Physical treatment and reinoculation of soil: Effects on microorganisms and enzyme activities

Soil Biul. Biuchem. Vol. 14. pp. 57 to 61, 1982 Prmted in Great Britain. All rights reserved

Copyright

0038-07l7/82/010057u)3.00/0 0 1982 Pergamon Press Ltd

PHYSICAL TREATMENT AND REINOCULATION OF SOIL: EFFECTS ON MICROORGANISMS AND ENZYME ACTIVITIES C. M. Tu Research Centre. Agriculture Canada, University Sub Post Office, London, Ontario, Canada N6A 5B7 (Accepted 1 July 1981) Summary-Three soils, sandy loam, clay loam, and muck were subjected to different physical treatments, reinoculated with fresh soil and the effect of these treatments on the numbers of microorganisms and soil enzyme activities was studied. Soils were subjected to air-drying, freeze-drying, freezing, dry

ice-freezing, autoclaving and oven drying. The results indicated that the microbial populations increased with some of the physical treatments after 2 or 7 days incubation, while, some of the treatments decreased the populations. Air-drying the clay loam inhibited urease and phosphatase activities. None of the treatments inhibited dehydrogenase activity in either the clay loam or the muck. However, a stimulatory effect after 4 days was evident in the muck with heat treatments. Heat treatments inhibited phosphatase activity in all soils and urease activity throughout the experiment in loams, whereas after 14 days, there was a rapid recovery of urease activity in the muck soil. Autoclaving resulted in a pronounced increase in CsH2 reduction in the three types of soils. Heating appears to have an effect in an organic soil where the formation of 2,35triphenyltetrazolium formazan (formazan) and C2H, were significantly increased.

INTRODUCTION

lOO-ml serum bottles (Wheaton Glass Co., Brampton, Ontario). Samples were air-dried at room temperature at 22°C or freeze-dried by immersing the soil-serum bottles in a mixture of dry ice and acetone at -8O’C, evacuating to dryness and storing the samples at - 15°C. To determine the effect of cold temperature, soil samples were stored in a freezer at - 15’C for 3 weeks. A portion of the soil samples which had been dry ice-frozen was stored in a freezer. Some samples were heated by autoclaving at 121°C for 7 h every day for 5 days and others were oven-dried once at 105°C for 6 h. Non-treated soils were used for controls. All data are expressed on an oven-dry basis and are averages of triplicate determinations. Each soil after treatment received 17; by weight of fresh soil of the same kind, and was rewetted to 600/Aof the moisture holding capacity (MHC) with sterile distilled water. Soil microorganisms were counted by a soil-dilution plate technique, using sodium albuminate agar for bacteria and actinomycetes (Waksman and Fred, 1922) and Martin’s rose bengal streptomycin agar for fungi (Allen, 1959). Plates were incubated at 28°C. Activity of soil urease was determined using a steam distillation method (Bremner and Keeney, 1966) after 2 and 14 days at 28’C. To test the effects of physical treatments and reinoculation on phosphatase activity, 1 g soil in 20-ml serum bottles was added to p-nitrophenyl disodium orthophosphate (British Drug House, Toronto, Ontario) and hydrolysis determined after 2 h (Tabatabai and Bremner, 1969). To determine soil N,ase, 20 g soil samples were placed in lOO-ml serum bottles, stoppered with red butyl rubber stoppers and sealed with aluminum seals using a hand crimper. Samples in the serum bottles were held at 28’C for 24 h in the dark and then evacuated and flushed for 20min with a gas mixture of 0.4”,, COZ, 22% Ot and 77.6% Ar. To assay C,H,-production.

Physical treatments such as freezing, drying or heating of soil have been shown to change the environment in the soil. Freezing or drying of soils results in increased exposure of the surface. area of the soil colloids providing a greater availability of nutrients (Soulides and Allison, 1961). When soil is partially sterilized by heat, it is very favourable to colonization by microbes because nutrients are liberated and competition is temporarily limited (Waksman, 1932). Conditions during physical treatments of soil greatly affect the apparent enzymatic activities. Freezing appears to increase urease activity (Tagliabue, 1958), while invertase activity initially decreases but further change is very slight on prolonged storage (Ross, 1965). Amylase, on the other hand decreases more both initially and with length of storage (Ross, 1965). During air-drying, there may be considerable losses in enzymatic activities, but once the soil is airdry further losses usually are minimal (Skujins, 1967). Little is known of the significance of the effect of reinoculation of fresh soils into physically treated soils on number of microorganisms and enzyme activities. Results reported here provide data on tests on microbial populations and enzyme activities in soils that have been physically treated and reinoculated. MATERIALS AND METHODS

The soils used were a sandy loam, a clay loam and a muck from southwestern Ontario (Table 1). Random samples were collected in later spring to a depth of 15 cm and sieved ( c2 mm). The procedures for microbial, chemical, and physical analyses of the soils were given by Tu (1970). Physical treatments of the soils were carried out on 20-g of the samples in 57

c.

58

M. Tt

Table 1. Characterlsticr

.

of soils

Organic* matter (“,A

NO; (egg-“1

Nitrogen NOT (tigg-‘)

KJeldahi (“,,I

C:N

0.64 0.17

21.5 8.8

0.21 0.12

5.4 9.7

Sample

PH

MHC (:‘;,I

Sandy loam Clay loam

7.9 7.4

53 48

3.04 I.88

Muck

7.6

176

46.60

1.68

L52.5

1.92

14.1

* Organic matter = organic carbon x 1.724.

samples received 1 ml purified CzHz and CzH4 for- sandy loam with treatments of air-drying, freeze-drymation was determined using a CC method (Tu, ing, freezing and oven-drying. A similar effect was ob1978). The capacity of soil samples to reduce CzH2 to served after 7 days in the clay loam with dry icefreezing or autoclaving and with freeze-drying or CzH4 provided evidence for potential N2 fixation (Hardy et ul., 1973). Dehydrogenase activity was *oven-drying. Autoc~aving and oven-drying of the muck soil reduced fungai numbers. An increase on the measured by incubating the soil at 28’C with 2.35 triphenyitetrazotium chloride (TTC) (Casida et ul.. fungal numbers was observed in the sandy loam with autoclaving after 7 days. in the clay loam with air1964). drying and in the muck with freezing at 2 days, and with air-drying after 2 and 7 days. RESULTS AND DISCUSSION During the 7-day incubation, plate counts in the The effect of different treatments on populations of controls showed a decrease in the populations of microRora in the three soils was measured after 2 and fungi and bacteria (Table 2) in the soils. These de7 days (Table 2). Plate counts indicated that bacterial clines probably resulted from the fact that during a counts were not affected 2 days after the treatments in prolonged incubation aeration was inadequate. the sources of available nutrients were depleted, and the clay loam. However, after 7 days a stimulatory waste metabolites had accumulated (Eno. 1960; effect was evident with autoclaving and oven-drying. A stimulatory effect was again observed with treatStotzky et a/.. 1962). Reinoculation with fresh soil and subsequent incuments of freeze-drying, and heating of the sandy loam bation tended to equalize the microbial populations and with air-drying of the muck. At 2 days bacterial in general. but in some of the heated soil samples numbers in the muck soil were decreased by autocalving and oven-drying treatments but after 7 days with there was an increase in microbial numbers. This was more apparent for bacteria than fungi (Table 2). oven-drying populations had recovered and increased Autoclaving resulted in greater changes than ovento a level greater than those found in the control. Reinoculation with fresh soil and incubation of the drying but both initially destroyed the bacteria and remoistened soils showed that after 2 days there was a fungi (Tu. 1977). Reinoculation with fresh soil caused an increase in bacterial numbers in many samples significant decrease in fungal numbers (Table 2) in the

Table 2. Changes in populations of microorganisms

with incorporation soils after different physical treatments

Sandy loam

Clay loam

of fresh

Muck

Incubation time (days) Treatment Control Air-drying Freeze-drying Freezing Dry ice-freezing Autoclaving Oven-drying

2

7

27 SO 107” 42 45 129* 114*

41 42 143* 52 75 180* 184*

Control Air-drying Freeze-drying

2f03g‘L) 13* 9*

Freezing Dry ice-freezing Autoclaving Oven-drying

12* 16 21 8*

* Significantly

different

7 7 6 10 7 15* 5

from control

2

7

Bacteria (IO’ g- ‘I 39 17 30 16 47 22 35 14 32 14 25 35* 49* 59 Fungi (102 g- ‘) 80 62 140* 62 36* 22* 96 94 5s 12’ at the 0.05 level.

47 26* 36* 7*

2

7

80 96 60 97 96 34, 45*

100 141* 79 118 108 34+ 124*

(103g-‘) 19 13 30* 39* 12 6 31* 15 6* 6”

15 9 5’ 5*

Soil physical treatments, microbes and enzymes Table 3. Enzymatic

Treatment Sandy loam Control Air-drying Freeze-drying Freezing Dry ice-freezing Autoclaving Oven-drying Clay loam Control Air-drying Freeze-drying Freezing Dry ice-freezing Autoclaving Oven-drying Muck Control Air-drying Freeze-drying Freezing Dry ice-freezing Autoclaving Oven-drying

activities in 3 soils with incorporation treatments Urease (pegNH:-Ng-’ soil h-‘) Incubation time (days) 2 14

13.8 17.5* 15.4* 17.18 17.9* 12.5; l2.9*

15.2 18.5* 15.5 15.5 15.9 10.9* 7.0*

15.0 12.1* 15.0 15.4 15.0 12.9* 10.4* 37.1 42.1* 45.4* 37.1 36.7 18.3* l8.3*

59

of fresh soils after different physical

Phosphatase (IOOpg pnitrophenol released g -I soil 2 h-‘)

Nitrogenase @mole C,H2 CsH,, g- ’ soil) Incubation time (days) 2 7

10.0 5.5* 6.1; 9.1 8.5 2.4* 2.9*

3.2 3.6 2.7 2.6 3.1 5.2* 3.3

3.4 4.1 3.1 4.4 3.5 7.1* 4.3

3.9 2.4* 2.1* 2.9; 2.7* .1.7* 2.1;

6.9 3.6* 6.3 6.3 6.0 3.6; 3.9*

3.4 2.6 6.2* 2.4 3.0 5.2* 3.5

3.7 4.7 6.8* 2.8 3.1 7.7* 4.8

25.7 26.9* 26.4* 25.7 25.7 29.2* 29.7*

43.6 29.2* 26.6* 37.3 32.3 6.4* 8.1*

3.4 3.5 3.2 4.2 4.4 8.9: 6.3*

3.7 4.8 3.8 5.4 5.1 13.6* 7.8

* Significantly different from control at the 0.05 level. over the control in the sandy loam and with ovendrying in the clay loam and in the muck after 7 days. Goodding and McCalla (1945) have found that airdrying a soil converts the microorganisms into an inactive form such as spores. In soil, microbes are in a heterogeneous state, and the logarithmic growth phase is rare (Alexander, 1961). However. due to the physical treatments of the soils, there is a significant release of amino acids and organic matter which are susceptible to microbial utilization and decomposition (Jenkinson and Powlson, 1976; Paul and Tu, 1965). It is possible that the soil microflora were selectively decreased leaving species tolerant of the conditions produced by the physical treatments. Table 3 shows the effect of different physical treatments and inoculation of fresh soils on urease activity. Heat treatments of muck reduced urease activity at 2 days and with loams throughout the 1Cday incubation. Air-drying of the clay loam was also inhibitory for the same period of the experiment. Recovery of inhibition of urease was rapid with autoclaving and oven-drying of the muck after 14 days. Urease in the frozen samples was still inhibited after 14 days in the clay loam, while a stimulatory effect was evident in the sandy loam after 2 days. A stimulatory effect was also observed with air-drying in the sandy loam and muck and with freeze-drying in the muck after 2 and 14 days. Increase of enzymatic activity depends largely upon microbial populations, and the amount and type of organic materials (Skujins, 1967). Phosphatase in soils is largely responsible for mineralization of organic P (Cosgrove. 1967). Hy-

drolysis of an incorporated substrate, p-nitrophenyl disodium orthophosphate by phosphatase (Table 3) was depressed in all soils with treatments of air-, oven-drying and autoclaving, and with freeze-drying in the sandy loam and the muck. Acetylene reduction in the three soils following different physical treatments and the incorporation of fresh soil is shown in Table 3. The data were obtained by subtracting the CzH4 produced endogenously. With the exception of autoclaving, all treatments showed the same pattern in C2H., production in the sandy loam after 2 and 7 days. Autoclaved soils resulted in a pronounced increase in the formation of C2H4. Similarly freeze-drying and autoclaving increased C2H, formation in the clay loam. C2H4 production was again increased in the muck soil with autoclaving and oven-drying. A similar effect on Nz fixation in heated soils was demonstrated by Pfeiffer et al. (1909, 1910), and on C2H2 reduction by Tu (1977, 1978, 1979). None of the treatments inhibited dehydrogenase activity in the clay loam and muck (Fig. 1). However, after 2 weeks an increased effect was apparent in the clay loam with air-drying, autoclaving and freezing, and in the muck with autoclaving and oven-drying after 4 days, and with dry ice-freezing after 3 weeks. In sandy loam treated by air-drying, dehydrogenase activity was greater after 3 weeks. However, with autoclaving and freeze-drying, dehydrogenase was inhibited for 2 weeks and with oven-drying for 3 weeks respectively. Because of the low C and N contents in the loam (Table l), enzyme activities were also low with most treatments (Ladd, 1978). Drying and rewetting treat-

60

C. M. Tu Sandy

Clay Loam

Loam

Muck t ,\

: ’ *1 ‘\\ I ‘+

;:

--_# A

AD

Incubation

Time

( Weeks)

Fig, 1. Changes in dehydrogenase activity with incorporation of fresh soils after different physical treatments. CK = control. AD = air-drying. FD = freeze-drying, F = freezeing. DF = dry ice-freezing, A = autociaving. GD = oven-drying. *Significantly different from control at 0.05 level.

ments enhanced C and N mineralization and microbial activity in many soils. There was generally greater N release when incubation after drying was included than when omitted (Paul and Tu, 1965). Physical treatments increase the rate of mineralization of biomass C by killing and damaging organisms. Heating and drying processes appear to have an effect on soluble organic constituents. This was obvious in an organic soil where the TTC (Fig. 1) and CzH2 (Table 3) reduction were both markedly increased. The sources of TTC and CzH2 reducing substances are not clear. They could possibly have come from the organic or mineral fractions released during the very rapid heating and drying (Birch 1958. 1960: Dhar. 1959; Jenkinson and Powlson. 1976; Lyon and BizzeII, 1910, 1913; Paul and Tu, 1965: Powlson and Jenkinson, 1976: Robinson, 1920; Schreiner and Lathrop, I912; Waksman, 1932). The chemical reaction taking place through heat treatments is far in excess of that occurring during other physical treatments. Acknonleciy~meirt-The technical assistance Mr G. Heitkamp is gratefully acknowledged.

provided

by

ALLEN0. N. (1959) Experiments in Soil Bucteriology. 3rd edn. Burgess. Minneapolis. BIRCH H. $. (19.58) The effect of soil drying on humus decomposition and nitrogen availability. Plant & Soil 10. 9-31. . BIRCH H. F. (1960) Nitrification in soils after different periods of drying. Phnt & Soil 12, 81-98. BREMNERJ. M. and KEENEYD. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils. 3. Exchangeable ammonium, nitrate, and nitrite by extraction-distillation methods. Proceedings Soil Science

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