Environmental effects on microbial turnover of some mineral elements

Soil Viol. Biorhem.Vol. 1, pp. 167-176. PergamonPress 1969. Printedin Great Britain

ENVIRONMENTAL EFFECTS ON MICROBIAL OF SOME MINERAL ELEMENTS PART I-ABIOTIC

TURNOVER

FACTORS

M. WITKAMP

Health Physics Division, Oak Ridge National Laboratory,

Oak Ridge, Tennessee

Summary-The effects of some abiotic environmental factors on mineral cycling in leaflitter and in artificial media were measured using radionuclides. Leaching and microbial immobilization of various elements increased in the order Co < Fe < Ca=Sr < Cs and were proportional to their concentrations. The microbial accumulation of Co was curtailed as a result of Co poisoning when there was more than 1000 ppm CoClt in the substrate; and the microbial accumulation of Cs was greatly increased in a K-deficient substrate. The acidity of the substrate did not affect the uptake of Cs but the uptake of Co was lowest around neutrality. Leaching of Cs from litter increased with the rate of leaching but leaching etTiciency was highest at a rate equivalent to 50 cm of precipitation/yr. Immobilization of Cs at 20% leaf moisture was nil. Over the range from 60 to 200% leaf moisture, immobilization was significant and rather constant. Raising litter temperature from 2°C to 50°C increased leaching of Co by 40% and of Cs by 3 to 6 times. Immobilization of Cs in oak litter increased with increasing temperature. Freezing (and drying) mobilized over 95% of the Cs and over 85% of the Co in microbial tissue, but only one third of the Cs in decaying litter was mobilized as a result of resorption onto the litter. Resorption was even greater (-85 “/.) onto charred leaves after fire, and raised the Cs concentration per unit weight by a factor of four. INTRODUCTION

microflora plays an important role in the cycling process of mineral nutrients from plant tissue back to plant tissue via herbivore-carnivore, or saprophagous foodchains. Many elements can be mobilized from organic materials during microbial decomposition of tissues. This process of mineralization enhances leaching of the mobilized minerals by rain, and subsequent sorption by soil or uptake by plants. Simultaneous with mineralization, a part of the mineral content of the decomposed dead tissue becomes incorporated in living microbial tissue which results in decreased mobility of mineral nutrients. This microbial immobilization of minerals has received most attention in agriculture where the microflora may render nutrients in fertilizers unavailable to plants; and in forestry, where immobilization becomes important when large portions of the cycling minerals accumulate in dead organic matter and active cells of microorganisms. This study reports certain aspects of the mineralization and immobilization of various minerals in forest litter and some effects of various environmental factors on these processes. The study is meant to provide a first approximation to the effects of the environment on microbial turnover of some mineral elements. Forest litter was chosen because of considerable microbial development, uninterrupted mineral cycling, and practical implications. The minerals used were radionuclides which can be easily traced even in minute quantities. The results also may be of interest to health physicists since long-lived nuclides were used, which may accumulate to hazardous levels when the rates of nuclide immobilization surpass those of nuclide mineralization. Because it was impractical to separate the microbial cells and tissues from their litter substrates, direct measurements of the nuclide content of microbial tissue was prevented. Instead the difference in quantity of nuclide leaching from THE

167 S.&B.1/3-A

168

M. WITKAMP

litter, with and without an active microflora, was accepted as the measure for microbial immobilization. This immobilization is the net effect of microbial activity on retention and release of a nuclide by living and dead microbial tissue as well as by the decomposing substrate. The need for indirect methods and for environmental control to evaluate the influence of single ecological factors on mineral turnover necessitated laboratory experiments rather than field measurements. In these experiments field conditions were simulated as much as was practical. MATERL4LS

AND METHODS

In the experiments designed to measure microbial immobilization, parallel series of substrates tagged with a radionuclide and with or without an active microflora were leached with distilled water. The difference in radioactivity leaching from the two series was the measure for net microbial immobilization. The difference in microbial activity between both series was obtained either by refrigeration of one series and incubation of the other series at 25”C, or by initial sterilization of both series and subsequent inoculation of only one series while maintaining sterility of the other. Microorganisms used in this study were isolated from litter and soil in the Oak Ridge, USAEC reservation. Fungi were kept on peptone-dextrose agar. Inocula for fungi were 1 ml spore suspensions obtained from agar slants or plates. Inocula for natural mixed flora were 1 ml of supernatant of 10 g of air-dried litter shaken for 15 min in 100 ml of sterile water. Leaf litter was used as microbial substrate as much as feasible. The use of artificial substrate was primarily limited to experiments designed to clarify the results obtained on natural substrate. Artificial media enabled better quantitative separation of microbial tissue and substrate; homogenous environment for microorganisms (nuclide concentration, aeration, nutrient supply); and control of environmental factors (mineral concentration, pH) than the natural substrate. The artificial media were peptone-dextrose broth with 30 ppm rose bengal and streptomycin (Martin, 1950), and sucrose nitrate medium (sucrose, 15 g; NaNOJ 2 g; KH2P04, 1 g; (NH&H P04, 1 g; Mg SO,, O-5 g in 1 liter of water). Cultures were grown on 100 ml medium in 250 ml Erlenmeyer flasks incubated at room temperatures on an orbital shaker at 100 rpm. The leaf litter was collected directly before or after the time of leaf fall from trees that had been inoculated with radionuclide. Tree trunk inoculations had been made early in the growing season, ensuring natural distribution of the nuclides in the leaf tissue. Leaf litter was mainly from a stand dominated by tulip poplars (Liriodendron tulipifera L.) which had been tagged with 13’Cs. Various other tags were applied to the trunks of white oak (Quercus alba L.), black alder (Alms ghtinosa L.) Gaertn. and sycamore (Pseudoplatanus occidentalis L.). Either entire leaves or leaf and litter fragments that were sufficiently small to fit the containers were used. Fragmented leaves were made by crushing dry leaves by hand to particles of about 3 x 8 mm. The decomposing leaves were incubated at room temperatures in funnels with fritted glass filters unless specified otherwise. The funnels were closed on top with rubber stoppers to prevent desiccation and contamination and to enable measurement of microbial activity as the rate of CO2 evolution. This was done by exposing a dish containing 1 ml of 0. Iru’ KOH in each incubated funnel for 1 or 2 hr, depending on the rate of CO, evolution. The amount of CO2 absorbed was measured titrimetrically (Conway, 1950). Measurements were made before leaching or at daily intervals. Leaching of decomposing materials was done by dripping water from a pipet onto the

169

MICROBIAL TURNOVER OF MINERALS. PART I

leaves or litter at 2- or 3-day intervals at about the rate of average rainfall in Oak Ridge 2.5 cm/week). For l- and 5-g samples in funnels with fritted glass filters of 2- and 4-cm diameter, 2 - 5 and 10 ml of H,O were used at each leaching, respectively, unless specified otherwise. The funnels were seated on 125-ml suction flasks. During leaching, suction of 10 cm Hg was applied until no leachate was left on the titers. Controls with natural microflora were incubated at 2°C and leached at 28°C. Radionuclides used were 45Ca, 5gFe, 6oCo, g”Sr , lj4Cs and r3’Cs. All were 98-99 per cent carrier-free. Samples containing y-emitters were counted at appropriate energy peaks by scintillation counting and p-emitters were measured as integral counts using Geiger-Miiller equipment. Radionuchde concentration in microbial tissue from liquid shaking cultures and solid agar plates was counted directly. Fungal mycelium from shaking cultures was collected on fritted glass filters (4 cm diam. medium porosity) on suction flasks at a suction of 10 cm of Hg. Unless otherwise indicated data are means of duplicate series. Significance between means or regressions was assessed by Student’s t-test.

Efects of different n&ides Immobilization of four cationic elements with widely differing biochemical properties was measured in two experiments. Experiment I. A mixed microflora on fragmented tulip poplar leaves (1 g) immobilized nuclides in the order 6oCo < 5gFe < 45Ca-g0Sr < lJ4Cs (Table 1). The leachability of these minerals from controls kept for two weeks at 2°C and then leached at 25°C increased in the same order. TABLE 1. LEACHING AND MICFtOEIALIMMOBILIZA-IION OF RADIONUCLIDES

I-Natural microflora incubated for 14 days at 25°C on lg of fragmented leaves of tulip poplar. II-Phycomycete and Penicilfium sp. incubated for 26 days at 28°C after inoculating lg of steam-sterilized fragments of tulip poplar leayes

I-_% nuclide leached (6 x 5 ml) from control kept at 2°C % nuclide immobilized by natural microflora

II- % nuclide leached (11 x 2.5 ml) from sterile control

10

16

34

41

3

11

22

24

25

56

62

97

aAnuclide immobilized by a phycomycete

1.7

0.5

3.5

2.8

O!!nuclide immobilized by Penicilhm

5.1

1.9

6.2

19.4

sp.

Experiment II. In a similar experiment with fragmented tulip poplar leaves (1 g) but now reinoculated with cultures of a Penicillium and a phycomycete, leachability again increased in the order Co < Fe < Ca-Sr c Cs (Table 1). The amounts leached from sterile controls were larger than in the first experiment possibly as a result of longer leaching and preceding steam sterilization at 121°C (15 psi) for 20 min of the leaves. The amounts of immobilized radionuclides were smaller than in the first experiment and increased in the same order

170

M. WITKAMP

with the exceptions that less 5gFe was immobilized than 6oCo and that the phycomycete immobilized more 45Ca-goSr than 134Cs. These results indicate that there is a degree of proportionality between solubility and immobilization and that the percentages immobilized may differ by one order of magnitude for the elements used in the experiments. Eflects of mineral concentrations

Subsequent to the effect of differences among various elements, the influence of nuclide concentration on microbial uptake was studied on leaf substrate but also in shaking liquid cultures in order to assure homogeneous concentration distribution. Amounts of 13’Cs immobilized by a ten-day-old growth of Trichoderma viride on steamsterilized fragmented tulip poplar leaves (1 g, leached once with 10 ml H20) were highly correlated (r = O-996, n = 12) with the initial lJ7Cs concentrations (3,25,69 and 242 nCi 13’Cs/g) in the leaves. This proportionality between immobilization and the amount of 13’Cs in the substrate was also demonstrated in shaking cultures (Fig. 1). For concentrations of 6oCo ranging from 0.06 to 35 nCi/ml and of l 34Cs ranging from 0.045 to 4 - 5 nCi/ml in 100 ml of broth, concentration factors for 6oCo were between 12 and 14, and for 134Cs, between 2.3 and 2.9, respectively. The additions of 6oCo and 134Cs are negligible as com1

001

04 6oC~

AND

IO

1 ‘34C~

IN

MEDIUM

(nCi/ml

100 1

FIG. 1. Concentrations of ‘%o and lJ4Cs in fungal mycelium and surrounding medium. Three-day-old growth of Trichoderma uiride in 100 ml peptonedextrose broth at 28°C.

pared to the concentrations of stable Co and Cs that naturally occur in the broth. Thus the results show that for concentrations ranging over two orders of magnitude the fungus does not discriminate between stable Co and 6oCo or between stable Cs and 134Cs. This result allows use of the radioisotopes as a tag to study fungal uptake of stable Co and Cs from media with various concentrations of these minerals. This observation was utilized in an experiment in which rJ7Cs and 6oCo were used as tags to measure concentration factors for

171

MICROBIAL TURNOVER OF MINERALS. PART I

stable Cs and Co by Aspergillus niger after one week of growth in shaking cultures with peptone-dextrose broth. Presence of 0, 1, 10, 100 and 1000 ppm of the chlorides of these stable isotopes in the broth did not significantly (P < 0 ‘9) affect the concentration factors of the tags and thus of the stable isotopes. Mean concentration factors for Co and Cs were 56 *O& 8 -2 and 12 -0 & 0 -52, respectively. The cultures of the Cs and Co series grew about equally well (428 & 8 mg for Cs and 399 i- 10 mg for Co) with the exception that in the Co series poisoning occurred at 1000 ppm CoCl, and mycelium production (28 mg) as well as Co concentration factor (23 ‘7) in the mycelium was reduced. Thus microbial uptake and immobilization of stable as well as radioactive Co and Cs and presumably of several other elements appears to be proportional to their concentration in a given substrate. Chemical similarities between potassium and cesium prompted an experiment on the influence of potassium concentration in the substrate on Cs uptake and accumulation in microbial tissue. Shaking cultures were used for close control of experimental conditions. Trichodermu viride was grown in sucrose-nitrate broth (pH 6) with various concentrations of potassium and one concentration of 134Cs (13 - 5 nCi/ml). When no potassium was added growth of the fungus was suppressed. After six days the concentration factor for 134Cs was 67 (Table 2). With more than 10 mg of potassium in 100 ml of medium, mycelium growth increased tenfold, concentration factors for lJ4Cs varied from O-7 to 0.9. Flame photometry showed a rather high concentration of potassium in the mycelium when no potassium had been added, and a twofold increase of potassium in the fungus with a hundredfold increase in the amount of potassium added. Thus, concentration of Cs was only strongly affected by potassium when there was a potassium deficiency. TABLE2. EFFECTOF POTASSNMON “‘Cs Six-day-old cultures in sucrose-nitrate

UPTAKE

BY Trichodermaviriak

broth with 13.5 &i 134Cs/l at 28°C

Mycelium oven-dry after 6 days growth (me)

mgK g mycelium

345

1.22 O-67 O-72

29.474 o-364 o-324

66.6 0.7

:z

349 342

O-89 0.91

o-337 O-367

0.8 0.9

2.64 2.48

22:;

1000

301

1.41

O-336

O-8

4.19

;::

K (mg) added as KCl/lOO ml of broth 0

::

Efects

&i

’ Ws

SiiTun

Concentration

mg K

factor 13%s p’3rCS

Final PH

O-041 2.22 1.84

of pH

Acidity of the environment may affect mineral turnover through effects on composition and activity of the microflora as well as on the availability of minerals. In experiments on uptake and immobilization of 6oCo and lJ4Cs shaking cultures were used to obtain even pH throughout the medium. Initial acidity of kptonedextrose broth with ‘j°Co and l34Cs was adjusted by adding IN NaOH after sterilization. The initial pH did not greatly affect final pH, mycelium production, or 134Cs uptake by T. viride in three days. Uptake and accumulation of 6oCo, however, was more than four times higher at initial pH 4-O and almost two times higher at initial pH 8 -2 than around neutrality (Table 3; see also Fig. 1). Presumably, high solubility in acid medium and smaller colloid size in the basic medium than in the neutral medium caused the differences in 6oCo accumulation. Uptake of lJ4Cs

172

hf.WITJUMP

which appeared in ionic form over the entire pH range was not influenced by the initial pH of the medium. TABLE3. EFFECT OF pH ON FUNGAL Three-day-old

UPTAKE

OF

soCo

AND

137Cs

cultures of Trichoderma viride on peptone+lextrose broth with l-7 nCi 134Cs/mi or O-7 nCi 60Co/ml and different initial pH 4-o

5.4

6.4

7-l

8-2

Final pH Concentration factor Mycelium (g oven-dry)

3-9 3.3 0.42

4-o 3.6 O-42

3-9 3.6 0.35

3-9 3.7 o-33

4-O 4.3 OS35

6Wo Final pH Concentration factor Mycelium (g oven-dry)

3.6 48 o-45

4.6 12 0.45

4.4 I1 O-40

4.4 19 O-40

Initial pH ‘340

4.3 37 0.45

The effect of pH of natural substrate on microbial immobilization of 6oCo was explored in a leaching experiment. The acidity of steam-sterilized tulip poplar leaves (1 g) had been adjusted to pH 4, 5,6, and 7 by addition of 2 ml of the appropriate phosphate buffer. After six days the amount of 6oCo leached by 10 ml water from the sterile controls was slightly higher at pH 4 and 5 (both 28 per cent) than at pH 6 and 7 (24 and 25 per cent respectively). Immobilization of ‘j°Co by T. uiride after six days was 12 per cent at pH 4,8 per cent at pH 5, and -9 per cent at pH 6 and 7. Negative immobilization or mineralization at pH 6 and 7 indicates faster release of 6oCo from leaf tissue by decomposition than uptake of 6oCo by the fungus. This faster release was not a result of faster decay at pH 6 and 7 than at pH 4 and 5 because all rates of COZ production after ten days were between 0~44 and 0.46 ml/g hr. Thus, the effect of pH on 6oCo uptake showed the same trend for leaf substrate as for the liquid substrate, viz. increasing uptake of 6oCo when the pH decreases starting from around neutrality. The pH of the forest floor is directly affected by the species composition of the litter and its microflora. Consequently, these factors will influence the turnover of various mineral elements. Eflecfs of moisture

Moisture affects mineral turnover in leaf litter by the rate at which the leaves are leached (precipitation) and by the moisture content of the leaves which influences microbial activity as well as the amount of mineral in solution. Effects of rate of leaching on mineral turnover was measured using tulip poplar leaves tagged with 13’Cs, The leaves (1 g moist weight) were kept at 2°C to minimize microbial activity. They were leached for two weeks at rates of 5, 10, 35, and 100 ml/week, which are the equivalents of 15, 30, 100 and 300 cm of precipitationlyr. As a result 14, 36, 43 and 60 per cent of the initial 137Cs contents (65 nCi 13’Cs/g dry), were removed in 15 days respectively. The corresponding efficiencies of leaching (percentage of initial ‘37Cs/ml of leachant) were l-35, 1*44,0*57 and 0.27. This result suggests an optimum rate of leaching of around 10 ml/week. At lower rates part of the leachant may run off before the leaves are re-wetted and no 13’Cs diffuses out of the dry leaves between leachings. Thus losses of Cs and presumably other easily soluble mineral elements by litter, strictly as a result of physicochemicai leaching, may differ greatly in various climates.

MICROBIAL

TURNOVER

OF MINERALS.

173

PART 1

The effects of leaf moisture on microbial immobilization by T. viride were measured using 1 g dry wt. of steam-sterilized fragments of white oak leaves which were tagged with iJ4Cs and which contained 20, 60, 100, 150 and 200 per cent moisture relative to dry wt. Loss of 134Cs upon leaching with 10 ml of water after two weeks of incubation at 26°C was around 10 % of the initial tag for all moisture levels in the sterile series. Immobilization at that time was highly significant (P
of temperature

Temperature can influence movement of minerals in an ecosystem by affecting mineral solubility and thus leachability; by affecting microbial growth and activity and thus net immobilization; and at temperature extremes such as freezing, thawing, and burning by destruction of microbial tissue which will abruptly terminate immobilization. The influence of temperature on leaching of Co and Cs was measured using various leaf species. Leaching of 6oCo from 1 g dry wt. fragmented leaves of alder and sycamore with 10 ml water at 50°C was 39 and 32 per cent of the initial content, respectively. Corresponding values with water at 2°C were 29 and 23 per cent. Loss of lJ7Cs from tulip poplar and white oak upon leaching with 10 ml of water was 85 and 28 per cent at 5O”C, and 25 and 5 per cent at 2”C, respectively. Thus, leaching of Co may increase by 40 per cent and that of Cs may increase three to sixfold by raising the temperature from 2°C to 50°C. Consequently in the field, leaching of minerals may be considerably higher on warm days than on cold days. Temperature effects on immobilization of Cs were investigated in experiments involving: (1) incubation and leaching of decaying white oak leaves at 5, 15 and 25°C (2) freezing and drying of decaying white oak leaves (3) burning of white oak and tulip poplar leaves. Immobilization of lJ4Cs by T. viride growing on fragmented white oak leaves (5 g) increased with increasing temperature (Table 4). On the other hand, the percentage of ‘j4Cs and the quite similar percentage of initial weight leached from the leaves decreased with increasing temperature. Microbial respiration increased with temperature increase at a much faster rate than did immobilization. Development of T. viride at 25°C was profuse TABLE 4. EFFECTOF TEMPERATURE ON FUNGAL IMMOBILIZATION OF 134Cs

Twelveday-old cultures of Trichoderma viride on 5 g dry wt. of steam-sterilized fragments of white oak leaves incubated at 5,15 and 25°C and leached with 25 ml of water. Parallel cultures incubated at 25°C were dried or frozen prior to leaching Sterile % 134Cs immobilized ‘A 1s4Cs in leachate 0%weight loss ml COJ12 days

;:; -

so 0.9 8.7

15” 2.2 ;:; 5.6

25” 4.2 5.4 5.1 101.8

Dried ‘;‘;I .

Frozen ‘;‘;I . 6.9 104.6

174

M. WITKAMP

but growth at 5 and 15°C lagged and CO, evolution was slow. These results seem to indicate that with increasing temperature there is increasing fungal retention of 134Cs and mass. The effects of temperatures harmful to microbial tissue and microbial immobilization of minerals were investigated together with the influences of likewise destructive moisture contents below the wilting point. Effects of temperature and moisture were combined because they are difficult to separate and act simultaneously under field conditions. In the field, desiccation at elevated temperatures rather than heat will cause microbial death. Fragmented leaves of white oak with 12-day-old growth of T. viride were subjected to freezing or drying. The results of freezing and subsequent thawing, and of dessication, on immobilization are presented in the right-hand columns of Table 4. The untreated controls for this experiment were the cultures incubated at 25°C in the previous temperature gradient experiment. The percentage of initial 134Cs leached after drying of the cultures for 16 hr in a dry air stream at 40°C was 2’4 per cent higher than in the controls and after freezing for 16 hr at - 10°C the percentage leached was 1 a8 per cent higher than in the controls. Thus, after drying of the decomposing litter more than half of the immobilized 134Cs (4.2 per cent) was leached out. After freezing and thawing, leaching removed more than one third of the immobilized lJ4Cs. Similar data were obtained with fragmented and steam-sterilized tulip poplar leaves (1 g air dry) inoculated with Peniciflium sp. and leached 11 times with 2 - 5 ml of water during 22 days of incubation at 28°C. After the 22 days 42 per cent of the initial 134Cs was immobilized. Upon air drying and freezing (2 hr at - 10°C) of the leaves, one leaching with 10 ml of water removed 16 per cent of the initial 134Cs, or more than one third of the immobilized lJ4Cs. Drying and freezing had no effect on the leaching of 134Cs from the sterile leaves. Thus freezing and drying, which are common phenomena in the surface litter in many forest areas, have a profound itiuence on the movement of Cs and presumably of other mineral elements as well. The results of the freezing and drying experiments suggest a sudden release of immobilized mineral elements upon leaching of damaged microbial tissue. The magnitude of this release relative to mineral retention by the tissue before the insult was assessed in vitro. Release of 134Cs and 6oCo upon shaking (24 hr at 2°C) of 3-day-old mycelium (Z 100 mg dry wt.) of T. viride with 100 ml of distilled water in 250 ml Erlenmeyer flasks was 10 per cent and 11 per cent of the initial content, respectively. Corresponding values for frozen mycelium (8 hr at -10°C) were 97 and 89 per cent, and for dried mycelium (8 hr at 28°C) 96 and 85 per cent, respectively. The difference between the almost complete release of immobilized 134Cs from mycelium and the only partial leaching of immobilized 134Cs from mycelium + litter after freezing and drying may be the result of incomplete leaching of the mycelium on the leaves and sorption of 134Cs onto litter after release from damaged mycelium. Thus, in the forest loss of minerals from litter by rain following dry or freezing weather will be incomplete because of resorption of minerals released from damaged microbial tissue onto the litter. The influence of fire on 134Cs movement in leaf litter was assessed by leaching ashed leaves of tulip poplar and white oak. The leaves (about 3 g dry wt.) were sprinkled with ethanol and ignited in drying dishes until they had lost about 80 per cent by weight. The ashed and charred leaves were then washed onto filter paper with 25 ml of water. The leachates of the ashes of tulip poplar and white oak contained 19 per cent and 7 per cent of the initial lj4Cs whereas 64 per cent and 87 per cent of the 134Cs remained in the ashes on the filters. Presumably the quickly burning tulip poplar material, which had lost 82 per cent of its weight, lost 17 per cent of the initial l 34Cs in smoke or by volatilization. The slower burning oak material (flame temperature 770°C) lost 79 per cent by weight and only 6 per

MICROBIAL

TURNOVER

OF MINERALS.

PART I

175

cent of the initial 134Cs into the air. Thus, more 134Cs disappeared into the air and could be leached from tulip poplar than from oak. As a result, a relatively high percentage of lJ4Cs remained on the oak ashes presumably adsorbed onto charcoal. On a weight basis the concentration of lJ4Cs was more than 4.2 times higher in burned oak leaves and 3 -6 times higher in burned tulip poplar leaves than in the original leaves. Thus, burning of litter destroyed its associated microflora and its substrate but losses of l 34Cs in leachate and into the air were considerably lower than loss of weight of the litter. DISCUSSION

The immobilization of more than half of the leachable Fe, Cs and Ca, and almost one fourth of the total Cs and Ca by the natural flora of a natural litter substrate (Table 1) indicates the large effect of microbes on mineral turnover in the field. The immobilization of fertilizer minerals, especially after organic amendments (Jansson, 1958 ; Swaby, 1962), is well known. In forest litter evidence of immobilization of Mg and P (Burges, 1956), and N (Bocock, 1964) has been demonstrated. The percentage of the initial tag that was immobilized increased with increasing leachability for the four elements tested. The effect of pH on uptake of Co, and of K on uptake of Cs however, indicate the complexity of the environmental interactions with microbial turnover of mineral elements. More research has to be done before the net immobilization of different mineral elements can be predicted. Immobilization appears to be mainly controlled by temperature as long as moisture conditions are adequate. In warm weather leaching and immobilization of Cs can be several times higher than during cold weather. Where the temperature may drop periodically below freezing, subsequent thaw and rain will remove most of the immobilized minerals from microbial tissue. Drying of surface litter on sunny spots or during dry periods followed by rain will have a similar effect. In these cases of microbial death due to the weather and even more so after tie, any subsequent leaching of mineralized elements by rain into deeper layers is reduced by sorption onto the organic substrate. Thus, both the microflora and the substrate have a buffering effect. Release of nutrient minerals from dried and rewetted litter and humus is well known (Birch, 1964) and is important to primitive agriculture. For the major biological elements N, P and S, microbial immobilization becomes of practical importance to agriculture when concentrations of their available anions in the substrate are below thresholds of roughly 2,0-2 and O-02 per cent respectively (Swaby, 1962). These values coincide with the concentrations of these elements in microbial tissue. Excess available N, P and S in the substrate will be mineralized and thus may be available to crops. Immobilization of Cs and Co, however, seems for all practical purposes unlimited and proportional to the concentrations of these elements in the substrate. Consequently, radioactive forms of Cs and Co from fallout may accumulate in microbial tissue to appreciable concentrations. Acknowledgment-This research was sponsored with the Union Carbide Corporation.

by the U.S. Atomic Energy Commission

under contract

REFERENCES BIRCH H. F. (1964) Mineralization of plant nitrogen following alternate wet and dry conditions. PI. Soil 20, 43-50. B~COCK K. K. (1964) Changes in the amount of nitrogen in decomposing leaf litter of sessile oak (Quercus petraeu). J. Ecol. 51, 555-556.

176

M. WITKAMP

BURGESA. (1956) The Release of Cations During the Decomposition of Forest Litter, Rap. VI’ Congr. Sci. du Sol, Vol. 2, pp. 741-745. COMNAY El J. (1950)Microdifliion and Volumetric Error, C. Lockwood, London. JANSSON S. L. (1958) Tracer studies on nitrogen transformations in soil with special attention to mineralisation-immobilisation relationships. K. LantbrHb’gsk. Annlr 24, 101-361. MARTINJ. P. (1950) Use of acid, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69, 213-232. SWAJSYR. J. (1962) Effects of micro-organisms on nutrient availability. In Transactions of the Joint Meeting Commissions IV and V of the International Society of Soil Science, pp. 159-172.