JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen Aikakauskirja Vol. 58: 9—17, 1986 Extracellular protease-producing actinomycetes and other bacteria in cultivated soil RAINA NISKANEN 1 and EVA EKLUND 2 1 Department of Agricultural Chemistry, University of Helsinki, SF-00710 HELSINKI, Finland 2 Department of Microbiology, University of Helsinki, SF-00710 HELSINKI, Finland Abstract. The occurrence and properties of extracellular protease-producing actinomycetes and other bacteria in cultivated soil were studied. Experimental soils consisted of three mineral soil samples and one Sphagnum peat sample from a greenhouse. The mineral soil samples represented arable, pasture and uncultivated soils. From experimental soils, 240bacterial strains were isolated, 68 strains thereof were proteo- lytic. A greater number ofproteolytic strains originated from pasture soil than from the other soils. Actinomycetes accounted for 70 % of the proteolytic strains isolated from pasture soil. Several proteolytic bacteria were isolated also from peat, but only few of them were typical actinomycetes. Many strains with high extracellular protease activity proved to be fermentative bacilli. Production of oxidase enzymes, significant in the humification processes, occurred fre- quently among strains isolated from pasture soil and peat. The ability to produce dark raelanoid pigments was a frequently noted characteristic of the proteolytic actinomycetes. Index words: soil proteases, soil phenoloxidases, soil oxidases, humus polymerization Introduction Transformation of organic matter in soil is partially catalyzed by enzymes found outside the living soil organisms (SkujinS 1967). Many soil microorganisms release extracellular en- zymes into the soil, and these enzymes are in- volved in the decomposition of plant, animal and microbial residues. Proteases are enzymes which hydrolyze residues containing peptide bonds. Energy and nutrients are formed in the degradation process of organic residues, and the degradation products are also involved in the formation of humic matter. Soil is a very complicated environment and enzymes acting therein are supposed to tolerate numerous environmental factors. However, it is well known that part of the enzymes remain 9 https://www.c-info.fi/en/info/?token=tbARFFeAejVOR4Xv.hzRZvvkPIpcNSbOLKMEb2Q.DA_TW6APYlI9v_wUWn8dYk9nX_ByJJW10UNxmZysNsDF_vfNTxU-obmzA1fS9Br5KZX0Pua1S3UMqQCIrbSVnFiDdnWpJN1-Vbr4rd48H00v6Tg1eDXQAuIW_ZPFgLCjpEfMwH942JfLtO-8YiauupU4fkKLFcNSzsqLBsB9Ts27t8R7tCFTzQRRgw in the soil in an active state for a certain period (SkujinS 1967). The persistence of extracellular proteases in soil is enhanced by their adsorp- tion to soil organic matter and clay particles, lonically bound proteases might represent readily mobilizable reserves, while covalently bound proteases form a more stable but im- mobilized reserve (Ladd 1972). The composition and abundance of soil microbial flora producing extracellular pro- teases are affected by environmental factors such as moisture, temperature, aeration, acid- ity and type and content of organic matter. These factors are remarkably affected by cul- tivation. In arable soil, various measures re- lated to cultivation bring about considerable fluctuations in environmental conditions. In view of soil microbes, regularly repeated ploughing is a radical action, while in pasture soil which is not tilled every year, undisturbed development of microbial flora may continue. When decomposable organic residues occur in abundance in soil, microbial flora proliferates in the presence of favourable environmental conditions. In this respect, excess acidity is a usual limiting factor. Well humified soil or- ganic matter is not primarily important as a source of energy, but it has favourable effects on the environment, e.g. increasing the water- holding capacity of the soil. The aim of this study was to investigate the occurrence and properties of extracellular protease-producing actinomycetes and other bacteria in cultivated soil. Material and methods Experimental soils. For isolation of pro- teolytic soil organisms, four soil samples were collected. Three of the samples representing arable, pasture and uncultivated soil were from the surface layer of mineral soils and one sample was a Sphagnum peat sample from a greenhouse. The pH of the soil was measured in a soil 0.01 M CaCl2 suspension (1 : 2.5) (Ryti 1965). The organic carbon content of the samples was determined using a modifi- cation (Graham 1948) of Alten’s wet com- bustion method. The samples, by increasing pH, were as follows: Sample No. Origin Soil class pH Org.C, % 1 Uncultivated soil Muddy clay 3.5 3.1 2 Arable soil Coarser finesand 5.0 3.1 3 Greenhouse peat Sphagnum peat 5.7 43.4 4 Pasture soil Coarser finesand 6.0 4.9 Isolation of soil organisms. Samples of 5 g undried soil were dispersed in 100 ml of steri- lized phosphate buffer solution (pH 7.2) using a sterile Ultra Turrax K homogenizer. Three successive hundredfold dilutions of soil sus- pensions were prepared with sterilized phos- phate buffer solution, shaken by hand, and five replicates of 1 ml and 0.1 ml of the two latter dilutions were plated in the following medium: soluble starch, Bacto peptone and yeast extract 0.5 g each, glycerol 1 ml, K 2 HP04 0.2 g, MgS04.7H 20 0.05 g, 0.01 % FeClj water solution 4 drops, agar 15 g, soil extract 250 ml, distilled water 750 ml, acti- dione 20 mg, pH 6.9. The medium was dis- tributed in 10 ml portions into test tubes and sterilized at 120°C for 20 minutes. Ten grams of milk powder was dissolved in 100 ml distilled water and sterilized at 118°C for 15 min. and supplied into the medium (10 % v/v). The plates were incubated at room tem- perature for one week and counted after two and six days. The criterion of identification of caseolytic organisms was a clear zone sur- rounding the colony. For isolation of actino- mycetes, five replicates of 1 ml of the three dilutions were plated in chitine agar (Sker- man 1969). The plates were incubated at room temperature for one week and counted after incubation. Testing of physiological characteristics. Proteolytic and chitinolytic colonies were transferred to test tubes containing 2.5 ml semisolid medium (7 g agar per 1000 ml) 10 which was prepared in the same way as the medium for isolation of soil organisms except that it contained no actidione. The cultures were incubated at room temperature until good growth was found. Thereafter the strains were transferred to liquid media: tryptone and yeast extract 0.5 g each, KH 2P04 0.4 g, MgS04 ■ 7H,0 0.05 g, NaCl 0.1 g, Fe€l3 0.01 g, soil extract 250 ml, distilled water 750 ml, pH 6.8, sterilized at 120°C for 20 min. The cultures were incubated at 28°C until good growth was found and then the strains were transferred with a multipoint inoculator to the media used for testing physiological charac- teristics. The ability to denitrify was indicated by gas bubbles in the test tubes containing the liquid medium described above supplied with 7 g agar and 1 g KNO a per 1000 ml and closed with vaspar. The utilization of glucose was tested with OF-medium (Hugh and Leifson 1953). The oxidase activity was indicated by red colour brought about by 0.5 % (w/v) water solution of dimethyl-p-phenylendiamine- hydrochloride (Klinge 1960) in liquid medium cultures containing tryptone and yeast extract 5 g each and agar 7 g per 1000 ml. The production of melanine was noted in this medium. The proteolytic properties of the strains were tested with litmus milk: 20 ml 2 % solution of litmus, 1000 ml skimmed milk, sterilized at 114°C for 15 min. All tests were checked after one week of incubation at room temperature. After five days of incubation, growth was checked in three successive pas- sages in synthetic medium with citrate as the sole carbon source (Eklund 1970). Determination ofprotease activity. Proteo- lytic strains were transferred to slants: tryp- tone 5 g, glycerol 1 ml, yeast extract 0.5 g, KH 2 P04 0.2 g, MgS0 4.7H 20 0.05 g, FeCl 3 0.01 % water solution 4 drops, agar 15 g, distilled water 1000 ml, pH 6.9, sterilized at 121°C for 15 min. The ability of strains to uti- lize casein was retested, and the strains showing good proteolysis were chosen for pro- tease production. For cultivation of inoculum, strains were transferred to test tubes contain- ing 2 ml of yeast extract-malt extract liquid medium (Pridham et al. 1956—1957, ref. Shirling and Gottlieb 1966) and incubated for two days. Inocula were transferred to 250 ml conical flasks containing a medium pre- sented by Eklund et al. (1971) and shaken at 28°C (200 rpm). Samples for the determina- tion of protease activity were taken after 3, 4 and 5 days of incubation. Protease activity was determined according to Eklund et ai. (1971). The growth solutions were allowed to hydrolyze casein at 40°C and pH 8.0. The reaction time was 20 min for the samples incubated for three days and 10min for the samples incubated for four or five days. The reaction was stopped by precipi- tating the unhydrolyzed casein with 5 % tri- chloroacetic acid. The content of tyrosine produced in casein hydrolysis was determined in the filtrates by measuring the absorbances of the assayed solutions and blanks at the wavelenght 280 nm, using a Beckman DB spectrophotometer. One enzyme unit (EU) is the amount of enzyme which releases hydro- lysis products at a rate equivalent to 1 meq of tyrosine per minute under the conditions spe- cified (Jönsson 1969). Results The number of soil organisms grown on soil extract-milk agar plates are given in Table 1. Table 1. Number of proteolytic and other soil orga- nisms. Soil sample Organism Number of organisms (g -1 of soil)* 106 No. Origin After After 2 days of 6 days of incubation incubation 1 Uncultivated Proteolytic 0.48 soil Total 0.56 2 Arable soil Proteolytic 0.16 Total 0.88 3 Greenhouse Proteolytic 3.76 30.0 peat Total 7.76 115.5 4 Pasture soil Proteolytic 1.44 48.0 Total 9.24 143.6 11 After two days of incubation, growth was found only among the organisms isolated from greenhouse peat and pasture soil. Near- ly 50 °7o of the colonies derived from peat and 16 % of those derived from pasture soil were proteolytic. After six days of incubation, the percentages of proteolytic colonies isolated from these soil samples were 26 and 33, re- spectively. After six days of incubation, growth was found also on plates representing other soil samples, but the numbers of colo- nies were much smaller. The percentages of proteolytic colonies isolated from uncultivated and arable soil samples were 86 and 18, re- spectively. After seven days of incubation, growth was found on all chitine agar plates (Table 2). The Table 2. Number of actinomycetes and other soil orga- nisms counted on chitine agar plates. Soil sample No. Origin Organism Number of organisms (g-1 soil) x 106 after 7 days of incubation 1 Uncultivated Typical soil actinomycetessoil actinomycetes 0.035 Total 0.055 2 Arable soil Typical actinomycetes 0.54 3 Peat Typical actinomycetes Other* 3.50 4 Pasture soil Typical actinomycetes 13 * mainly nocardioforms and mycobacteria colonies grown on plates representing peat and pasture soil were more numerous than those representing other soil samples. All the colo- nies isolated from arable and pasture soil samples and 64 % of those from the uncul- tivated soil sample were actinomycetes, while typical actinomycete colonies were not found on plates representing peat. However, the bac- teria grown on these plates included Nocar- dia and Mycobacterium strains. The number of bacterial strains isolated from soil extract-milk agar plates was 124. The majority of the strains originated from peat and pasture soil samples (Table 3). Acti- nomycetes were isolated mainly from pasture soil. On the basis of physiological tests, pro- teolytic strains were isolated from all soils, but oxidase-producing and denitrifying strains only from peat and pasture soil (Table 3). The ability to produce melanine was associated exclusively with strains originating from pas- ture soil. The glucose utilization was mainly fermentative among strains originating from uncultivated and arable soil samples, but mainly oxidative among strains originating from peat. Strains originating from peat ex- hibited the poorest ability to grow in synthetic medium. The number of strains isolated from chitine agar was 116. The proportion of actinomycetes was greatest among strains originating from pasture soil and lowest among those originat- ing from peat (Table 4). Proteolytic and oxidase-producing strains originated more fre- Table 3. Properties of all strains isolated from soil extract-milk agar plates. Soil sample 12 3 4 Uncultivated Arable Peat Pasture Number of strains 6 6 46 66 % of isolated strains Typical actinomycetes 26 Proteolytic 33 67 15 23 Oxidase-producing 72 56 Melanine-producing 12 Fermentative glucose utilization 66 67 5 26 Oxidative » » 17 33 72 32 Growth in synthetic medium 67 100 40 58 Denitrifying 5 6 12 13 quently from peat and pasture soil than from the other soil samples. Melanine-producing strains were isolated from all soil samples. The majority of strains were able to grow without organic nitrogen, while the proportion of denitrifying strains was low. Oxidative utili- zation of glucose was more common than fer- mentative utilization. The number of proteolytic strains was 68, 30 of which were isolated from soil extract- milk agar plates (Table 5) and 38 from chitine agar plates (Table 6). The majority of proteo- lytic strains originated from pasture soil, ac- tinomycetes accounting for 70 %. The protease activity of 30 strains showing good proteolysis was determined (Fig. 1). Of Table 4. Properties of strains isolated from chitine agar. Soil sample 12 3 4 Uncultivated Arable Peat Pasture Number of strains 45 16 13 42 % of isolated strains Typical actinomycetes 47 50 23 74 Proteolytic 13 19 54 50 Oxidase-producing 9 19 46 33 Melanine-producing 13 31 8 30 Fermentative glucose utilization 64 6 8 26 Oxidative » » 7 75 92 41 Growth in synthetic medium 76 63 85 67 Denitrifying 7 6 8 Table 5. Properties of proteolytic strains isolated from soil extract-milk agar plates. Soil sample 12 3 4 Uncultivated Arable Peat Pasture Number of strains 2 4 8 16 % of isolated strains Typical actinomycetes 63 Oxidase-producing 75 25 Melanine-producing 31 Fermentative glucose utilization 100 75 25 31 Oxidative » » 25 63 50 Growth in synthetic medium 100 100 50 88 Denitrifying 6 Table 6. Properties of proteolytic strains isolated from chitine agar. Soil sample 12 3 4 Uncultivated Arable Peat Pasture Number of strains 7 3 7 21 % of isolated strains Typical actinomycetes 43 14 76 Oxidase-producing 33 43 24 Melanine-producing 43 48 Fermentative glucose utilization 43 14 14 Oxidative » » 29 100 86 67 Growth in synthetic medium 57 100 86 81 Denitrifying 14 33 14 14 Fig. I. Protease activity of actinomycetes (a) and other bacteria in uncultivated soil (b), arable soil (c), greenhouse peat (d) and pasture soil (e). 15 these strains, 11 were actinomycetes originat- ing chiefly from pasture soil (Fig. la). Many actinomycetes strains showed moderate pro- tease activity. The bacterial strains from un- cultivated soil showing high protease activity were all Bacillus cereus strains (Fig. lb). Pro- tease activity of strains originating from arable soil was relatively low (Fig. 1c). Two of these strains were bacilli (strain 8 Bacillus cereus var. mycoides, strain 11 Bacillus cereus) (Fig. 1c). Strains from peat represented both low and high protease activity (Fig. Id). Three strains showed high protease activity: strain 123 was Bacillus subtilis, 190 and 196, not being identified, produced melanine and their utilization of glucose was oxidative. The four bacterial strains from pasture soil showing high protease activity were Bacillus coagulans strains, except strain 92 (Fig. le). Figure 1 shows that protease activity tended to be high- est on the fourth day of protease production. Discussion In this study, pasture soil was superior to arable soil in terms of number of proteolytic bacteria, especially actinomycetes. This is in accordance with observations that protease ac- tivity is higher in pasture soil than in arable soil (Ladd 1972) and availability of organic nitrogen is better in pasture soil than in tilled soil (Huntjens 1972).Because the proteolytic activity is concentrated in the surface layer of soil (Hoffmann and Teicher 1957), the dis- turbing effect of tillage on microbial flora may arise from the fact that surface soil is mixed into deeper layers. Increased proportions of actinomycetes in the microbial flora of grass- land soil, as compared to that of tilled soil, have been reported also by Wieringa (1958) and Woldendorp (1963). Furthermore, ac- cording to Speir et al. (1982), the number of actinomycetes and other bacteria increases with pasture age. Soil pH is an important factor in the main- tenance of soil microbial flora. Generally, casein hydrolysing activity is low in acid soils (Ambroz 1965). In this study, the number of proteolytic bacterial strains was greater in peat and pasture soil than in the other soils with lower pH. The abundance ofproteolytic actinomycetes in pasture soil may partly be explained by the pH level of the soil (6.0) which was higher than the pH of the other soils examined. In greenhouse peat, however, typical actinomy- cetes were uncommon despite its high pH lev- el (5.7). The low pH (3.5) of uncultivated mud- dy clay soil might explain the minute number of proteolytic actinomycetes and other bacte- ria as well. However, actinomycetes in general are known as active degraders of microbial biomass. Therefore, they may also reflect an rich microbial flora in pasture soil, as com- pared to the other soils investigated. The richer proteolytic flora in peat and pas- ture soil, as compared to that of the other ex- perimental soils, may also be related to the higher content of organic matter. This is sup- ported by the observations of Hoffmann and Teicher (1975). They reported fivefold pro- teolytic activity in a peat soil (pHKCI 6.0) as compared to that in a sand soil (pH KCI 5.8). According to them, proteolytic activity in three sand soils with nearly the same pH (6.7 —6.9) increased with increasing content of organic matter. In this study, many strains with high pro- tease activity were bacilli. The ability to utilize both inorganic and organic nitrogen might en- hance the maintenance of bacilli in different soils (Mishustin and Mirsoeva 1968). The ability to form endospores may enable bacilli to survive in an inactive state in acid soils (Goodfellow et al. 1968, Holding et al. 1965). Oxidase enzymes, especially peroxidases and phenoloxidases, play an important role in humification processes (SkujinS 1967). Oxidase-producing bacteria have also been found in fertile soil (Sundman 1970). Various organisms, e.g. common rhizosphere orga- nisms like pseudomonads (Eklund 1970), are included in oxidase producers. In this study, several strains isolated from peat and pasture soil were capable of producing oxidase en- zymes. Production of dark melanoid pigments re- sembling humic polymers is often found in cultures of fungi, actinomycetes and other bacteria (Von Plotho 1950, Kuster 1952, 1955, Martin and Haider 1969, 1971, Ek- Lund 1970, Huntjens 1972). In this study, the production of melanoid pigments seemed to be associated chiefly with the strains isolated from pasture soil. Especially actinomycetes, which accounted for 70 % of the proteolytic strains isolated from pasture soil, were mela- noid producers. This is in accordance with the observation of Flaio and Kutzner (1960) that actinomycetes which produce melanoid pigments are more numerous in grassland soil than in tilled soil of the same type. In soil, the phenolic compounds occur to- gether with other oxidable compounds like proteins and carbohydrates, all involved in References Ambroz, Z. 1965. O proteolyticken komplexy slepicim bilkoviny piide. Rostlinnä Vyroba 2: 161—170. Zusam- menfassung; Über den proteolytischen, die Eiweisstoffe im Boden spaltendenKomplex (ref. Skujins, J.J. 1967). Eklund, E. 1970. Secondary effects of some pseudo- monads in the rhizoplane of peat grown cucumber plants. Acta Agr. Scand. Suppl. 17: 1—57. —, Backman, T. & Gyllenbero, H.G. 1971. Extracel- lular proteases from soil actinomycetes I. Comparison with commercial Pronase B. Zbl. Bakt, Abt. 11, 126: 725—734. Flaio, W. & Kutzner, H.J. 1960. Beitrag zur Ökologie der Gattung Slreplomyces Waksman et Henrici. Arch, Mikrobiol. 35: 207—228. Goodfellow, M., Hill, I.R. & Gray, T.G.R. 1968. Bac- teria in pine forest soil. The ecology of soil bacteria (eds. Gray, T.R.G. & Parkinson, D.), p. 500—515. Liver- pool. Graham, E.R. 1948. Determination of soil organic matter by means of a photoelectric colorimeter. Soil Sci. 65: 181—183. Haider, K., Frederick, L.R. & Flaio, W. 1965. Reac- tions between amino acid compounds and phenols dur- ing oxidation. Plant and Soil 22: 49—64. Hoffmann, G. & Teicher, K. 1957. Das Enzymsystem unserer Kulturböden VII. Proteasen 11. Z. Pflanzener- nähr. Diing. Bodenk. 77: 243—251. metabolic processes including formation of humic material. In the formation of humic material, metabolism of proteins and phenolic compounds are interrelated in such a way that amino acids released by the action of extra- cellular proteases react with phenols in the presence of phenoloxidases (e.g. Haider et al. 1965). Phenoloxidases also catalyze poly- merization reactions of phenolic and humic compounds. Presence of proteolytic actinomycetes in abundance in pasture soil may also be an in- dication of rich microbial biomass in the de- gradation of which actinomycetes hold a key position (Webley and Jones 1971). The re- sults confirm from a certain microbial point of view the favourable influence of pastures (and leys in general) on the microbial activity in soil closely associated with mobilization of nitrogenous compounds and a restoring effect on the stable organic matter in soil. Holding, A.J., Franklin, D.A. & Watlino, R. 1965. The microflora of peatpodzol transitions. J. Soil Sci. 16: 44—59. Hugh, R. & Leifson, E. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbo- hydrates by various Gram negative bacteria. J. Bact. 66: 24—26. Huntjens, J.L.M. 1972. Immobilization and mineraliza- tion of nitrogen in pasture soil, Diss. Wageningen. Jönsson, A.G. 1969, Proteases from fungi of the genera Ademaria and Entomophora. Diss. Uppsala. Klinge, K. 1960, Differential techniques and methods of isolation of Pseudomonas. J. Appi. Bact. 23: 442 462. Kuster, E. 1952. Umwandlung von Mikroorganismen- Farbstoffen in Humusstoffe. Z. Pflanzenernähr. Diing. Bodenk. 57: 51—57. 1955. Humusbildung und Phenoloxydasen bei Slrep- tomyceten. Z. Pflanzenernähr. Diing. Bodenk. 69: 137—142. Ladd, J.N. 1972. Properties of proteolytic enzymes extracted from soil. Soil Biol. Biochem. 4: 227—237. Martin, J.P. & Haider, K. 1969. Phenolic polymers of Slachybolrys alra, Stachybotrys chartarum and Epicoc- cum nigrum in relation to humic acid formation. Soil Sci. 107: 260—270. & Haider, K. 1971. Microbial activity in relation to 16 2 soil humus formation. Soil Sci. Ill: 54—63. —, Richards, S.J. & Haider, K. 1967. Properties and decomposition and binding action of »humic acid» syn- thetisized by Epicoccum nigrum. Proc. Soil Sci. Soc. Am. 31: 657—662. Mishustin, E.N. & Mirsoeva, V.A. 1968. Sporeforming bacteria in the soils of the USSR. The ecology of soil bacteria (eds. Gray, T.R.O. & Parkinson D.), p. 458— 473. Liverpool. Plotho, O. Von 1950. Die Humusbildung der Mikro- organismen. Z. Pflanzenernähr. Diing. Bodenk. 51: 212—224. Ryti, R. 1965. On the determination of soil pH. J. Scient. Agric. Soc. Finl. 37: 51—60. Skirling, E.B. & Gottlieb, D. 1966. Methods for char- acterization of Streptomyces species. Int. J. Syst. Bact. 16: 313—340. Skerman, V.B.D. 1969. Abstracts of microbiological methods. 883 p. New York. SkujinS, J.J. 1967. Enzymes in soil. Soil Biochemistry 1 (eds, McLaren, A.D. & Peterson, G.H.), p. 371—414. SELOSTUS Ekstrasellulaarisia proteaaseja tuottavat sädesienet ja muut bakteerit viljelysmaassa Raina Niskanen 1 ja Eva Eklund 2 1 Maanviljelyskemian laitos, Helsingin yliopisto, 00710 Helsinki 71 2 Mikrobiologian laitos, Helsingin yliopisto, 00710 Helsinki 71 Ekstrasellulaariset proteaasit ovat entsyymejä, jotka maassa osallistuvat typellisten orgaanisten jätteiden ha- jotukseen. Proteaaseja tuottavan mikrobiston koostumus ja runsaus riippuu ympäristötekijöistä kuten kosteudes- ta, lämpötilasta, ilmavuudesta, happamuudesta ja orgaa- nisen aineksen määrästä ja laadusta. Viljelytoimenpitei- den kuten muokkauksen avulla näihin ympäristötekijöi- hin voidaan vaikuttaa. Tarkoituksena oli tutkia proteo- lyyttisten bakteerien määrää, laatua ja fysiologisia omi- naisuuksia laidunmaassa ja säännöllisesti muokatussa pel- tomaassa. Lisäksi koemaina olivat orgaanista maata edus- tava kasvuturve ja viljelemätön hapan liejusavi. Koemaista New York. Speir, T.W., Ross, D.J., Orchard, V.A., Cairns, A. & Pansier, E.A. 1982. Biochemical and microbiological properties of a West Coast wet land soil at different stages of pasture development. New Zealand J. Sci. 25: 351—359. Sundman, V. 1970. Four bacterial soil populations char- acterized and compared by a factor analytical method. Can. J. Microbiol. 16: 455—464. Webley, D.M. & Jones, D. 1971. Biological transforma- tion of microbial residues in soil. Soil Biochemistry 2 (eds. McLaren, A.D. & Skujins, J.), p. 446—485. New York. Wierinoa, K.T. 1958. The problems of standardization of methods in use in microbiological soil research. Neth. J. Agr. Sci. 6: 61—67. Woldendorp, J.W. 1963. The influence of living plants on denitrification. Diss. 100 p. Wageningen. Ms received November 6, 1985 eristetyistä 240 bakteerikannasta 68 oli proteolyyttistä. Eniten proteolyyttisiä kantoja eristettiin laidunmaasta, jossa näistä kannoista 70 % oli sädesieniä. Myös turpeesta eristettiin runsaasti proteolyyttisiä bakteerikantoja, joi- den joukossa ei kuitenkaan juuri ollut tyypillisiä sädesie- niä. Useat kannat, joiden proteaasiaktiivisuus oli voima- kas, osoittautuivat fermentatiivisiksi bacilluksiksi. Lai- tumen ja turpeen bakteerikannoille oli tavallista kyky muodostaa oksidaasientsyymejä, joilla on merkitystä hu- mifioitumisprosesseissa. Tutkituille proteolyyttisille säde- sienille oli tyypillistä kyky tuottaa tummia melanoidipig- menttejä. 17