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Changes in microbial populations following the application of cattle slurry to soil at two temperatures
Soil Bid. Biochem. Vol. 21. No. 2. pp. 263-268. Printed in Great Britain. All rights rescrwd
1989 Copyright
0038-0717 89 S3.00 + 0.00 C 1989 Pcrgamon Rcu plc
CHANGES IN MICROBIAL POPULATIONS FOLLOWING THE APPLICATION OF CATTLE SLURRY TO SOIL AT TWO TEMPERATURES M. H. OPPERMAN, M. WOOD and P. J. HARRIS Department of Soil Science, University of Reading, London Road, Reading, Berkshire RGI SAQ. England (Accepted 5 July 1988) Summary-Fresh cattle slurry applied to a sandy, free-draining soil and stored for 135 days at 23 or 5’C resulted in an immediate increase in numbers of viable bacteria and presumptive coliforms followed by increases in numbers of protozoa and nematodes. Amounts of fungal hyphae were not affected by temperature or presence of slurry. Temperature had no effect on total microbial activity. however the composition of the protozoan population changed with flagellates dominating at 5’C and amoebae at 23’C. After initial fluctuations, microbial numbers stabilized and populations of bacteria, total protozoa and nematodes in slurry-amended soil remained significantly higher than those of unamended soils for up to I35 days.
(2) to study the effect of soil temperature on the interactions between these groups of microorganisms and hence on the rate of slurry decomposition.
INTRODUCI’ION Addition of slurry to soil introduces large numbers of viable bacteria (Opperman ef al., 1987a,b) along with dead bacteria and readily-utilizable energy sources. Under natural conditions the soil microflora is ready to make use of any available substrates (Stotzky and Norman, 1964) and the presence of larger bacterial populations, whether introduced directly in the slurry or indirectly by rapid multiplication of the soil microflora, provides potential food for bacterial feeders such as protozoa and nematodes. Danso and Alexander (1975) and Habte and Alexander (1977) reported that protozoa could decrease the number of Rhizobium in soil, Clarholm (1981, 1984) suggested that amoebae could regulate numbers of soil bacteria and Abrams and Mitchell (1980) suggested that nematode-bacterial interactions affected the decomposition of sewage sludge. Such interactions may play an important role in the decomposition of cattle slurry. Observations of microbial interactions in the field are difficult to interpret due to variation in climate, soil fauna and vegetation, therefore microcosms have been used (Elliott er al., 1979; Danso and Alexander, 1975; Habte and Alexander, 1977; Griffiths, 1986). However, conditions are often different from those found under field conditions. For example, incubation temperatures higher than 20°C are unrepresentative of temperate soils which rarely rise above this value. In the U.K. soil temperatures at the time of slurry application (autumn or spring) are usually between I and 10°C at IOcm depth, and surface temperatures can fluctuate widely. We have compared microbial populations in soils after the surface application of cattle slurry at 23°C with those found in similarly treated soils maintained at S’C (approximate soil temperature during spring slurry application). The main objectives were:
(I) to study the populations of bacteria, fungi, protozoa and nematodes in soil in the presence or absence of cattle slurry; and SBB?I 2-r
MATERIALS AND iMETHODS A sandy soil of the Rowland series (Kay, 1936) was collected from an unsown area of the University Farm, Sonning, Berkshire. On collection the soil was at 2°C and contained 16% moisture. In the laboratory the soil was hand-sorted to remove stones, earthworms and plant debris, and 80g sub-samples were placed in 250 ml white, opaque plastic bottles with wide necks. Half the bottles were placed at 5 f 0.6”C and the remainder at 23 + I’C for 5 days before the addition of slurry. Cotton wool covers were used to permit gas diffusion, and all bottles were stored in the dark throughout the experiment. Cattle slurry (88% moisture) was obtained from beneath the slatted floor of cattle sheds at the Bernard Weitz Centre, Shinfield, Berkshire. The slurry was at 6’C on collection and was maintained at this temperature until used (within 24 h of deposition). Slurry (IO g), equivalent to an application of 75 kg N ha-’ (area based), was added to half of the bottles at each temperature. Deionized water (9 ml) was added to the remaining control (soil only) samples. Each bottle was weighed every 4 days and any weight loss replaced by distilled water to keep the moisture content constant in all treatments. Bottles were arranged randomly at both temperatures. Numbers of viable bacteria and presumptive coliforms were estimated by plate counts (Opperman et ol., 1987a). Total lengths of fungal hyphae were estimated using the agar-film technique (Jones and Mollison, 1948) followed by staining in Trypan-blue lactophenol, mounting in DPX mountant (BDH Chemicals Ltd) and counted at x400 magnification using a Weibel graticule. Nematodes were extracted from a soil-water matrix by migration through a fine mesh screen into water. Numbers were counted di263
OPPERMAN
M. H.
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et uf.
Viable bacteria 23
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140 The
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(days)
Fig. I. Effect of slurry addition on the total numbers of viable bacteria (log,,, no. g-’ fresh wt) at 5 and 23°C. Values are means of 10 replicates, S, = 0.89.
rectly at x 40 magnification. Protozoa populations were enumerated by the microtitre method of Darbyshire et al. (I 974) using most-probable-number (MPN) tables (Rowe ef al., 1977). Microtitre dishes were stored at 23°C for up to 14 days and each well was examined after 3, 7 and 10 days for the presence of protozoa. Organisms were grouped into flagellates, amoebae or ciliates (the sequential observations ailowed slower-growing organisms to develop). Soils (two replicates) were sampled periodically for up to 135 days. All data were analysed using a multifactorial analysis of variance (ANOVA). Numbers of bacteria and protozoa were transformed using a log,, transformation before analysis.
RESULTS For the populations of bacteria, presumptive coliforms and protozoa all three-factor interactions (treatment x temperature x time) were significant at P c 0.0001 (F values being 7.86 for bacteria, 3.85 for coliforms and 4.10 for protozoa). Differences between populations of flagellates, amoebae and ciliates were significant at P < 0.05 for each treatment and throughout the experimental period there were significantly more bacteria, total protozoa and nematodes in slurry-amended soil than in the control soils (P < 0.05). Addition of slurry resulted in an immediate increase in bacterial numbers at both temperatures (Fig. I) due to the bacteria added with the slurry, followed by a further increase. In unamended soil numbers of bacteria remained constant and were the same at both temperatures. After 135 days the bacterial oooulation of amended soil was still si~ni~cantlv gre;e; than that of unamended soil anz had ndt decreased sinnificantlv from the value obtained 14 days after slurry ad&ion. There was no obvious effect of temperature on total bacterial populations. Numbers of presumptive coliforms declined below the detection limit (10 g-* soil) after 85 days (Fig. 2).
in soil
At both temperatures, numbers of coliforms increased on the addition of slurry. After addition, numbers in treated and untreated soils declined at similar rates. There was no significant difference between numbers of coliforms obtained at both temperatures. Total numbers of protozoa were higher at 23°C than at 5’C and there was a significantly higher population (P < 0.05) in slurry-amended soil than in unamended soil. Addition of slurry did not cause an immediate increase in numbers of protozoa but numbers in amended soils gradually increased, reaching maximum values after 13 days at 23’C and I6 days at S’C. However, individual groups of protozoa responded differendy. At both temperatures flagellate numbers increased initially but in slurry-amended soil at 23’C numbers had declined to that of unamended soil after 26 days. In amended soil at 5’C numbers remained high throughout the experiment (Fig. 3). Numbers of amoebae increased rapidly in amended soil stored at 23°C and became signi~cantly higher than the population in amended soil at S’C. At 5’C populations in amended and unamended soil were similar and remained stable throughout the experiment (Fig. 4). Ciliate numbers fluctuated throughout the experiment at both temperatures (Fig. 5). Higher numbers of ciliates were found at 23°C than at 5’C in amended soils. Peak values were obtained 60-80 days after addition of slurry. At 5’C ciliate numbers started to increase after 80 days but never became significantly higher than in unamended soil. Nematode numbers increased slowly after slurry addition at both temperatures (Fig. 6) but after 51 days populations began lo increase rapidly. After 92 days the number of nematodes in amended soil at 5°C was greater than that in amended soil at 23”C, however, after 121 days numbers at both temperatures were similar. In unamended soil at both temperatures nematode populations remained lower than 2Og-I fresh weight soil throughout the experiment.
Effects of temperature and slurry on micro-organisms
265
Colitarms
Time kiaysf Fig. 2. Effect of slurry addition on the number of presumptive coliforms (log,, no. g-’ fresh wt) in soil at 5 and 23°C. Values are means of 10 replicates, S, = 1.80. No significant
differences
were apparent
between
lengths of fungal hyphae in amended and unamended soil at both temperatures. DISCUSSION
Statistical analysis of the data suggested that temperature did not have a significant effect on the
IO'
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lx)
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micr&ora of sturry-amended soils. The addition of slurry atfected the ~~u~at~ons more than temperature, although the composition of the protozoan population was different at the two temperatures. It was impossible to determine whether some of the peaks observed in the data were actual population fluctuations or merely due to the sampling techniques employed, as increases observed after the initial
0
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Time Mays)
Fig_ 3. Efi’ect of sfurry addition on Bagellate numbers (IogtOno. g-’ fresit wt) in soil at 5 and 23°C. Values are means of 4 rephcates, g, = 0.80.
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OPPEKUS
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Amoebae r
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Fig. 4. Effect of slurry addition on numbers of amoebae (log,, no. g-r fresh wt) in soil at 5 and 23°C. Values are means of 4 replicates, S, = 0.80.
Ciliotes IO'
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Time
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Fig. 5. Effect of slurry addition on ciliate numbers (log,, no. g-r fresh wt) in soil at 5 and 23’C. Values are means of 4 replicates, Sd = 0.80.
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Fig. 6. Effect of slurry addition on nematode numbers in soil at 5 and 23’C. Values are means of 2 replicates, S, = 3 1.2.
Effects of temperature and slurry on micro-organisms fluctuations were not statistically significant. Cycling of microbial populations has been reported for both marine and soil ecosystems. Fenchel (1982) reported that in marine ecosystems an observed bacterial peak was followed by an increase in flagellate numbers 4 days later; a flagellate-ciliate cycle occurred with a frequency of about 30 days. Clarholm (1981) reported peaks in protozoa activity following bacterial activity in soils. In our work, the initial peaks observed after slurry addition are significant at both tem~ratur~, and protozoan numbers peaked a few days later than bacteria1 numbers suggesting that cycling of the populations may have occurred. The addition of slurry to soil introduced both dead and live bacteria; this was reflected in the immediate increase in numbers of viable bacteria following the addition of slurry due to a direct input of bacteria from slurry. No immediate increase in numbers of protozoa and nematodes was observed. Previous experiments had suggested that any nematodes in slurry were either dead or present as eggs, and that the protozoan population was reduced to c 10: ffagellates g-’ fresh wt, with no amoebae or ciliates being detected (Oppcrrnan ef af., 1987b). However, slurry also contains readily-utilizable substrates {for example, approx. 0.7% glucose (Opperman ef af., 1987b)) which could promote the development of a large, mixed microbial population. Addition of slurry to soil probably introduced at least 300 fig C g-’ soil which was sufficient to account for the increase in bacterial numbers observed up to day 4 of the experiment at both temperatures (based on figures reported by Dash et ui., 1985; Schniirer et al., 1985). Stotzky and Norman (1964) observed that the addition of 1% substrate resulted in maximum respiratory activity 24-36 h after addition, which correlates with the observations made during our work, although the substrate concentration was < 1%. Numbers of protozoa may have increased in response to the increase in bacterial biomass which occurred after slurry addition, as well as in response to nutrients and dead micro-organisms introduced with the slurry. Flagellate populations were the first to increase and it is suggested that they are capable of utilizing readily-available C sources (Clarholm, 1984) and stimulating the breakdown of polysaccharides (Sherr et al., 1982). At 5’C they remained the most numerous protozoan group, whereas at 23°C amoebae soon became more numerous and dominated for the remainder of the experiment. This population shift may have been due to the difference in temperatures since amoebae and ciliates are reported to be absent from soils at fow temperature or with alternate freezing and thawing cycles (Stout, 1980). Schniirer et al. (1985) reported numbers of amoebae in soil amended with animal manure, at a site with a mean annual temperature of 5.4’C, similar to those observed in soil stored at 5°C during this work. However acidity may also be a contributory factor affecting protozoan populations. Schniirer ef al. (1985) did not record acidity changes foIlowing manure application but we observed that slurry caused increased pH values at both temperatures. At 23°C pH values soon returned to those found in unamended soil but at 5’C remained high for 60 days following slurry addition (Oppe~an et a!., 1989).
267
Therefore, a combination of both pH and temperature differences may have accounted for the observed differences in protozoan populations. In slug-amended soil at 23X numbers of amoebae were significantly higher than in amended soil at 5°C. Cutler (1927), Singh (1946) and Clarholm (1981) have reported that amoebae are more numerous than flagellates in agricultural soils, and it is suggested that they are more important than flagellates in regulating bacteria1 populations (Clarholm, 1981, 1984). Consumption by bacte~al-fading flagellates may account for only 0.2% of bacterial production (Umorin, 1976). However, these observations are not sup ported by our data showing similar bacterial numbers at both temperatures, although amoeba1 populations remained low at 5’C. There are reports of flagellates regulating bacteria1 numbers (Gude, 1979; Fenchel, 1982), and Clarholm f 1981) also observed that flagellates peaked soon after an increase in bacterial activity. We suggest that because bacterial populations were the same at both temperatures, both flagellates and amoebae may regulate bacterial populations in soil, and that group dominance is determined by soil conditions. Numbers of ciliates fluctuated throughout the experiment and very few were observed in unamended soil, possibly because the bacterial population was too low to maintain a substantial ciliate population. Berk er al. (1976) and Taylor (1978) reported that bacterial densities between 106 and IO* ml-’ water were required to maintain populations of marine citiates. In unamended soil the bacterial population was in the region of IO6g-’ soil and this, coupled with the comparatively large size of individual ciliates which makes movement in the water films around soil particles difficult, could have accounted for the low numbers observed. Nematode populations increased more slowly than the other microbial groups after the addition of slurry, in contrast to the report by Guiran et af. (1980) who found increases in nematode numbers within 24 h of spreading pig manure. The lower temperature initially delayed development of the nematode population, possibly by delaying hatching of eggs or the lowering of the reproduction rate (Abrams and Mitchell, 1980). However, after 90 days populations at both temperatures were large, This rapid increase late in the experiment could have been associated with changes in the N status of the soil (Oppetman et al., 1989) as nematodes have been reported to increase N mineralization in soil (Grifliths, 1986). Overall these data indicate the dynamic nature of responses by the soil microflora to the addition of cattle slurry. Maximum respdnses occurred between 10 days for bacteria and 100 days for nematodes. Only protozoan groups were affected by different temperatures.
Ackno&dgemenu--We thank Mr C. Cherrctt for preparing the figures. This work was performed as part of a “Link Programme” with the institute of Grassland and Animal Production, Hurley, Berkshire, and was financed by the Agricultural and Food Research Council.
M. H. OPPERMAN et al.
268
Habte M. and Alexander M. (1977) Further evidence for the regulation of bacterial populations in soil by protozoa.
REFERENCES Abrams B. 1. and Mitchell M. J. (1980) Role of nematodebacterial interactions in heterotrophic systems with emphasis on sewage-sludge decomposition. Oikos 35, 404-410. Berk S. G., Colwell R. R. and Small E. B. (1976) A study of feeding responses to bacterial prey by estuarine ciliates. Transactions of the American Microscopic 5 14-520.
Society 95,
Clarholm M. (1981) Protozoan grazing of bacteria in soilimpact and importance. Microbiul Ecology 7, 344-350. Clarholm M. (1984) Heterotrophic, free-living protozoa: neglected organisms with an important task in regulating bacterial populations. In Current Perspecrioes in Microbial Ecology (M. J. Klug and C. A. Reddy. Eds), pp. 321-326. American Society for Microbiology, Washington. Cutler D. W. (1927) Soil protozoa and bacteria in relation to their environment. -Journal of fhe Quekefr Microscooical Club. Series 2 15. 309-330.
Dansb S. K. A. and Alexander M. (1975) Regulation of predation by prey density: The protozoan-Rhizobium relationshio. Aoohed Microbiolony 29. 5 15-52 I. Darbyshire J: F., ‘Wheatley R. F.. Greaves M. P. and lnkson R. H. E. (1974) A rapid method for estimating bacterial and protozoan populations in soils. &rue d’Ecologie et de Biologie du Sol It, 465-475.
Dash M. C.. Behera B., Satpathy B. and Pati D. P. (1985) Comparison of two methods for microbial biomass estimation in tropical soils. Recue d’Ecologie et du Biologie du Sol 22, 13-20. Elliott E. T., Cole C. V., Coleman D. C.. Anderson R. V., Hunt H. V. and McClellan J. F. (1979) Amoeba] growth in soil microcosms. A model system of C, N and P trophic dynamics. Inrernarional Journal qf Encironmenfal Studies 13. 169-174. Fenchel T. (1982) Ecology of heterotrophic microflagellates IV. Quantitative occurrence and importance as bacterial consumers. Murine Ecology-Progress Series 9, 3542. Griffiths B. S. (1986) Mineralization of nitrogen and phosphorus by mixed cultures of the ciliate protozoan Colpoda sreinii, the nematode Rhabdiris sp. and the bacterium Pseudomonasjuorescens. 637642.
Soil Biology & Biochemisrry 18,
Gude H. (1979) Grazing by protozoa as a selection factor for activated sludge bacteria. Microbial Ecology 5, 225-237.
Guiran G. de, Bonnel L. and Abirached M. (1980) Landspreading of pig manures IV. Effect on soil nematodes. In Efluenrsfrom Licesrock (J. E. Gasser, Ed.), pp. 109-I 19. Applied Science Publishers, London.
Archices of Microbiology 113. 181-183.
Jones P. C. T. and Mollison J. E. (1948) A technique for the quantitative estimation of soil micro-organisms. Journal of General .Wcrobiology 2, 54-69.
Kay F. F. (1936) A Soil Survey of Ihe Unieersily Farm, Sonning, Berkshire. University of Reading Faculty of Agriculture and Horticulture Bulletin XLIX. Bradley, Reading. Opperman M. H., McBain L. and Wood M. (1987a) Movement of cattle slurry through soil by Eisenia foerida (Savigny). Soil Biology & Biochemisrry 19, 741-745. Opperman M. H., Wood M., McBain L. and Harris P. J. (1987b) The characterization of slurry and the response of a range of crop plants to slurry application. In Animal Manure on Grassland and Fodder Crops, Ferrilizer or Waste? (H. G. van der Meer, R. J. Unwin. T. A. van Dijk
and G. C. Ennik, Eds), pp. 329-331. Nijhoff, Dordrecht. Omxrman M. H., Wood M. and Harris P. J. (1989) ‘Changes in inorganic N following the application ofcattle slurry at two temperatures. Soil Biology & Biochemisrry 21, 319-321.
Rowe R., Todd R. and Waide J. (1977) Microtechnique for most-probable-number analysis. Applied and Enuironmenial Microbiology 33, 675-680.
Schniirer J., Clarholm M. and Rosswall T. (1985) Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biology & Biochemistry 17, 61 l-618.
Sherr B. F.. Sherr E. B. and Berman T. (1982) Decomposition of organic detritus: a selective role for microflagellate protozoa. Limnology and Oceanography 27, 765-769.
Singh B. N. (19-16)A method of estimating the numbers of soil protozoa, especially amoebae, based on their differential feeding on bacteria. Annuls of Applied Biology 33. 112-l 19. Stotzky G. and Norman
A. G. (1964) Factors limiting microbial growth activities in soil III. Supplementary substrate additions. Canadian Journal of Microbiology 10, 143-147. Stout J. D. (1980) The role of protozoa in nutrient cycling and enerev flow. Advances in Microbial Ecology 4. l-50. Taylor W. 5. (1978) Growth responses of cilia6 protozoa to the abundance of their bacterial prey. Microbial Ecology 4, 207-214.
Umorin P. P. (1976) Relationships between bacteria and flagellates in destruction of organic matter. Zhurnal Obshchei Biologii 37, 83 l-835.
1989 Copyright
0038-0717 89 S3.00 + 0.00 C 1989 Pcrgamon Rcu plc
CHANGES IN MICROBIAL POPULATIONS FOLLOWING THE APPLICATION OF CATTLE SLURRY TO SOIL AT TWO TEMPERATURES M. H. OPPERMAN, M. WOOD and P. J. HARRIS Department of Soil Science, University of Reading, London Road, Reading, Berkshire RGI SAQ. England (Accepted 5 July 1988) Summary-Fresh cattle slurry applied to a sandy, free-draining soil and stored for 135 days at 23 or 5’C resulted in an immediate increase in numbers of viable bacteria and presumptive coliforms followed by increases in numbers of protozoa and nematodes. Amounts of fungal hyphae were not affected by temperature or presence of slurry. Temperature had no effect on total microbial activity. however the composition of the protozoan population changed with flagellates dominating at 5’C and amoebae at 23’C. After initial fluctuations, microbial numbers stabilized and populations of bacteria, total protozoa and nematodes in slurry-amended soil remained significantly higher than those of unamended soils for up to I35 days.
(2) to study the effect of soil temperature on the interactions between these groups of microorganisms and hence on the rate of slurry decomposition.
INTRODUCI’ION Addition of slurry to soil introduces large numbers of viable bacteria (Opperman ef al., 1987a,b) along with dead bacteria and readily-utilizable energy sources. Under natural conditions the soil microflora is ready to make use of any available substrates (Stotzky and Norman, 1964) and the presence of larger bacterial populations, whether introduced directly in the slurry or indirectly by rapid multiplication of the soil microflora, provides potential food for bacterial feeders such as protozoa and nematodes. Danso and Alexander (1975) and Habte and Alexander (1977) reported that protozoa could decrease the number of Rhizobium in soil, Clarholm (1981, 1984) suggested that amoebae could regulate numbers of soil bacteria and Abrams and Mitchell (1980) suggested that nematode-bacterial interactions affected the decomposition of sewage sludge. Such interactions may play an important role in the decomposition of cattle slurry. Observations of microbial interactions in the field are difficult to interpret due to variation in climate, soil fauna and vegetation, therefore microcosms have been used (Elliott er al., 1979; Danso and Alexander, 1975; Habte and Alexander, 1977; Griffiths, 1986). However, conditions are often different from those found under field conditions. For example, incubation temperatures higher than 20°C are unrepresentative of temperate soils which rarely rise above this value. In the U.K. soil temperatures at the time of slurry application (autumn or spring) are usually between I and 10°C at IOcm depth, and surface temperatures can fluctuate widely. We have compared microbial populations in soils after the surface application of cattle slurry at 23°C with those found in similarly treated soils maintained at S’C (approximate soil temperature during spring slurry application). The main objectives were:
(I) to study the populations of bacteria, fungi, protozoa and nematodes in soil in the presence or absence of cattle slurry; and SBB?I 2-r
MATERIALS AND iMETHODS A sandy soil of the Rowland series (Kay, 1936) was collected from an unsown area of the University Farm, Sonning, Berkshire. On collection the soil was at 2°C and contained 16% moisture. In the laboratory the soil was hand-sorted to remove stones, earthworms and plant debris, and 80g sub-samples were placed in 250 ml white, opaque plastic bottles with wide necks. Half the bottles were placed at 5 f 0.6”C and the remainder at 23 + I’C for 5 days before the addition of slurry. Cotton wool covers were used to permit gas diffusion, and all bottles were stored in the dark throughout the experiment. Cattle slurry (88% moisture) was obtained from beneath the slatted floor of cattle sheds at the Bernard Weitz Centre, Shinfield, Berkshire. The slurry was at 6’C on collection and was maintained at this temperature until used (within 24 h of deposition). Slurry (IO g), equivalent to an application of 75 kg N ha-’ (area based), was added to half of the bottles at each temperature. Deionized water (9 ml) was added to the remaining control (soil only) samples. Each bottle was weighed every 4 days and any weight loss replaced by distilled water to keep the moisture content constant in all treatments. Bottles were arranged randomly at both temperatures. Numbers of viable bacteria and presumptive coliforms were estimated by plate counts (Opperman et ol., 1987a). Total lengths of fungal hyphae were estimated using the agar-film technique (Jones and Mollison, 1948) followed by staining in Trypan-blue lactophenol, mounting in DPX mountant (BDH Chemicals Ltd) and counted at x400 magnification using a Weibel graticule. Nematodes were extracted from a soil-water matrix by migration through a fine mesh screen into water. Numbers were counted di263
OPPERMAN
M. H.
264
et uf.
Viable bacteria 23
0
0
lC
0 0 0
0
P
ii
10’1 ’ 0 20
’
40
0
cl
0.
’
60
0
SoiL
WC
1
80
’
too
’
120
’
140 The
I
I
I
I
I
I
0
20
40
60
80
100
I
I
120 140
(days)
Fig. I. Effect of slurry addition on the total numbers of viable bacteria (log,,, no. g-’ fresh wt) at 5 and 23°C. Values are means of 10 replicates, S, = 0.89.
rectly at x 40 magnification. Protozoa populations were enumerated by the microtitre method of Darbyshire et al. (I 974) using most-probable-number (MPN) tables (Rowe ef al., 1977). Microtitre dishes were stored at 23°C for up to 14 days and each well was examined after 3, 7 and 10 days for the presence of protozoa. Organisms were grouped into flagellates, amoebae or ciliates (the sequential observations ailowed slower-growing organisms to develop). Soils (two replicates) were sampled periodically for up to 135 days. All data were analysed using a multifactorial analysis of variance (ANOVA). Numbers of bacteria and protozoa were transformed using a log,, transformation before analysis.
RESULTS For the populations of bacteria, presumptive coliforms and protozoa all three-factor interactions (treatment x temperature x time) were significant at P c 0.0001 (F values being 7.86 for bacteria, 3.85 for coliforms and 4.10 for protozoa). Differences between populations of flagellates, amoebae and ciliates were significant at P < 0.05 for each treatment and throughout the experimental period there were significantly more bacteria, total protozoa and nematodes in slurry-amended soil than in the control soils (P < 0.05). Addition of slurry resulted in an immediate increase in bacterial numbers at both temperatures (Fig. I) due to the bacteria added with the slurry, followed by a further increase. In unamended soil numbers of bacteria remained constant and were the same at both temperatures. After 135 days the bacterial oooulation of amended soil was still si~ni~cantlv gre;e; than that of unamended soil anz had ndt decreased sinnificantlv from the value obtained 14 days after slurry ad&ion. There was no obvious effect of temperature on total bacterial populations. Numbers of presumptive coliforms declined below the detection limit (10 g-* soil) after 85 days (Fig. 2).
in soil
At both temperatures, numbers of coliforms increased on the addition of slurry. After addition, numbers in treated and untreated soils declined at similar rates. There was no significant difference between numbers of coliforms obtained at both temperatures. Total numbers of protozoa were higher at 23°C than at 5’C and there was a significantly higher population (P < 0.05) in slurry-amended soil than in unamended soil. Addition of slurry did not cause an immediate increase in numbers of protozoa but numbers in amended soils gradually increased, reaching maximum values after 13 days at 23’C and I6 days at S’C. However, individual groups of protozoa responded differendy. At both temperatures flagellate numbers increased initially but in slurry-amended soil at 23’C numbers had declined to that of unamended soil after 26 days. In amended soil at 5’C numbers remained high throughout the experiment (Fig. 3). Numbers of amoebae increased rapidly in amended soil stored at 23°C and became signi~cantly higher than the population in amended soil at S’C. At 5’C populations in amended and unamended soil were similar and remained stable throughout the experiment (Fig. 4). Ciliate numbers fluctuated throughout the experiment at both temperatures (Fig. 5). Higher numbers of ciliates were found at 23°C than at 5’C in amended soils. Peak values were obtained 60-80 days after addition of slurry. At 5’C ciliate numbers started to increase after 80 days but never became significantly higher than in unamended soil. Nematode numbers increased slowly after slurry addition at both temperatures (Fig. 6) but after 51 days populations began lo increase rapidly. After 92 days the number of nematodes in amended soil at 5°C was greater than that in amended soil at 23”C, however, after 121 days numbers at both temperatures were similar. In unamended soil at both temperatures nematode populations remained lower than 2Og-I fresh weight soil throughout the experiment.
Effects of temperature and slurry on micro-organisms
265
Colitarms
Time kiaysf Fig. 2. Effect of slurry addition on the number of presumptive coliforms (log,, no. g-’ fresh wt) in soil at 5 and 23°C. Values are means of 10 replicates, S, = 1.80. No significant
differences
were apparent
between
lengths of fungal hyphae in amended and unamended soil at both temperatures. DISCUSSION
Statistical analysis of the data suggested that temperature did not have a significant effect on the
IO'
0
20
40
60
60
IQ0
lx)
140
micr&ora of sturry-amended soils. The addition of slurry atfected the ~~u~at~ons more than temperature, although the composition of the protozoan population was different at the two temperatures. It was impossible to determine whether some of the peaks observed in the data were actual population fluctuations or merely due to the sampling techniques employed, as increases observed after the initial
0
20
40
60
60
100
120
940
Time Mays)
Fig_ 3. Efi’ect of sfurry addition on Bagellate numbers (IogtOno. g-’ fresit wt) in soil at 5 and 23°C. Values are means of 4 rephcates, g, = 0.80.
H.
M.
266
OPPEKUS
et
al.
Amoebae r
10'
0
I
I
20
40
11 60
80
1
’
100
120
1
23-c
Time
1
1
120
140
II
III1 0
140
20
40
60
60
100
(days)
Fig. 4. Effect of slurry addition on numbers of amoebae (log,, no. g-r fresh wt) in soil at 5 and 23°C. Values are means of 4 replicates, S, = 0.80.
Ciliotes IO'
23.C
5-c
.
_I 5
5
2
sol1
l
*wry
0
10'
II
10 0
20
40
I
I
I
I
I
60
60
100
120
140
I
I
l
l
l
l
40
60
60
100
120
140
II 20
Time
(days1
Fig. 5. Effect of slurry addition on ciliate numbers (log,, no. g-r fresh wt) in soil at 5 and 23’C. Values are means of 4 replicates, Sd = 0.80.
5
=
600
z 5
500
i r
400
3 B
300
E 2
200
'ii ; 100 "E IO
10
20
30
40
50
60
TO Tlme
60
90
100
110
120
130
140
(days1
Fig. 6. Effect of slurry addition on nematode numbers in soil at 5 and 23’C. Values are means of 2 replicates, S, = 3 1.2.
Effects of temperature and slurry on micro-organisms fluctuations were not statistically significant. Cycling of microbial populations has been reported for both marine and soil ecosystems. Fenchel (1982) reported that in marine ecosystems an observed bacterial peak was followed by an increase in flagellate numbers 4 days later; a flagellate-ciliate cycle occurred with a frequency of about 30 days. Clarholm (1981) reported peaks in protozoa activity following bacterial activity in soils. In our work, the initial peaks observed after slurry addition are significant at both tem~ratur~, and protozoan numbers peaked a few days later than bacteria1 numbers suggesting that cycling of the populations may have occurred. The addition of slurry to soil introduced both dead and live bacteria; this was reflected in the immediate increase in numbers of viable bacteria following the addition of slurry due to a direct input of bacteria from slurry. No immediate increase in numbers of protozoa and nematodes was observed. Previous experiments had suggested that any nematodes in slurry were either dead or present as eggs, and that the protozoan population was reduced to c 10: ffagellates g-’ fresh wt, with no amoebae or ciliates being detected (Oppcrrnan ef af., 1987b). However, slurry also contains readily-utilizable substrates {for example, approx. 0.7% glucose (Opperman ef af., 1987b)) which could promote the development of a large, mixed microbial population. Addition of slurry to soil probably introduced at least 300 fig C g-’ soil which was sufficient to account for the increase in bacterial numbers observed up to day 4 of the experiment at both temperatures (based on figures reported by Dash et ui., 1985; Schniirer et al., 1985). Stotzky and Norman (1964) observed that the addition of 1% substrate resulted in maximum respiratory activity 24-36 h after addition, which correlates with the observations made during our work, although the substrate concentration was < 1%. Numbers of protozoa may have increased in response to the increase in bacterial biomass which occurred after slurry addition, as well as in response to nutrients and dead micro-organisms introduced with the slurry. Flagellate populations were the first to increase and it is suggested that they are capable of utilizing readily-available C sources (Clarholm, 1984) and stimulating the breakdown of polysaccharides (Sherr et al., 1982). At 5’C they remained the most numerous protozoan group, whereas at 23°C amoebae soon became more numerous and dominated for the remainder of the experiment. This population shift may have been due to the difference in temperatures since amoebae and ciliates are reported to be absent from soils at fow temperature or with alternate freezing and thawing cycles (Stout, 1980). Schniirer et al. (1985) reported numbers of amoebae in soil amended with animal manure, at a site with a mean annual temperature of 5.4’C, similar to those observed in soil stored at 5°C during this work. However acidity may also be a contributory factor affecting protozoan populations. Schniirer ef al. (1985) did not record acidity changes foIlowing manure application but we observed that slurry caused increased pH values at both temperatures. At 23°C pH values soon returned to those found in unamended soil but at 5’C remained high for 60 days following slurry addition (Oppe~an et a!., 1989).
267
Therefore, a combination of both pH and temperature differences may have accounted for the observed differences in protozoan populations. In slug-amended soil at 23X numbers of amoebae were significantly higher than in amended soil at 5°C. Cutler (1927), Singh (1946) and Clarholm (1981) have reported that amoebae are more numerous than flagellates in agricultural soils, and it is suggested that they are more important than flagellates in regulating bacteria1 populations (Clarholm, 1981, 1984). Consumption by bacte~al-fading flagellates may account for only 0.2% of bacterial production (Umorin, 1976). However, these observations are not sup ported by our data showing similar bacterial numbers at both temperatures, although amoeba1 populations remained low at 5’C. There are reports of flagellates regulating bacteria1 numbers (Gude, 1979; Fenchel, 1982), and Clarholm f 1981) also observed that flagellates peaked soon after an increase in bacterial activity. We suggest that because bacterial populations were the same at both temperatures, both flagellates and amoebae may regulate bacterial populations in soil, and that group dominance is determined by soil conditions. Numbers of ciliates fluctuated throughout the experiment and very few were observed in unamended soil, possibly because the bacterial population was too low to maintain a substantial ciliate population. Berk er al. (1976) and Taylor (1978) reported that bacterial densities between 106 and IO* ml-’ water were required to maintain populations of marine citiates. In unamended soil the bacterial population was in the region of IO6g-’ soil and this, coupled with the comparatively large size of individual ciliates which makes movement in the water films around soil particles difficult, could have accounted for the low numbers observed. Nematode populations increased more slowly than the other microbial groups after the addition of slurry, in contrast to the report by Guiran et af. (1980) who found increases in nematode numbers within 24 h of spreading pig manure. The lower temperature initially delayed development of the nematode population, possibly by delaying hatching of eggs or the lowering of the reproduction rate (Abrams and Mitchell, 1980). However, after 90 days populations at both temperatures were large, This rapid increase late in the experiment could have been associated with changes in the N status of the soil (Oppetman et al., 1989) as nematodes have been reported to increase N mineralization in soil (Grifliths, 1986). Overall these data indicate the dynamic nature of responses by the soil microflora to the addition of cattle slurry. Maximum respdnses occurred between 10 days for bacteria and 100 days for nematodes. Only protozoan groups were affected by different temperatures.
Ackno&dgemenu--We thank Mr C. Cherrctt for preparing the figures. This work was performed as part of a “Link Programme” with the institute of Grassland and Animal Production, Hurley, Berkshire, and was financed by the Agricultural and Food Research Council.
M. H. OPPERMAN et al.
268
Habte M. and Alexander M. (1977) Further evidence for the regulation of bacterial populations in soil by protozoa.
REFERENCES Abrams B. 1. and Mitchell M. J. (1980) Role of nematodebacterial interactions in heterotrophic systems with emphasis on sewage-sludge decomposition. Oikos 35, 404-410. Berk S. G., Colwell R. R. and Small E. B. (1976) A study of feeding responses to bacterial prey by estuarine ciliates. Transactions of the American Microscopic 5 14-520.
Society 95,
Clarholm M. (1981) Protozoan grazing of bacteria in soilimpact and importance. Microbiul Ecology 7, 344-350. Clarholm M. (1984) Heterotrophic, free-living protozoa: neglected organisms with an important task in regulating bacterial populations. In Current Perspecrioes in Microbial Ecology (M. J. Klug and C. A. Reddy. Eds), pp. 321-326. American Society for Microbiology, Washington. Cutler D. W. (1927) Soil protozoa and bacteria in relation to their environment. -Journal of fhe Quekefr Microscooical Club. Series 2 15. 309-330.
Dansb S. K. A. and Alexander M. (1975) Regulation of predation by prey density: The protozoan-Rhizobium relationshio. Aoohed Microbiolony 29. 5 15-52 I. Darbyshire J: F., ‘Wheatley R. F.. Greaves M. P. and lnkson R. H. E. (1974) A rapid method for estimating bacterial and protozoan populations in soils. &rue d’Ecologie et de Biologie du Sol It, 465-475.
Dash M. C.. Behera B., Satpathy B. and Pati D. P. (1985) Comparison of two methods for microbial biomass estimation in tropical soils. Recue d’Ecologie et du Biologie du Sol 22, 13-20. Elliott E. T., Cole C. V., Coleman D. C.. Anderson R. V., Hunt H. V. and McClellan J. F. (1979) Amoeba] growth in soil microcosms. A model system of C, N and P trophic dynamics. Inrernarional Journal qf Encironmenfal Studies 13. 169-174. Fenchel T. (1982) Ecology of heterotrophic microflagellates IV. Quantitative occurrence and importance as bacterial consumers. Murine Ecology-Progress Series 9, 3542. Griffiths B. S. (1986) Mineralization of nitrogen and phosphorus by mixed cultures of the ciliate protozoan Colpoda sreinii, the nematode Rhabdiris sp. and the bacterium Pseudomonasjuorescens. 637642.
Soil Biology & Biochemisrry 18,
Gude H. (1979) Grazing by protozoa as a selection factor for activated sludge bacteria. Microbial Ecology 5, 225-237.
Guiran G. de, Bonnel L. and Abirached M. (1980) Landspreading of pig manures IV. Effect on soil nematodes. In Efluenrsfrom Licesrock (J. E. Gasser, Ed.), pp. 109-I 19. Applied Science Publishers, London.
Archices of Microbiology 113. 181-183.
Jones P. C. T. and Mollison J. E. (1948) A technique for the quantitative estimation of soil micro-organisms. Journal of General .Wcrobiology 2, 54-69.
Kay F. F. (1936) A Soil Survey of Ihe Unieersily Farm, Sonning, Berkshire. University of Reading Faculty of Agriculture and Horticulture Bulletin XLIX. Bradley, Reading. Opperman M. H., McBain L. and Wood M. (1987a) Movement of cattle slurry through soil by Eisenia foerida (Savigny). Soil Biology & Biochemisrry 19, 741-745. Opperman M. H., Wood M., McBain L. and Harris P. J. (1987b) The characterization of slurry and the response of a range of crop plants to slurry application. In Animal Manure on Grassland and Fodder Crops, Ferrilizer or Waste? (H. G. van der Meer, R. J. Unwin. T. A. van Dijk
and G. C. Ennik, Eds), pp. 329-331. Nijhoff, Dordrecht. Omxrman M. H., Wood M. and Harris P. J. (1989) ‘Changes in inorganic N following the application ofcattle slurry at two temperatures. Soil Biology & Biochemisrry 21, 319-321.
Rowe R., Todd R. and Waide J. (1977) Microtechnique for most-probable-number analysis. Applied and Enuironmenial Microbiology 33, 675-680.
Schniirer J., Clarholm M. and Rosswall T. (1985) Microbial biomass and activity in an agricultural soil with different organic matter contents. Soil Biology & Biochemistry 17, 61 l-618.
Sherr B. F.. Sherr E. B. and Berman T. (1982) Decomposition of organic detritus: a selective role for microflagellate protozoa. Limnology and Oceanography 27, 765-769.
Singh B. N. (19-16)A method of estimating the numbers of soil protozoa, especially amoebae, based on their differential feeding on bacteria. Annuls of Applied Biology 33. 112-l 19. Stotzky G. and Norman
A. G. (1964) Factors limiting microbial growth activities in soil III. Supplementary substrate additions. Canadian Journal of Microbiology 10, 143-147. Stout J. D. (1980) The role of protozoa in nutrient cycling and enerev flow. Advances in Microbial Ecology 4. l-50. Taylor W. 5. (1978) Growth responses of cilia6 protozoa to the abundance of their bacterial prey. Microbial Ecology 4, 207-214.
Umorin P. P. (1976) Relationships between bacteria and flagellates in destruction of organic matter. Zhurnal Obshchei Biologii 37, 83 l-835.