Dyke M. I., Lee H. and Trevors J. T. (1991) Applications of microbial surfactants. Biotechnology Advances 9, 241-252. Van Elsas J. D., Dijkstra A. F., Govaert J. M. and van Veen J. A. (1986) Survival of Pseudomonas fluorescens and Bacillus subfilis introduced into two soils of different texture in field microplots. FEMS Microbiology Ecology 38, 151-160. Van Elsas J. D., Trevors J. T., van Overbeek L. S. and Starodub M. E. (1989) Survival of Pseudomonasjuorestens containing plasmids RP4 or pRK2501 and plasmid stability after introduction into two soils of different texture. Canadian Journal of Microbiology 35, 951-959. Van Loosdrecht M. C. M., Lyklema J., Norde W. and Zehnder A. J. B. (1990) Influence of interfaces on microbial activity. Microbiological Reviews 54, 75-87. Varaas R. and Hattori T. (1986) Protozoan oredation of bacterial cells in soil aggregates. FEMS Microbiology Ecology 38, 233-242. Wood M. (1988) Soil Biology. Chapman & Hall, New York.
0038-0717/93 $6.00 + 0.00 Copyright0 1993 PergamonPressLtd
REVIEW BACTERIAL
SURVIVAL AND
IN SOIL: EFFECT PROTOZOA
OF CLAYS
LAURA S. ENGLAND, HUNG LEE and JACK T. TREVORS*
Department
of Environmental
Biology,
University
(Accepted
of Guelph,
30 November
Guelph,
Ontario,
Canada
NIG 2W1
1992)
Summary-There are numerous interacting biological, physical and chemical factors that affect survival of both indigenous and introduced bacteria in soil. Three important factors arc clay type, clay content and predation by soil protozoans. These factors are important when considering release of non-engineered and genetically engineered microorganisms (GEMS) into soil. In this review we examine the influence of clays and predatory activity of protozoans on bacteria1 survival. These aspects are related because protozoa may ingest clay particles as well as bacteria. In addition, clays may protect bacteria from predation by protozoans by increasing the number of protective microhabitats available to bacteria in soil.
INTRODUCTION
Selected factors
content and predation by protozoans. The effects of nutrient availability, temperature, pH, microbial interactions, redox potential, salinity, radiation, light, antibiotics, capsules, slime layers and toxic compounds have been reviewed by Babich and Stotzky (1985), Clark (1965), Duxbury (1981), Gadd and Griffiths (1978), Henis (1987), Roszak and Colwell (1987) Shales et al. (1989) and Smit et a/. (1992).
that influence bacterial survival
Interest in using microorganisms for controlling plant diseases (Fuxa, 1991) enhancing productivity of agricultural crops (Keeler and Turner, 1991; Pimentel, 1985), mineral leaching (Brierley, 1985; Hughes and Poole, 1989), emulsifying (Berg et al., 1990; Jain et al., 1992; Van Dyke et al., 1991) and degrading toxic chemicals (Alexander, 1981; Briglia et al., 1990; Horvath, 1972; Jain et al., 1992; Leahy and Colwell, 1990; Schwien and Schmidt, 1982; Seech et al., 1991) has led to increased research into factors that affect bacterial survival, activity, dispersal and gene transfer in soil (Levin and Strauss, 1991; Trevors, 1991; Trevors and van Elsas, 1989). Both naturally occurring and genetically engineered microorganisms (GEMS) may be released deliberately or accidentally into soil and aquatic environments. It is therefore necessary to understand survival and activity of these organisms in the environment. The study of bacterial survival in soil is a challenge to researchers due to complex physical, chemical and biological interactions that occur in soil (Paul and Clark, 1989). A recognized problem is bacteriostasis, defined as a physiological resting state of bacteria characterized by inactivity (Brown, 1973; Ho and Ko, 1982; Ko and Chow, 1977). Another problem is the viable but non-culturable cells phenomenon (Roszak and Colwell, 1987). Bacteria may be surviving and growing in their environment but can not be recovered and cultured by conventional plating methods (Stotzky, 1989). Bacterial survival in soil can be influenced by a number of factors (Table 1) and also by clay type and
*Author
Selected clay characteristics Clays are silicate minerals belonging to the phyllosilicates, which have a layered structure. Two main types may be distinguished: the 2: 1 type (e.g. illite, bentonite, montmorillonite and vermiculite, the latter three with a strong swelling capacity after wetting) consisting of two layers of Si04 tetrahedrons and an octahedral layer of, e.g. AlO, (OH),; the 1: 1 type (e.g. kaolinite, a non-expansible clay) with one SiO, tetrahedral layer and one octahedral layer of, e.g. AlOz (OH), (Borchardt, 1979; Dixon, 1979; Fanning and Keramidas, 1979; Harter, 1979). Some commonly studied clays with regard to their influence on microorganisms include bentonite, montmorillonite, kaolinite, illite and vermiculite. The cation exchange capacity (CEC) of expansible clays is greater than non-expansible clays due to exchangeable cations on internal as well as external surfaces of the clay particles. For example the CEC of montmorillonite is 8&100me lOOg_‘, of illite 15-40 me 100 gg’, while that of kaolinite is 3-5 me lOOg-’ (Buckman and Brady, 1964). The type and amount of clay present in a soil can affect the soil matrix, and therefore, bacterial survival and activity. Clay particles generally impart a fine texture and a heavy nature to soil. The fine texture produces a large amount of total pore space but pore sizes are small. Clay particles generally have a ce-
for correspondence. 525
526
LAURA S. ENGLAND et al.
menting effect on soil aggregation and clays of smaller particle sizes were found to form more stable aggregates than clays of larger particle sizes (Mazurak, 1950). Mazurak also suggested clay particle size has a role in the permeability of aggregates to water and gases, and pore size is controlled by clay particle size. The ranges of particle sizes of montmorillonite, illite and kaolinite are 0.01-1.0, 0.1-2.0 and 0.1-5.0 pm, respectively (Buckman and Brady, 1964). The roles of clays and protozoans on bacterial survival have not been fully investigated in a wide range of soils, even though they influence microbial populations (Heijnen et al., 1992; Kuikman, 1990; Stotzky and Rem, 1966). Information on claymicrobe interactions has come from laboratory and field studies (Heijnen et al., 1988; Marshall, 1968; Marshall and Roberts, 1963; Stotzky, 1966; Stotzky and Rem, 1966). We review the influence of clays and predation by protozoa on bacterial survival in soil. EFFECT OF CLAYS ON BACTERIAL
SURVIVAL
Some excellent studies by Marshall and colleagues provided evidence that bacterial survival is influenced
by clays. Marshall and Roberts (1963) examined the influence of fine particle materials (fine silica, montmorillonite and fly ash consisting mainly of burnt clay) on survival of Rhizobium trifolii in sandy soil. Both fly ash and montmorillonite increased Rhizobium survival as determined by 73 and 54%, respectively, nodulation of clover compared to 21% in the silica-amended and 28% in non-amended soil. The nature of this increased nodulation was not described, although it may be due to one or more of the microbe-clay interactions reported by Marshall (1968). When coated with montmorillonite, the electrophoretic mobility of Rhizobium cells was similar to montmorillonite. Secondly, an “edge-to-face” association between clay platelets and bacterial cell surfaces may account for the large amount of montmorillonite absorbed per unit area. The amount of montmorillonite absorbed per unit area was higher with cells possessing surface carboxyl groups. Thirdly, a montmorillonite envelope which surrounded bacterial cells may confer a protective effect from desiccation, high temperatures, X-rays and metabolically induced pH changes (Marshall, 1968). Stotzky and Rem (1966) reported that montmoril-
Table I. Some factors that affect bacterial survival in soil Factors Surfaces
Moisture
Soil texture
Plants (rhizosphere)
Bacterial movement
lnoculum density
comments Review of influence of surfaces on microbial activity Discussion of terrestrial subsurface with respect to microbial ecology Review of microbial adhesion to clays Discussion of interfaces and interfacial interactions with respect to microbial ecology Rhizobium sp. exhibit better survival with lower initial moisture content Rewetting dried soil increased bacterial population Water potential influenced survival of four rhizobial strains Short review on water activity in the soil and its effect on bacterial survival Rhizobium sp. final cell numbers were higher m silt loam than in loamy sand Recombinant PseudomonasPuorescens survived better in a loam than in a loamy sand f?. subrilis cell numbers declined rapidly in loamy sand and silt loam, PseudomonasPuoresrens cell numbers declined slowly in both soils but survival was better in silt loam than loamy sand Soil texture affected survival of rhizobial strains, better survival in sandy loams. silt loams and sandy clay loams Bacteria-plant species specificity, probable cause of rhizosphere effect: root exudates and growth factors synthesized by other bacteria, generalization that rhizosphere effect increases with plant age Bacteria-plant species specificity, amount of bacterial growth possibly related to quantity of substrates released Review-bacterial species present/absent in the rhizosphere, factors affecting bacterial survival in the rhizosphere Recombinant Pseudomonas sp., limited lateral migration, vertical movement to a depth of 30 cm (after 5 weeks) (possible association with roots of inoculated plants) Transport of bacteria with water through soil, six genera of bacteria tested, concluded cell size plus other unknown factors affect transport Recombinant Pseudomonas sp. downward transport through soil dependent on flow rate and number of times water was added Addition of water found to affect bacterial distribution, Pseudomonas strain inoculated on wheat roots was recovered 3.5 cm below inoculation site (after 24 h) when water was added to soil surface, 0.5 cm without water Rhizobium japonicum and Pseudomonas purida, both strains were not found in nonsterile soil below 2.7 cm in absence of a transporting agent or in the presence of plant roots, earthworm and combinations of a transporting agent and earthworm or plant roots enhanced transport Rhizobium sp., as inoculum density increased final population sizes increased in both a silt loam and a loamy sand
Sources Van Loosdrecht et al. (1990) Ghiorse and Wilson (1988) Stotzky (1985) Marshall (1976) Postma ef al. (1989) Lund and Goksoyr (1980) Mahler and Wollum (1981) Morita (1992) Postma et al. (1990) Van Elsas ef al. (1989) Van Elsas ef al. (1986)
Mahler and Wollum (1981) Curl and Truelove (1986) Bennett and Lynch (1981) Katznelson
(I 965)
Kluepfel er al. (1991)
Gannon er al. (1991) Trevors er al. (1990) Parke et cl. (1986)
Madsen and Alexander ( 1982)
Postma et al. (1990)
Bacterial survival in soil lonite stimulated bacterial respiration by maintaining the pH at a value suitable for sustained growth. Due to the higher CEC of montmorillonite, more H + ions can be exchanged with adsorbed basic cations. The low CEC of kaolinite could not provide this buffering capacity. They also attributed this respiratory stimulation in part to mineral nutrients provided by clays. They suggested both montmorillonite and kaolinite were equally effective in providing minerals for bacteria. Stotzky (1985) later suggested that exchangeable inorganic nutrients adsorbed on clays may be available to microbes. It is not known how bacterial survival or activity in soil may be affected due to greater nutrient availability from clays exhibiting higher CEC values. Roper and Marshall (1974) presented evidence that Escherichia coli was protected from phage lysis by sediments, montmorillonite or organic matter at salinity values below or above the required point for desorption and dispersal. At low electrolyte concentrations, this protection was provided by an envelope of colloidal materials sorbed to cells. At high electrolyte concentrations, protection resulted from both the colloidal envelope around cells and sorption of cells and phages to solid particles. The electrolyte (salt) concentration was varied by using different concentrations of sea water. Stutz et al. (1989) demonstrated that survival of Pseudomonas fluorescens in vermiculite was better than in montmorillonite, which was better than in illite. For example, after 6 weeks, survival in pure vermiculite was 2 x lo5 cfu gg’ whereas in pure montmorillonite, survival was 2 x lo3 cfu g-‘. No cfu were detected in pure illite. The experiment was conducted at 20°C and 70% relative humidity. It is not understood how different clay types influenced bacterial survival. This may be related to particle size-surface area, moisture retention, cation exchange capacity (CEC) or nutrient status. In recent years, the effect of adding bentonite to a loamy sand inoculated with R. Ieguminosarum biovar trifofii was examined by Heijnen et al. (1988). It was observed that cell numbers in non-sterile soil at 15°C and 18% moisture decreased from 10’ to 3 x lo5 cfu g-r dry soil after 60 days. In soil amended with 10% (w/w) bentonite, cell numbers remained at 10’ cfu g-r during the experiment. When inoculated into sterile soil, Rhizobium cell numbers increased from 10’ to 3 x lo* cfu g-’ dry soil during the first week, and remained unchanged for the remainder of the 60 day incubation. A similar trend was observed with the same microorganism in sterile soil amended with bentonite. These results showed the protective effect of bentonite depended on whether the soil was sterile or not. A decrease in introduced Rhizobium cells in non-sterile soil may have been due to competition by indigenous organisms or predation primarily by protozoa. These interactions would not be present in sterile soil. A Tn5 mutant of R. leguminosarum biovar trifolii survived better in a non-sterile
527
loamy sand agricultural soil amended with 10% (w/w) bentonite or 10% (w/w) kaolinite compared to unamended soil (Heijnen and van Veen, 1991). Bacteria were introduced into soils at log 7.4 to 7.7 cfu gg’ dry soil, and the indigenous clay content of unamended loamy sand soil was 3% (van Elsas et al., 1986). After 57 days, the log cfu gg’ soil were 8.05, 6.41 and 5.49 for soil amended with bentonite, kaolinite and unamended soil, respectively. Immunofluorescence counts for the same treatments were 8.09, 6.42 and 5.62, respectively, showing an excellent correlation between two different enumeration methods. One suggestion to explain increased cell survival in clay amended soils was that clays shielded microorganisms from predation by protozoans by forming protective microhabitats (Heijnen et al., 1988; Heijnen and van Veen, 1991). It is noteworthy that introduced bacteria survived better in soil amended with bentonite than with kaolinite. It is possible that addition of clay to soil creates new microhabitats from those initially present, especially if the soil had a low clay content (e.g. loamy sand soil). Heijnen and van Veen (1991) suggested the number of protective pore spaces increased with addition of bentonite. These authors used moisture retention curves to show increased pore spaces in the following order: loamy sand < loamy sand + 10% kaolinite < loamy sand + 5% bentonite < loamy sand + 10% bentonite. PROTOZOAN-BACTERIAL
INTERACTIONS
Predation was recognized as a significant factor affecting bacterial survival in soil more than 60 years ago (Cutler, 1927, cited in Fenchel, 1987). Of soil organisms that feed on bacteria, protozoa appear to be the group that has the most significant effect (Acea et al., 1988; Acea and Alexander, 1988; Danso et al., 1975; Heijnen et al., 1988). The possibility that slime molds, bacteriophages, Bdellovibrios and myxobacteria might be partially responsible for decline of bacteria in soil has not been shown to be the case (Acea et al., 1988). Bacteria are a major food source for protozoans such as flagellates, naked amoebae and some ciliates (Fenchel, 1987). Some amoebae exhibit definite bacterial preferences, for example, between Gram-positive and Gram-negative and between old and young cultures (Oehler, 1919, cited in Sandon, 1927). Within the free living phagotrophic protozoan group there are three feeding habits: (a) filter feeding, (b) raptorial feeding and (c) diffusion feeding. Filter feeding is the filtering of suspended food particles (e.g. bacteria) by some flagellates and ciliates. A filter-feeding ciliate such as Paramecium caudatum can ingest about 400 particles s-r (Fenchel, 1980). Raptorial feeding involves the individual ingestion of bacteria, both suspended and surface-bound, by small flagellates and amoebae. Diffusion feeding by some amoeboid protozoa, such as foraminifera and
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small heliozoans, involves extension of the pseudopodium to engulf the prey, followed by food vacuole formation (Fenchel, 1987). The number of protozoans in soil has been estimated at between 1000 and 500,000 organisms per g of moist soil (Sleigh, 1973). This agrees with the estimate of Campbell (1983) that protozoan numbers range from log 4 gg ’ soil or 38 g m-3 in the top 15 cm of an agricultural soil. Protozoans are important in carbon and nitrogen cycling in soil. The presence of protozoans in soil has been shown to increase the turnover and mineralization of carbon (Kuikman et al., 1990a) and nitrogen (Kuikman et al., 1990a, b; Kuikman and van Veen, 1989). Kuikman et al. (1990a) have concluded from experimental observations that protozoa increase the turnover rate of carbon and nitrogen by (a) consuming bacteria and releasing nitrogen as a byproduct and (b) increasing activity of bacterial populations and this in turn increases the amount of carbon metabolized by bacteria. A number of studies have been performed to assess if protozoans have an effect on introduced bacteria. When bacteria were added to sterile soil no decline in numbers occurred (Danso et al., 1975; Heijnen et al., 1988). If protozoans were also introduced into sterile soil, bacterial numbers declined (Heijnen et al., 1988). Generally, results have shown that when bacteria are added to non-sterile soils, bacterial populations decline (Chao and Alexander, 1981; Danso et al., 1975; Heijnen et al., 1988), while protozoan numbers increase (Chao and Alexander, 1981; Danso et al., 1975). The addition of 1% glucose to sterile soil, with added protozoa, delayed decline of Agrobacterium, Pseudomonas and Corynebacterium spp for approximately 2 days, while the protozoan population grew for the first 4-6 days then declined (Acea and Alexander, 1988). Danso et al. (1975) studied Rhizobium cells and protozoa in nonsterile soil. Rhizobial cell numbers declined, but were not eliminated, whereas protozoan numbers increased. The inability of protozoans to remove all prey was not attributed to another predator attacking the protozoa. In salt solution, protozoa were also incapable of eliminating the bacterial prey (Danso and Alexander, 1975). In sterile soil inoculated with both bacteria and protozoa, protozoa also did not eliminate all the bacteria (Danso et al., 1975). It was also noted that protective microhabitats were not the reason for bacterial survival because results were the same in sterile pond water as in soil (Danso et aI., 1975). It was suggested the inability of the predators to eliminate their prey was a result of protozoan numbers reaching a density where the energy used in finding the prey equalled that from feeding. Amoebae populations in both field and pot experiments were found to be present in large enough numbers and fluctuated in a manner that indicated their involvement in regulating bacterial numbers
(Clarholm, 1981). Flagellates and ciliates did not have a similar role. In the field study, the presence of amoebae resulted in a 60% decrease in bacterial populations. For example, during a rainfall, amoebae density was lo5 gg’ dry soil weight. The large rainfall caused a IO-fold increase in bacterial biomass after 2 days. Four days later, the amoebae population increased 20-fold while bacterial biomass declined. In pot studies not containing plants, amoebae numbers increased 6-fold over the initial value of 104g-’ dry soil weight. However, in pot studies containing wheat seedlings, this increase was 30-fold (Clarholm, 198 1). These populations were transient as 2 days later, amoebae numbers decreased to 3 times the initial value in planted soil and 2 times in unplanted pots. Bacteria are not evenly distributed throughout soil. Instead cells are found in small colonies (usually less than 10 cells) (Campbell, 1983) that cover about 0.1% of the available surface area (Wood, 1988). This suggests that protozoa must move through soil to seek bacteria and to avoid unfavourable conditions. Cultivation of soils will assist in bringing bacterial and protozoan populations into contact with each other (Wood, 1988). Vargas and Hattori (1986) reported that cell numbers of Aerobacter aerogenes in the presence of Colpodu sp. decreased from lo* to about lo4 cells gg’ soil in the outer zone of soil aggregates (1-2 mm) in 12 days. By contrast, 10’ cells gg’ soil remained in the inner aggregate zone after the same period of exposure. This suggests susceptibility of bacterial cells to predation is influenced by their spatial location in soil. Protozoa do not always respond to bacteria added to soil. Casida (1989) did not observe an increase in protozoan numbers on addition of Arthrobacter globiformis and Bacillus thuringiensis cells to soil. However, B. mycoides spores added to soil caused a marked increase in numbers of selected protozoans. It was not known if this response was associated with spore germination. The results suggest some preferential feeding. Even though E. coli is not a common soil microorganism, the protozoans increased in response to its addition in a manner similar to that when B. mycoides was added. Heijnen et al. (199 1) observed that Bodo sultans fed almost solely on R. leguminosarum biovar trifolii despite the presence of other bacterial species present in demineralized water. It should be noted that prior to inoculation into water, B. sultans was maintained exclusively on R. leguminosarum biovar trifolii. It is not known if this maintenance regime influenced subsequent feeding behaviour of protozoans during the experiment. In their study, addition of protozoans to 1O’cfu ml-’ rhizobial cells in distilled water resulted in a decline in cell numbers by 2-3 log units over 3540 days. Addition of 2.5% (w/v) bentonite led to little change in Rhizobium cell numbers in the presence of B. sultans. The protozoan population was not significantly affected by bentonite for the first 7
Bacterial survival in soil days, whereas after 15 days, protozoa were undetectable. It is noteworthy that clay particles were found to interfere with microscopic detection of this organism. They also reported that bentonite did not release substances toxic to protozoans (Heijnen et al., 1991). Free-living phagotrophic protozoa may not be able to distinguish between clay particles and bacteria, and ingest both. Fenchel (1980) observed that filter feeding ciliates ingested latex beads (dia studied 0.09-5.7 pm) as easily as bacteria. The optimal size ingested by 14 different ciliates ranged from 0.3 to 1 pm dia. Interestingly, this size range is very similar to the size of some bacterial cells. For example, Rhizobium sp. are rod shaped ranging in size from 0.5-0.9 by 1.2-3.0pm in length (Holt et al., 1977). Dimensions of the genus Pseudomonas are straight or curved rods of 0.5-1.0 by 1.54.0pm (Holt et al., 1977). The ranges of particle size of montmorillonite, illite and kaolinite are 0.01-1.0, 0.1-2.0 and 0.1-5.0 pm, respectively (Buckman and Brady, 1964). It is noteworthy that the l-5-pm clay sizes are in the same magnitude as some common soil bacteria. Fenchel (1980) suggested size selection may be a function of the size of the mouth apparatus. Selective feeding behaviour by protozoans has been documented (Sandon, 1927). If selective feeding behaviour by indigenous protozoans is common, it may be possible to select bacterial strains that are not ingested, or ingested at a low frequency, for use in soil. Bacterial cells adsorbed to clay particles exhibit an increase in mass and volume. This can affect the feeding behaviour of predatory protozoans because the combined clay-cell complex may be too large to ingest. Predation as a factor affecting bacterial survival in soil must be examined critically if introduced bacteria are to survive and perform the function they were introduced to do. Ramadan et al. (1990) suggested that bacterial populations added as inocula for biodegradation may fail to survive because of nutrient deficiencies or grazing by protozoans. CONCLUDING
REMARKS
Clays and protozoa play important roles in determining microbial survival and activity in soil. Clays may protect bacterial cells from predation by creating protective pore spaces. Also, protozoa may ingest clay particles indiscriminately, thereby sparing bacterial cells. This may reduce their food intake and decrease their reproductive potential. In recent years, information on clay-bacteria-protozoan interactions has been obtained using sterile and non-sterile soils amended with this mixture. Although this approach provides useful information, the laboratory conditions do not reflect those found in the field. There is a lack of information on whether the protective effects of added clays is also seen in soil with a high initial clay content. More studies are needed to determine if soil protozoa exhibit selective
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feeding behaviour with respect to indigenous or introduced bacterial strains. An interesting study would be to determine if a protozoan reared on a bacterial strain it is not known to feed on will exhibit preferential feeding of this strain when provided with its preferred bacterial prey. It is not understood how indigenous protozoa compete with introduced protozoans. In environmental applications, bacterial cells will be released into different soil types under a variety of conditions. A range of soil type, bacterial or protozoan numbers and species has not been studied in detail, and hence requires further investigation. For specific applications such as bioremediation of contaminated soils, it would be useful to know if indigenous protozoa may affect the survival and degradative activity of introduced cells. A knowledge of these factors will assist researchers when formulating and releasing microbial cells for introduction into soils (Trevors et al., 1993) which may contain active protozoan populations. Acknowledgements-Our research on bacterial survival was supported by a grant from the Ontario Ministry of the Environment. L. S. England was the holder of a Postgraduate scholarship award from the Natural Sciences and Engineering Research
Council
of Canada.
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