Microb. Ecol. 6:115-123 (1980)
MICIK~IAL ECO.OG'V
Effects of Water Fluctuations on Microbial Mass and Activity in Soil Vera Lund and Jostein Goksoyr Departmentof Microbiologyand Plant Physiology,Universityof Bergen,Norway Abstract.When previously dried soil was remoistened, a series of microbial events occurred. The bacterial plate count population increased rapidly, with a doubling time of 4-5 h. The length of fungal hyphae and microscopic counts of bacteria increased more slowly. The microscopically counted bacterial population was estimated to have a doubling time of about 90 h. The respiratory burst occurring after 2-3 days coincided with the maximal growth rate of the bacterial plate count population. From the respiratory data, plate count bacteria were estimated to have a cell mass of 0.4 pg dry weight, whereas the mass of microscopically counted bacteria was only 10% of this. Changes in bacterial DNA content corresponded to changes in the microscopic count, whereas changes in soil catalase activity mainly corresponded to changes in the fungal biomass, which was dominant. It is suggested that bacterial plate counts and microscopic counts represent two distinct populations of bacteria, which for practical purposes may be termed zymogenous and autochthonous, respectively.
Introduction In connection with a study of the effects of fertilization and lime application on microbial mass and activity in alpine meadows, it was noted that one of the most important factors influencing microbial activity under field conditions was the fluctuations in water activity of the soil. Lebedjantzev (16) observed that air-drying increased the fertility of a variety of soils in pot experiments. The effect was most evident on uncultivated soils and soils fertilized with phosphate or manure. He found that during drying there was a large increase in the solubility of organic substances and especially an increase in ammonia nitrogen. There was also a sharp reduction in the number of microorganisms. He considered the drying as a partial sterilization, and pointed to the role of drying and rewetting cycles in maintaining the fertility of the soil. Stevenson (20) studied the microbial activity of remoistened, air-dried soil, and in particular the relationship between the increased soil respiration observed after rewetting, and the bacterial counts. The size of the respiratory " b u r s t " was directly related to the amount of amino acids and other materials released by the drying process, and the conclusion drawn was that the increased respiration was 0095-3628/80/0006-0115 $01.60 O 1980 Springer-Verlag New York Inc.
116
v. Lundand J. Goks0yr
m a i n l y due to biological activity, although the bacterial counts (plate counts) reached a m a x i m u m some time after the respiration had reached a maximum. In studies of East African soils, Birch ( I - 3 ) followed soil respiration, humus decomposition, and nitrogen mineralization during a number of drying and rewetting regimes. Birch also concluded that the primary effect of drying and rewetting was on the microbial populations in the soil. The soil microorganisms which survived the drying responded to rewetting by temporarily entering a state of high metabolic activity, resulting in " c a n n i b a l i z a t i o n " of dead microbial cells and even a burst of humus decomposition. This was followed by liberation of ammonia and subsequent nitrification, which explained the increased fertility of dried soils when rewetted. The agricultural implications for countries with marked dry and wet seasons were emphasized. Jenkinson and collaborators (11-15) have demonstrated that by sterilization of soil, biological (mainly microbial) material is made available for mineralization, and that there is good correlation between the biomass in the soil before sterilization and the a m o u n t of CO 2 released during a 10-day period after reinoculation. Shields et al. (19) found in a similar study that after partial sterilization with chloroform and reinoculation, the plate count n u m b e r of bacteria increased rapidly to reach a peak value that was higher than before sterilization. This rapid increase coincided in time with a high rate of carbon mineralization. The direct count values of bacteria, which also showed a decrease by the sterilization, increased more slowly after reinoculation. Jager and Bruins (10) and Sorensen (21) found that repeated cycles of drying and rewetting enhanced the decomposition rates in soil, and that a considerable number of cycles were needed to deplete the soil for the substrates that took part in the process. The present study has been concentrated on a more detailed investigation of the effects of drying and rewetting on bacterial and fungal masses and activities in the soil, and especially on the differences in behavior between plate-counted and microscopicall3/ counted bacteria.
Materials and Methods Soil samples were collected from the top 10 cm (A horizon) of an unfertilizedhumus podzol from Field A, KjOlastolen,Oystre Slidre in Valdres, Norway, during late summer. This field is situated 1000 m above sea level in Central Norway in an area used for mountainfarming.The soil was broughtto the laboratory, sieved (mesh size 2 ram), and mixedcarefully.Afterremovalof samplesfor direct analyses,the soil was dried at room temperature until air-dry(4--5days). It was then stored in polyethylenebags for approximately 1month, when the rewettingexperimentswere started. For rewetting, weighed samples of soil were placed in 200 ml jars with screw-caps. Membrane-filtered distilled water was added to give the same watercontentas beforedrying, and the jars were rotated slowlyin a clinostat for 24 h in order to secure even distributionof moisture. The jars were then kept in the dark at room temperature for the experimentalperiod. Samples were taken daily for the first 10 days, and thereafter every second day. When necessary, they were analyzed immediately;otherwise, they were stored frozen until analysis. Bacterial Plate Counts.
The soil was homogenized in a Waring blender with Winogradsky'ssalt solution diluted 1:20 (18), and a dilution series was made with the same solution.Then0. I ml of suitabledilutionswas spreadon the surfaceof agar plates containingThornton's mediumwith 10% soilextract (22) witha bentglass rod whilethe plates were rotating. The plates were incubatedat 22~ for 10 days beforecounting.
Water Fluctuations and Microbial Mass and Activity in Soil
117
Microscopic Counts of Bacteria First, 1 ml of an appropriate soil dilution was made up as for plate counts and stained with I ml of acridine orange solutin (conc. 1:5000) for at least 5 rain. The suspension was then diluted with membrane-filtered distilled water to 15 ml and filtered through Unipore polycarbonate filters (filter dia. 22 mm, pore size 0.2/,tin), previously stained with Irgalan black (8). Nonfluorescent liquid paraffin was used between the filter and the coverslip. Counting was carried out with a Leitz Orthoplan microscope equipped with Ploemopak illumination system for epifluorescence, at a magnification of 9 0 0 x , with an immersion objective and an ocular supplied with a rectangular grid. All bacteria, regardless of fluorescent color, were counted. The count values were corrected for blank values, obtained by counting filters through which had been passed samples of the Winogradsky's salt solution stained with the same batch of acridine orange.
Hyphal Lengths of Fungi The total length of fungal hyphae was determined by the membrane filter technique (7). Safranine (0.25% aqueous solution) was used as the staining agent, and the suspension was filtered through Oxoid membrane filters (dia. 22 mm, pore size 0.45 .urn).
Respiration Measurements Soil respiration was measured with an infrared CO2-analyzer (type 225 MK2 from The Analytical Development Co, Ltd., England). 15 g (wet weight) of soil was placed in a glass tube with about 2 cm inner din. This was connected by PVC tubes and via an air reservoir of about 500 ml to the outlet and inlet systems oftbe CO 2 analyzer, so that the air was recirculated. The glass tube and air reservoir were placed in a water bath which was kept at 25~ After an equilibration period of about 2 h, the increase in the CO 2 content in the recirculated air was recorded during a 30 rain period. The rate of CO 2 formation could be calculated after determination of the total air volume of the system. This was done by injection of a known volume of CO 2, and determination of the resulting CO 2 concentration.
Bacterial DNA The bacterial fraction from 10 g soil (wet weight) was prepared (5). After suspension in Winogradsky's salt solution diluted 1:20 to a total volume of 60 ml and dividing in portions of 10 ml, the suspensions were stored frozen until analysis. DNA was determined fluorometrically by the reaction with 3,5-diamino benzoic acid 2 HCI (9) as described by Lid Torsvik and Gokscyr (23). Calf thymus DNA was used as internal and external standards. Fluorescence measurements were made in a Jasco FP-4 fluorescence spectrophotometer. The DNA content per microscopically counted bacterium in the bacterial fraction was calculated, and from this and the microscopic counts of bacteria in the soil, the total amount of bacterial DNA in the soil was also determined.
Catalase (E.C. I. I I. 1.6)Measurements First. 5 g of frozen soil was homogenized with 95 ml of Winogradsky's salt solution 1:20 in a Waring blender for 2 rain at low speed. Then 6 ml of the soil suspension was placed in the reaction chamber of a Rank Brothers (England) oxygen electrode. Next, 0.2 ml 3% hydrogen peroxide was injected and the production of oxygen recorded during a 5-min period. Boiled soil suspension was used as control, and catalase activity determined as the difference in oxygen evolution between the fresh and the boiled suspension. One catalase unit was defined as the amount of enzyme required to evolve I p,mole 02 per minute under the stated conditions.
118
V. Luridand J. Goks#yr
Results All values are given per gram dry soil, unless otherwise stated. Data for the soil before drying are shown in Table 1. Assuming an average diameter of 3 /am for the fungal hyphae (no accurate measurements were made) and an average bacterial dry weight of 0.06 pg (17), the microbial mass was 12.6 mg (I 1.5 mg fungi and 1. l mg bacteria). From the start data in Figures 1 and 3, and with the same assumptions, the total mass after the drying was 7.1 mg (6.8 nag fungi and 0.3 mg bacteria). It should be noted that no distinction between live and dead microorganisms was attempted. When the soil was rewetted, there was a rapid increase in the bacterial plate count, with a maximum about 5 days after rewetting, and a considerably later and slower increase in the microscopic count. This is shown in Figure 1. From this figure, it was estimated that the doubling time for the plate count bacteria during the first 2 days was 4-5 h, whereas it was about 90 h for the microscopically counted bacteria in the period of most rapid increase (sixth to tenth days). The ratio between microscopic and plate counts was 1400 in the soil before drying, and about the same just after rewetting. Five days later the ratio had decreased to 11, and then it slowly increased again, to reach 60 after 18 days. The amount of bacterial DNA in the soil showed an increase similar to that of the microscopic count (Fig. 2), demonstrating that the increase in microscopic count is accompanied by DNA synthesis. At the start of the rewetting period and at the end of the experiment, there was 12 fg (10-15 g) DNA per microscopically counted bacterium. In the period of most rapid increase, 5 fg was found. There was a steady increase the hyphal length of fungi after rewetting, from about 700 to 920 m after I0 days (Fig. 3). This corresponded to an increase in fungal mass of about 2 rag, and should be compared with the 5.5 mg microbial mass that disappeared as such during the drying and now should be available as nutrient in the soil. Respiration was measured on the whole soil (Fig. 4A), and thus represents the sum of fungal and bacterial activity. The peak in respiratory activity on the second and third days is the well-known respiratory "burst" described by several authors. The only parameter to which this can be correlated is the increase in bacterial plate counts. With constant growth yield and negligible maintenance respiration, the respiratory rate of a microbial culture is proportional to the growth rate. Maximum growth rate and hence maximum respiratory rate are found at the inflexion point of the growth curve, when the growth is expressed on a linear scale. This explains why the respiratory maximum is found at some time before maximum cell number is attained. From Fig. 4A, the peak respiratory activity was estimated to 15/.tl CO2-h-1 above the stable respiratory level. Assuming an economic coefficient (on carbon basis) of 0.5, and a carbon content of 50% in the cell material, the mass increase of the plate-counted bacteria at this period (2.5 Table I. Data for the soil beforedrying, calculatedper g dry weight Water content Organic matter(ignition loss) pH (in distilled water) Hyphal length Bacteria, platecounts Bacteria, microscopiccount Respiration (25~
180% 40% 4.5 1250 m 1.0 • 107 1.4 • I010 16.5/al CO2.h- I
Water Fluctuations and Microbial Mass and Activity in Soil
119
10 _.. 9
O~O~O
,._g
I:::
0
eoll J
o
8
J
Fig. 1. Plate counts (o) and microscopic counts (o) of bacteria per g soil dry weight. Abscissa: Days after rewetting.
5 I
,
10 I
,
,
,
15
,
I
,
Days 200
/
0
~ 100 1--0'
Fig. 2. Bacterial DNA per g soil dry weight after rewetting.
I
5
I
I
I
t
I
I
,
,
10
,
f
15
Days days after rewetting) will be 16/ag-h- i. The plate count number was 1.7 x 108, and the multiplication rate 3.8 x 107 h -1. This gives an average cell mass (dry weight) of the plate-counted bacteria of 0.4 pg, which corresponds quite well with estimated sizes for bacteria in soil forming colonies on solid media (6). After the peak of respiratory activity, the soil respiration was fairly stable at 15/.tl CO2"h- ~ for the rest of the experiment. Besides maintenance respiration of the platecounted bacteria, this respiration was caused by bacteria not forming colonies on solid media, and by fungi. The fungal growth rate during the first 8 clays was estimated to be
V. Lund and J. GoksOyr
120
-c I000 900
~o0 0-
,- 700
O
600
C
1.1,.
Fig. 3. Fungal hyphae per g soil dry
5OO
i
i
L
I
,
i
,
i
[
5
I
~
I
1
10
weight after rewetting.
,
15 Days
~ 30 20 3._
A e-~
10
tn l
o
V~
-t'-
Ul
l
l
l
l
l
,
,
l
l
i
,
30 9 9
i
l
l
l
0..-i 9 ~0 9
O ~
~-------~
20 oo
101 I
(J
l
i
l
I
J
5
I
I
I
I
I
I
i
I
i
10
I
i
J
Fig. 4. A Soil respiration. B Catalase activity per g soil dry weight. Abscissa: Days after rewetting.
15 Days
10.4/.tg.h-i.g dry soil -]. With an economic coefficient of 0.5, this corresponds to a respiratory rate of about 10 #1 C O 2 " h - 1 . During the period of active growth, the microscopically counted bacteria could thus be attributed a respiratory rate of 5 /~1 CO2.h -I . The doubling time for this population was 90 h, and the cell number at maximum growth rate was taken as 10 m. Using the same kind of calculation as for the plate-counted bacteria, the average cell mass of the microscopically counted bacteria can be estimated to be 0.04 pg, or 10% of the cell mass of bacteria-forming colonies on solid media. This value is in fair agreement with that of Nikitin (17), who estimated the mass of electron microscopically counted bacteria to be 0.06 pg. Catalase activity in soil could be determined quite easily by using an oxygen electrode. As catalase is an intracellular, constitutive enzyme in aerobic and facultative anaerobic organisms, it seems to be a promising enzyme to use for estimations of microbial mass. The increase in catalase activity (Fig. 4B) corresponded well with the increase in hyphal length or total microbial mass.
Water Fluctuations and Microbial Mass and Activity in Soil
121
3 r.
Fig. 5. Absorbtion spectra of the pyrophosphate washings from the preparation of bacterial fractions (5). --: prepared from soil 1 day after rewetting. ----: 7 days after rewetting. - - - - : the difference spectrum between the two former curves. The spectra were recorded on a Shimadzu MPS-50L spectrophotometer.
0
',. / "-.. /
\
\
"I,
\
/
",
2/
\ " \ ~.......
"',............ i
I
J
I
300
~
I
t.O0
i
I "~"
500
"'~J
~ -
nM
600
~3 "~2 Fig. 6. Optical density at 300 nm of the pyrophosphate washings (cf. Fig. O 5). Abscissa: Days after rewetting.
I
~ . . . .
I
5
. . . .
o [
9 . . . .
10
o . ~ I
15
Days When bacterial fractions were prepared from soil incubated for different periods of time after rewetting, it was observed that they contained less soluble, dark-colored material as the incubation period was increased. This could be shown more precisely by taking absorbtion spectra of the washing solutions (Fig. 5). Water-soluble humus compounds thus disappeared during the incubation of the rewetted soil. This indicated that drying and rewetting also stimulated the rate of breakdown of water-soluble humus components. The disappearance curve (Fig. 6) indicates that this breakdown was caused by fungi and/or microscopically counted bacteria.
Discussion Drying of the soil resulted in the death of microorganisms. Under natural conditions there may also be death of other organisms and small plant roots. After rewetting, nutrients will thus be available for growth and respiration of microorganisms. The respiratory "burst" following rewetting was found to be closely related to the growth kinetics of plate-counted bacteria. Fungi and microscopically counted bacteria continued to grow for about 10 days, and with a much lower rate than the growth rate of bacteria forming colonies on solid media. The shapes of the plate count and microscopic count growth curves show that it is unlikely that the microscopically counted bacteria originate from the plate count population. If this were the case, the increase in microscopic counts in Figure 1 would
122
V. Lund and J. Goks~yr
have to be explained as a continuation of the plate count growth curve. At the start of the stationary phase for plate-counted bacteria, their number was 4-108. From this time and onwards, about 5.109 microscopically counted bacteria were produced. For bacteria growing in batch cultures, the maximum microscopic count will as a rule never exceed the maximum plate count value (i.e., the plate count value at the start of the stationary phase). In the present case, it did exceed the maximum plate count by a factor of 10. Thus, either soil conditions must cause bacteria to enter a state where their offspring is defective in not being able to form colonies on solid media, or the two growth curves must represent different bacterial populations. The latter explanation seems more likely. With minor modifications of Winogradsky's (24) concepts, the bacteria determined by plate counts can for practical purposes be said to represent the zymogenous population. They are characterized by being saprophytic, by having a fairly high growth rate (average doubling time 4-5 h at room temperature), and they are of "normal" bacterial size, with an average cell mass of 0.4 pg dry weight. The bacteria determined by microscopic counts (more precisely, minus the plate count figures) can be said to represent the autochthonous population. They have a low growth rate (doubling time about 90 h), and are quite small, with an average cell mass only about 10% of that of the zymogenous bacteria. Their response to added nutrients seems to be indirect only. The microbial activity in soil after drying and rewetting can be considered to proceed in waves, where the two bacterial populations (the zymogenous and the autochthonous) and the fungi have different growth and activity patterns. The overall effect is an increase in the mineralization rate, where easily available nutrients from dead microorganisms are used first, but which finally ends with mineralization of humic substances. This explains why fluctuating water activity in the soil may result in a more rapid decomposition and mineralization than constant moisture under optimal conditions will do. Decomposition models based on constant water and temperature regimes (e.g., 4) should thus be used with care. The decomposition rate will be dependent not only on the temperature and moisture at the time of the measurement, but also on the previous climatic history of the site.
Acknowledgment. This work was supported by a grant from the Agricultural Research Council of Norway.
References 1. Birch, H. F.: The effect of soil drying on humus decomposition and nitrogen availability. Plant and Soil X, 9--31 (1958) 2. Birch, H. F.: Further observations on humus decomposition and nitrification. Plant Soil XI, 262-286
(1959) 3. Birch, H. F.: Nitrification in soils after different periods of dryness. Plant Soil XII, 81-96 (1960) 4. Bunnell, F. L., D. E. N. Tait, and P. W. Flanagan: Microbial respiration and substrate weight loss. I. A general model of the influence of abiotic variables. Soil Biol. B iochem. 9, 33--40 (1977) 5. Faegri, A., V. Lid Torsvik, and J. Goksflyr: Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil Biol. Biochem. 9, 105-112 (1977) 6. Gray, T. R. G., R. Hissel, and T. Duxbury: Bacterial populations of litter and soil in a decidous woodland. II. Numbers, biomass and growth rates. Rev. Ecol. Biol. Sol. I1, 15-26 (1974) 7. Hanssen, J. F., T. F. Thingstad, and J. GoksCyr: Evaluation of hyphal lengths and fungal biomass in soil by a membrane filter technique. Oikos 25, ! 02-107 (1974) 8. Hobbie, J. E., R. J. Daley, and S. Jasper: Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33, 1225-28 (1977)
Water Fluctuations and Microbial Mass and Activity in Soil
123
9. Holm-Hanssen, O., W. H. Sutcliffe, Jr., and J. Sharp: Measurement of deoxyribonucleic acid in the ocean and its ecological significance. Limnol. Oceanogr. 13,507-514 (1968) 10. Jager, G., and E. H. Bruins: Effect of repeated drying at different temperatures on soil organic matter decomposition and characteristics and on the soil microflora. Soil Biol. Biochem. 7, 153--59 (1975) 11. Jenkinson, D. S.: Studies on the decomposition of plant materials in soil. 1I. Partial sterilization of soil and soil biomass. J. Soil Sci. 17, 280-302 (1966) 12. Jenkinson, D. S.: The effects of biocidal treatments on metabolism in soil. IV. The decomposition of fumigated organisms in soil. Soil Biol. Biochem. 8,203-208 (I 976) 13, Jenkinson, D. S., and D. S. Powlson: Residual effects of soil fumigation on soil respiration and mineralization. Soil Biol. Biochem. 2, 99--108 (1970) 14. Jenkinson, D. S., and D. S. Powlson: The effects of biocidal treatments on metabolism in soil. I. Fumigation with chloroform. Soil Biol. Biochem. 8, 167-77 (1976) 15. Jenkinson, D. S., and D. S. Powlson: The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem. 8, 209-213 (1976) 16. Lebedjantzev, A. N.: Drying of soil, as one of the natural factors in maintaining soil fertility. Soil Sci. 18, 419-447 (1924) 17, Nikitin, D. I.: Direct electron microscopic techniques for the observations of microorganisms in soil. Modem methods in the study of microbial ecology. Bull. Ecol. Res. Commun. (Stockh.) 17, 85-92 (1973) 18. Pochon, J.: Manuel Technique d'Analyse Microbiologique du Sol. Masson et Cie, Paris (1954) 19. Shields, J. A., E. A. Paul, and W. E. Lowe: Factors influencing the stability of labelled microbial materials in soil. Soil Biol. Biochem. 6, 31-37 (1974) 20. Stevenson, J. L.: Some observations on the microbial activity in remoistened air-dried soils. Plant Soil 8, 170-82 (1956) 21. SCrensen, L. H.: Rate of decomposition of organic matter in soil as influenced by repeated air-dryingrewetting and repeated additions of organic matter. Soil Biol. Biochem. 6,287-292 (1974) 22. Thornton, H. G.: On the development of a standardized agar medium for counting soil bacteria, with especial regard to the repression of spreading colonies. Ann. Appl. Biol. 9,241-274 (1922) 23. Torsvik, V. Lid, and J. GoksCyr: Determination of bacterial DNA in soil. Soil Biol. Biochem. 10, 7-12 (1978) 24. Winogradsky, S.: Sur la microflore autochtone de la terre arable. C. R. Acad. Sci. [D] (Paris) 178, 1236-39 (1924)
MICIK~IAL ECO.OG'V
Effects of Water Fluctuations on Microbial Mass and Activity in Soil Vera Lund and Jostein Goksoyr Departmentof Microbiologyand Plant Physiology,Universityof Bergen,Norway Abstract.When previously dried soil was remoistened, a series of microbial events occurred. The bacterial plate count population increased rapidly, with a doubling time of 4-5 h. The length of fungal hyphae and microscopic counts of bacteria increased more slowly. The microscopically counted bacterial population was estimated to have a doubling time of about 90 h. The respiratory burst occurring after 2-3 days coincided with the maximal growth rate of the bacterial plate count population. From the respiratory data, plate count bacteria were estimated to have a cell mass of 0.4 pg dry weight, whereas the mass of microscopically counted bacteria was only 10% of this. Changes in bacterial DNA content corresponded to changes in the microscopic count, whereas changes in soil catalase activity mainly corresponded to changes in the fungal biomass, which was dominant. It is suggested that bacterial plate counts and microscopic counts represent two distinct populations of bacteria, which for practical purposes may be termed zymogenous and autochthonous, respectively.
Introduction In connection with a study of the effects of fertilization and lime application on microbial mass and activity in alpine meadows, it was noted that one of the most important factors influencing microbial activity under field conditions was the fluctuations in water activity of the soil. Lebedjantzev (16) observed that air-drying increased the fertility of a variety of soils in pot experiments. The effect was most evident on uncultivated soils and soils fertilized with phosphate or manure. He found that during drying there was a large increase in the solubility of organic substances and especially an increase in ammonia nitrogen. There was also a sharp reduction in the number of microorganisms. He considered the drying as a partial sterilization, and pointed to the role of drying and rewetting cycles in maintaining the fertility of the soil. Stevenson (20) studied the microbial activity of remoistened, air-dried soil, and in particular the relationship between the increased soil respiration observed after rewetting, and the bacterial counts. The size of the respiratory " b u r s t " was directly related to the amount of amino acids and other materials released by the drying process, and the conclusion drawn was that the increased respiration was 0095-3628/80/0006-0115 $01.60 O 1980 Springer-Verlag New York Inc.
116
v. Lundand J. Goks0yr
m a i n l y due to biological activity, although the bacterial counts (plate counts) reached a m a x i m u m some time after the respiration had reached a maximum. In studies of East African soils, Birch ( I - 3 ) followed soil respiration, humus decomposition, and nitrogen mineralization during a number of drying and rewetting regimes. Birch also concluded that the primary effect of drying and rewetting was on the microbial populations in the soil. The soil microorganisms which survived the drying responded to rewetting by temporarily entering a state of high metabolic activity, resulting in " c a n n i b a l i z a t i o n " of dead microbial cells and even a burst of humus decomposition. This was followed by liberation of ammonia and subsequent nitrification, which explained the increased fertility of dried soils when rewetted. The agricultural implications for countries with marked dry and wet seasons were emphasized. Jenkinson and collaborators (11-15) have demonstrated that by sterilization of soil, biological (mainly microbial) material is made available for mineralization, and that there is good correlation between the biomass in the soil before sterilization and the a m o u n t of CO 2 released during a 10-day period after reinoculation. Shields et al. (19) found in a similar study that after partial sterilization with chloroform and reinoculation, the plate count n u m b e r of bacteria increased rapidly to reach a peak value that was higher than before sterilization. This rapid increase coincided in time with a high rate of carbon mineralization. The direct count values of bacteria, which also showed a decrease by the sterilization, increased more slowly after reinoculation. Jager and Bruins (10) and Sorensen (21) found that repeated cycles of drying and rewetting enhanced the decomposition rates in soil, and that a considerable number of cycles were needed to deplete the soil for the substrates that took part in the process. The present study has been concentrated on a more detailed investigation of the effects of drying and rewetting on bacterial and fungal masses and activities in the soil, and especially on the differences in behavior between plate-counted and microscopicall3/ counted bacteria.
Materials and Methods Soil samples were collected from the top 10 cm (A horizon) of an unfertilizedhumus podzol from Field A, KjOlastolen,Oystre Slidre in Valdres, Norway, during late summer. This field is situated 1000 m above sea level in Central Norway in an area used for mountainfarming.The soil was broughtto the laboratory, sieved (mesh size 2 ram), and mixedcarefully.Afterremovalof samplesfor direct analyses,the soil was dried at room temperature until air-dry(4--5days). It was then stored in polyethylenebags for approximately 1month, when the rewettingexperimentswere started. For rewetting, weighed samples of soil were placed in 200 ml jars with screw-caps. Membrane-filtered distilled water was added to give the same watercontentas beforedrying, and the jars were rotated slowlyin a clinostat for 24 h in order to secure even distributionof moisture. The jars were then kept in the dark at room temperature for the experimentalperiod. Samples were taken daily for the first 10 days, and thereafter every second day. When necessary, they were analyzed immediately;otherwise, they were stored frozen until analysis. Bacterial Plate Counts.
The soil was homogenized in a Waring blender with Winogradsky'ssalt solution diluted 1:20 (18), and a dilution series was made with the same solution.Then0. I ml of suitabledilutionswas spreadon the surfaceof agar plates containingThornton's mediumwith 10% soilextract (22) witha bentglass rod whilethe plates were rotating. The plates were incubatedat 22~ for 10 days beforecounting.
Water Fluctuations and Microbial Mass and Activity in Soil
117
Microscopic Counts of Bacteria First, 1 ml of an appropriate soil dilution was made up as for plate counts and stained with I ml of acridine orange solutin (conc. 1:5000) for at least 5 rain. The suspension was then diluted with membrane-filtered distilled water to 15 ml and filtered through Unipore polycarbonate filters (filter dia. 22 mm, pore size 0.2/,tin), previously stained with Irgalan black (8). Nonfluorescent liquid paraffin was used between the filter and the coverslip. Counting was carried out with a Leitz Orthoplan microscope equipped with Ploemopak illumination system for epifluorescence, at a magnification of 9 0 0 x , with an immersion objective and an ocular supplied with a rectangular grid. All bacteria, regardless of fluorescent color, were counted. The count values were corrected for blank values, obtained by counting filters through which had been passed samples of the Winogradsky's salt solution stained with the same batch of acridine orange.
Hyphal Lengths of Fungi The total length of fungal hyphae was determined by the membrane filter technique (7). Safranine (0.25% aqueous solution) was used as the staining agent, and the suspension was filtered through Oxoid membrane filters (dia. 22 mm, pore size 0.45 .urn).
Respiration Measurements Soil respiration was measured with an infrared CO2-analyzer (type 225 MK2 from The Analytical Development Co, Ltd., England). 15 g (wet weight) of soil was placed in a glass tube with about 2 cm inner din. This was connected by PVC tubes and via an air reservoir of about 500 ml to the outlet and inlet systems oftbe CO 2 analyzer, so that the air was recirculated. The glass tube and air reservoir were placed in a water bath which was kept at 25~ After an equilibration period of about 2 h, the increase in the CO 2 content in the recirculated air was recorded during a 30 rain period. The rate of CO 2 formation could be calculated after determination of the total air volume of the system. This was done by injection of a known volume of CO 2, and determination of the resulting CO 2 concentration.
Bacterial DNA The bacterial fraction from 10 g soil (wet weight) was prepared (5). After suspension in Winogradsky's salt solution diluted 1:20 to a total volume of 60 ml and dividing in portions of 10 ml, the suspensions were stored frozen until analysis. DNA was determined fluorometrically by the reaction with 3,5-diamino benzoic acid 2 HCI (9) as described by Lid Torsvik and Gokscyr (23). Calf thymus DNA was used as internal and external standards. Fluorescence measurements were made in a Jasco FP-4 fluorescence spectrophotometer. The DNA content per microscopically counted bacterium in the bacterial fraction was calculated, and from this and the microscopic counts of bacteria in the soil, the total amount of bacterial DNA in the soil was also determined.
Catalase (E.C. I. I I. 1.6)Measurements First. 5 g of frozen soil was homogenized with 95 ml of Winogradsky's salt solution 1:20 in a Waring blender for 2 rain at low speed. Then 6 ml of the soil suspension was placed in the reaction chamber of a Rank Brothers (England) oxygen electrode. Next, 0.2 ml 3% hydrogen peroxide was injected and the production of oxygen recorded during a 5-min period. Boiled soil suspension was used as control, and catalase activity determined as the difference in oxygen evolution between the fresh and the boiled suspension. One catalase unit was defined as the amount of enzyme required to evolve I p,mole 02 per minute under the stated conditions.
118
V. Luridand J. Goks#yr
Results All values are given per gram dry soil, unless otherwise stated. Data for the soil before drying are shown in Table 1. Assuming an average diameter of 3 /am for the fungal hyphae (no accurate measurements were made) and an average bacterial dry weight of 0.06 pg (17), the microbial mass was 12.6 mg (I 1.5 mg fungi and 1. l mg bacteria). From the start data in Figures 1 and 3, and with the same assumptions, the total mass after the drying was 7.1 mg (6.8 nag fungi and 0.3 mg bacteria). It should be noted that no distinction between live and dead microorganisms was attempted. When the soil was rewetted, there was a rapid increase in the bacterial plate count, with a maximum about 5 days after rewetting, and a considerably later and slower increase in the microscopic count. This is shown in Figure 1. From this figure, it was estimated that the doubling time for the plate count bacteria during the first 2 days was 4-5 h, whereas it was about 90 h for the microscopically counted bacteria in the period of most rapid increase (sixth to tenth days). The ratio between microscopic and plate counts was 1400 in the soil before drying, and about the same just after rewetting. Five days later the ratio had decreased to 11, and then it slowly increased again, to reach 60 after 18 days. The amount of bacterial DNA in the soil showed an increase similar to that of the microscopic count (Fig. 2), demonstrating that the increase in microscopic count is accompanied by DNA synthesis. At the start of the rewetting period and at the end of the experiment, there was 12 fg (10-15 g) DNA per microscopically counted bacterium. In the period of most rapid increase, 5 fg was found. There was a steady increase the hyphal length of fungi after rewetting, from about 700 to 920 m after I0 days (Fig. 3). This corresponded to an increase in fungal mass of about 2 rag, and should be compared with the 5.5 mg microbial mass that disappeared as such during the drying and now should be available as nutrient in the soil. Respiration was measured on the whole soil (Fig. 4A), and thus represents the sum of fungal and bacterial activity. The peak in respiratory activity on the second and third days is the well-known respiratory "burst" described by several authors. The only parameter to which this can be correlated is the increase in bacterial plate counts. With constant growth yield and negligible maintenance respiration, the respiratory rate of a microbial culture is proportional to the growth rate. Maximum growth rate and hence maximum respiratory rate are found at the inflexion point of the growth curve, when the growth is expressed on a linear scale. This explains why the respiratory maximum is found at some time before maximum cell number is attained. From Fig. 4A, the peak respiratory activity was estimated to 15/.tl CO2-h-1 above the stable respiratory level. Assuming an economic coefficient (on carbon basis) of 0.5, and a carbon content of 50% in the cell material, the mass increase of the plate-counted bacteria at this period (2.5 Table I. Data for the soil beforedrying, calculatedper g dry weight Water content Organic matter(ignition loss) pH (in distilled water) Hyphal length Bacteria, platecounts Bacteria, microscopiccount Respiration (25~
180% 40% 4.5 1250 m 1.0 • 107 1.4 • I010 16.5/al CO2.h- I
Water Fluctuations and Microbial Mass and Activity in Soil
119
10 _.. 9
O~O~O
,._g
I:::
0
eoll J
o
8
J
Fig. 1. Plate counts (o) and microscopic counts (o) of bacteria per g soil dry weight. Abscissa: Days after rewetting.
5 I
,
10 I
,
,
,
15
,
I
,
Days 200
/
0
~ 100 1--0'
Fig. 2. Bacterial DNA per g soil dry weight after rewetting.
I
5
I
I
I
t
I
I
,
,
10
,
f
15
Days days after rewetting) will be 16/ag-h- i. The plate count number was 1.7 x 108, and the multiplication rate 3.8 x 107 h -1. This gives an average cell mass (dry weight) of the plate-counted bacteria of 0.4 pg, which corresponds quite well with estimated sizes for bacteria in soil forming colonies on solid media (6). After the peak of respiratory activity, the soil respiration was fairly stable at 15/.tl CO2"h- ~ for the rest of the experiment. Besides maintenance respiration of the platecounted bacteria, this respiration was caused by bacteria not forming colonies on solid media, and by fungi. The fungal growth rate during the first 8 clays was estimated to be
V. Lund and J. GoksOyr
120
-c I000 900
~o0 0-
,- 700
O
600
C
1.1,.
Fig. 3. Fungal hyphae per g soil dry
5OO
i
i
L
I
,
i
,
i
[
5
I
~
I
1
10
weight after rewetting.
,
15 Days
~ 30 20 3._
A e-~
10
tn l
o
V~
-t'-
Ul
l
l
l
l
l
,
,
l
l
i
,
30 9 9
i
l
l
l
0..-i 9 ~0 9
O ~
~-------~
20 oo
101 I
(J
l
i
l
I
J
5
I
I
I
I
I
I
i
I
i
10
I
i
J
Fig. 4. A Soil respiration. B Catalase activity per g soil dry weight. Abscissa: Days after rewetting.
15 Days
10.4/.tg.h-i.g dry soil -]. With an economic coefficient of 0.5, this corresponds to a respiratory rate of about 10 #1 C O 2 " h - 1 . During the period of active growth, the microscopically counted bacteria could thus be attributed a respiratory rate of 5 /~1 CO2.h -I . The doubling time for this population was 90 h, and the cell number at maximum growth rate was taken as 10 m. Using the same kind of calculation as for the plate-counted bacteria, the average cell mass of the microscopically counted bacteria can be estimated to be 0.04 pg, or 10% of the cell mass of bacteria-forming colonies on solid media. This value is in fair agreement with that of Nikitin (17), who estimated the mass of electron microscopically counted bacteria to be 0.06 pg. Catalase activity in soil could be determined quite easily by using an oxygen electrode. As catalase is an intracellular, constitutive enzyme in aerobic and facultative anaerobic organisms, it seems to be a promising enzyme to use for estimations of microbial mass. The increase in catalase activity (Fig. 4B) corresponded well with the increase in hyphal length or total microbial mass.
Water Fluctuations and Microbial Mass and Activity in Soil
121
3 r.
Fig. 5. Absorbtion spectra of the pyrophosphate washings from the preparation of bacterial fractions (5). --: prepared from soil 1 day after rewetting. ----: 7 days after rewetting. - - - - : the difference spectrum between the two former curves. The spectra were recorded on a Shimadzu MPS-50L spectrophotometer.
0
',. / "-.. /
\
\
"I,
\
/
",
2/
\ " \ ~.......
"',............ i
I
J
I
300
~
I
t.O0
i
I "~"
500
"'~J
~ -
nM
600
~3 "~2 Fig. 6. Optical density at 300 nm of the pyrophosphate washings (cf. Fig. O 5). Abscissa: Days after rewetting.
I
~ . . . .
I
5
. . . .
o [
9 . . . .
10
o . ~ I
15
Days When bacterial fractions were prepared from soil incubated for different periods of time after rewetting, it was observed that they contained less soluble, dark-colored material as the incubation period was increased. This could be shown more precisely by taking absorbtion spectra of the washing solutions (Fig. 5). Water-soluble humus compounds thus disappeared during the incubation of the rewetted soil. This indicated that drying and rewetting also stimulated the rate of breakdown of water-soluble humus components. The disappearance curve (Fig. 6) indicates that this breakdown was caused by fungi and/or microscopically counted bacteria.
Discussion Drying of the soil resulted in the death of microorganisms. Under natural conditions there may also be death of other organisms and small plant roots. After rewetting, nutrients will thus be available for growth and respiration of microorganisms. The respiratory "burst" following rewetting was found to be closely related to the growth kinetics of plate-counted bacteria. Fungi and microscopically counted bacteria continued to grow for about 10 days, and with a much lower rate than the growth rate of bacteria forming colonies on solid media. The shapes of the plate count and microscopic count growth curves show that it is unlikely that the microscopically counted bacteria originate from the plate count population. If this were the case, the increase in microscopic counts in Figure 1 would
122
V. Lund and J. Goks~yr
have to be explained as a continuation of the plate count growth curve. At the start of the stationary phase for plate-counted bacteria, their number was 4-108. From this time and onwards, about 5.109 microscopically counted bacteria were produced. For bacteria growing in batch cultures, the maximum microscopic count will as a rule never exceed the maximum plate count value (i.e., the plate count value at the start of the stationary phase). In the present case, it did exceed the maximum plate count by a factor of 10. Thus, either soil conditions must cause bacteria to enter a state where their offspring is defective in not being able to form colonies on solid media, or the two growth curves must represent different bacterial populations. The latter explanation seems more likely. With minor modifications of Winogradsky's (24) concepts, the bacteria determined by plate counts can for practical purposes be said to represent the zymogenous population. They are characterized by being saprophytic, by having a fairly high growth rate (average doubling time 4-5 h at room temperature), and they are of "normal" bacterial size, with an average cell mass of 0.4 pg dry weight. The bacteria determined by microscopic counts (more precisely, minus the plate count figures) can be said to represent the autochthonous population. They have a low growth rate (doubling time about 90 h), and are quite small, with an average cell mass only about 10% of that of the zymogenous bacteria. Their response to added nutrients seems to be indirect only. The microbial activity in soil after drying and rewetting can be considered to proceed in waves, where the two bacterial populations (the zymogenous and the autochthonous) and the fungi have different growth and activity patterns. The overall effect is an increase in the mineralization rate, where easily available nutrients from dead microorganisms are used first, but which finally ends with mineralization of humic substances. This explains why fluctuating water activity in the soil may result in a more rapid decomposition and mineralization than constant moisture under optimal conditions will do. Decomposition models based on constant water and temperature regimes (e.g., 4) should thus be used with care. The decomposition rate will be dependent not only on the temperature and moisture at the time of the measurement, but also on the previous climatic history of the site.
Acknowledgment. This work was supported by a grant from the Agricultural Research Council of Norway.
References 1. Birch, H. F.: The effect of soil drying on humus decomposition and nitrogen availability. Plant and Soil X, 9--31 (1958) 2. Birch, H. F.: Further observations on humus decomposition and nitrification. Plant Soil XI, 262-286
(1959) 3. Birch, H. F.: Nitrification in soils after different periods of dryness. Plant Soil XII, 81-96 (1960) 4. Bunnell, F. L., D. E. N. Tait, and P. W. Flanagan: Microbial respiration and substrate weight loss. I. A general model of the influence of abiotic variables. Soil Biol. B iochem. 9, 33--40 (1977) 5. Faegri, A., V. Lid Torsvik, and J. Goksflyr: Bacterial and fungal activities in soil: separation of bacteria and fungi by a rapid fractionated centrifugation technique. Soil Biol. Biochem. 9, 105-112 (1977) 6. Gray, T. R. G., R. Hissel, and T. Duxbury: Bacterial populations of litter and soil in a decidous woodland. II. Numbers, biomass and growth rates. Rev. Ecol. Biol. Sol. I1, 15-26 (1974) 7. Hanssen, J. F., T. F. Thingstad, and J. GoksCyr: Evaluation of hyphal lengths and fungal biomass in soil by a membrane filter technique. Oikos 25, ! 02-107 (1974) 8. Hobbie, J. E., R. J. Daley, and S. Jasper: Use of nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33, 1225-28 (1977)
Water Fluctuations and Microbial Mass and Activity in Soil
123
9. Holm-Hanssen, O., W. H. Sutcliffe, Jr., and J. Sharp: Measurement of deoxyribonucleic acid in the ocean and its ecological significance. Limnol. Oceanogr. 13,507-514 (1968) 10. Jager, G., and E. H. Bruins: Effect of repeated drying at different temperatures on soil organic matter decomposition and characteristics and on the soil microflora. Soil Biol. Biochem. 7, 153--59 (1975) 11. Jenkinson, D. S.: Studies on the decomposition of plant materials in soil. 1I. Partial sterilization of soil and soil biomass. J. Soil Sci. 17, 280-302 (1966) 12. Jenkinson, D. S.: The effects of biocidal treatments on metabolism in soil. IV. The decomposition of fumigated organisms in soil. Soil Biol. Biochem. 8,203-208 (I 976) 13, Jenkinson, D. S., and D. S. Powlson: Residual effects of soil fumigation on soil respiration and mineralization. Soil Biol. Biochem. 2, 99--108 (1970) 14. Jenkinson, D. S., and D. S. Powlson: The effects of biocidal treatments on metabolism in soil. I. Fumigation with chloroform. Soil Biol. Biochem. 8, 167-77 (1976) 15. Jenkinson, D. S., and D. S. Powlson: The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem. 8, 209-213 (1976) 16. Lebedjantzev, A. N.: Drying of soil, as one of the natural factors in maintaining soil fertility. Soil Sci. 18, 419-447 (1924) 17, Nikitin, D. I.: Direct electron microscopic techniques for the observations of microorganisms in soil. Modem methods in the study of microbial ecology. Bull. Ecol. Res. Commun. (Stockh.) 17, 85-92 (1973) 18. Pochon, J.: Manuel Technique d'Analyse Microbiologique du Sol. Masson et Cie, Paris (1954) 19. Shields, J. A., E. A. Paul, and W. E. Lowe: Factors influencing the stability of labelled microbial materials in soil. Soil Biol. Biochem. 6, 31-37 (1974) 20. Stevenson, J. L.: Some observations on the microbial activity in remoistened air-dried soils. Plant Soil 8, 170-82 (1956) 21. SCrensen, L. H.: Rate of decomposition of organic matter in soil as influenced by repeated air-dryingrewetting and repeated additions of organic matter. Soil Biol. Biochem. 6,287-292 (1974) 22. Thornton, H. G.: On the development of a standardized agar medium for counting soil bacteria, with especial regard to the repression of spreading colonies. Ann. Appl. Biol. 9,241-274 (1922) 23. Torsvik, V. Lid, and J. GoksCyr: Determination of bacterial DNA in soil. Soil Biol. Biochem. 10, 7-12 (1978) 24. Winogradsky, S.: Sur la microflore autochtone de la terre arable. C. R. Acad. Sci. [D] (Paris) 178, 1236-39 (1924)
