THE MICROBIOLOGY OF ACID SOILS

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1 [ 172 ] THE MICROBIOLOGY OF ACID SOILS II. RINGINGLOW BOG, NEAR SHEFFIELD BY J. G. BOSWELL AND J. SHELDON The University, Sheffield {Received 4 October 1950) (With I figure in the text) The surface vegetation of the Ringinglow Bog was described by Conway (1949). The peat samples described in this paper were taken from an area dominated by Eriophorum vaginatum. The samples were removed from the bare surface between the tussocks, each extending to the depth of about 5 cm. and containing the remains of Eriophorum. The samples were well mixed, the excess water was allowed to drain off and any roots were removed before analysis. Care was taken to avoid contamination with 'foreign' organisms of those samples examined microbiologically. CHEMICAL ANALYSES Table i gives the analysis of one sample which may be regarded as representative of the peat examined. Other samples were analysed on a slightly different basis. For example, one with dry weight and ash content of 11-2 and 0-9% of the fresh weight, respectively, gave values comparable to those in Table i for the fractions soluble in benzene, alcohol and water. The residue from these extractions was suspended in 50 ml. distilled water, treated with 0-50 ml. 2N-NaOH, raising the ph from 3-41 to 8-9, and was allowed to stand overnight. Part of the material dissolved, equal in amount to 7-4% of the dry weight of the original peat, and the residue contained a fraction insoluble in 72% H2SO4 and equal to 52-8 % of the dry weight of the original peat. Comparison of this value with the values recorded in Table i shows that much of this lignin-like fraction is soluble in o-25n-naoh. Of the fraction soluble at ph 8-9 about 80% was precipitated by the addition of acetic acid. After the removal of the acetic acid and sodium acetate by dialysis, the precipitate was suspended in distilled water and titrated with 2N-NaOH. Table i Dry weight Ash Soluble in benzene Soluble in 95 % alcohol Soluble in hot water Soluble in O'25N-NaOH i6'4 16'6% of fresh weight i-o8-i-46% of fresh weight i'6o% of dry weight 9-10% of dry weight S'SS o of dry weight 47'58% of dry weight A number of titration curves are given in Fig. i. The titration curve for the peat indicates the presence of a number of weak acids. The curves obtained for the extracted peat show that the weak acids are progressively removed until, after extraction at ph IO"3 with NaOH, the residue is almost wholly free from acids with pk values in excess of 7.

2 The microbiology of acid soils 173 In Table 2 there are set out the acidities of the different fractions of the peat. In calculating these values from the curves in Fig. i it was difficult to determine the point at which some of the weak acids were completely titrated and the curves had become the titration curve of sodium hydroxide. To obtain a value for the end-point of the acidtitration curves the following method was used: the ratio, change of ph value/volume of 2N-NaOH added (ph/ml.) was calculated from the titration curves, including one for N-NaOH (ml.) O8 09 Fig. I. Titration curves of peat and some residues after extraction. 5 g. peat or the residue thereof suspended in 50 ml. distilled water and titrated with 2N-NaOH using a glass electrode. A, residue after extraction with benzene, alcohol, water and NaOH to ph 103; B, as A, but final ph 89; C, peat. Table 2 Peat Residue from peat after extraction with benzene, alcohol, hot water and NaOH at ph 89 Residue from peat extracted as above but with NaOH at ph 103 Acidity, m-equiv./ioo g. fresh weight of initial peat O Acidity, m-equiv./ioo g. dry weight of initial peat sodium hydroxide, for values from ph 8 upwards. The end-point of each acid-titration curve was taken as the ph value at which the ph/ml. ratios for the acid and the NaOH curves first became equal. The values were determined graphically. Attempts were made to titrate the residues from the benzene and alcohol extractions but without much success. The residues were extremely hard when air-dried and even after soaking in water overnight failed to form satisfactory suspensions. On the addition of 0-03 ml. 2N-NaOH the initial ph of the suspension, 4-5, was raised to above 10 and drifted slowly downwards during

3 J- ^- BOSWELL AND J. SHELDON many hours to 5 08, a further addition of 0-05 ml. raised the ph to 8, again followed by a steady downward drift. Three days later the addition of 0-4 ml. raised the ph to a stable value of 97 and thereafter the titration was completed in the usual way. The air-dried residues after extraction with benzene, alcohol and water were also very hard and failed to form satisfactory suspensions in water; they behaved during titration in much the same manner as those described above. It would appear that the colloidal organic matter in the peat has, during the benzene and alcohol extractions, undergone a chemical change in which the acidic groups become masked. This would occur if lactone rings were formed during the extractions. These would be opened slowly by the action of dilute alkah in the cold with the restoration of the acidic properties. MICROBIOLOGY An attempt was made to estimate the bacterial population by plating out diluted samples of peat washings on selected synthetic media following the technique used by Boswell & Gover (1946). It was clear from an examination of the colonies which developed on the agar plates that some of the individual colonies were not the product of single cells. Examination of slides prepared from peat extracts showed that the bacteria were clumped in small masses of mucilage and on numerous fragments of plant remains. It was decided, therefore, to estimate the number of bacteria by the use of direct counting methods. Two methods were used, the 'indigotin technique' of Thornton & Gray (1934) and the 'agar film method' of Jones & Mollison (1948). The methods proved difficult to handle since the bacteria were very small, and almost coccoidal, and stained very feebly. In both methods control slides were prepared using distilled water in the place of the soil extract in order to make allowance for the bacteria-like particles in the indigotin suspension and in the agar. The results are set out in Table 3. No very great significance is attached to the exact values secured, since the variation from slide to slide was considerable and the labour and strain of counting make it impossible to obtain a large enough number of results for a useful statistical analysis to be made. Table 3 Date of sample 22 Aug. 3 Sept, 29 Sept, 10 Oct, Indigotin technique Dilution I 1-58 X lo' 1-19 X X X 10 Dilution 1x5 Agar film method Dilution i Dilution 1x5 Number of bacteria/g, peat, fresh weight 1-19 X X X X io» 4-21 X x X io» 2-60 X 10' 6-35 X X X 10" 6-55x10 Microscopic examination of the peat revealed fungal spores but little fungal hyphae. While it is not possible to estimate fungal activity in the soil by plate counts of colonies growing on synthetic media, it is possible to identify the genera which grow on the media used, and to estimate roughly the proportions of their reproductive cells. When peat samples were shaken with distilled water and the washings, after suitable dilution, had

The microbiology of acid soils 175 been plated out on malt agar, a number of different genera appeared. Cephalosporium spp. occurred on every plate and were the dominant fungi in every sample.

4 The microbiology of acid soils 175 been plated out on malt agar, a number of different genera appeared. Cephalosporium spp. occurred on every plate and were the dominant fungi in every sample. No attempt was made to distinguish between the species of Hyalopus and of Cephalosporium. Pullularia pullulans and Cladosporium herbarum were also present in every sample, yielding about equal numbers of colonies, but only a small fraction of the number of those of Cephalosporium. In view of the dominance of this genus estimates were made, using plate counts, of the number of colonies which could be grown from known amounts of peat at intervals during 4 months. The plate counts probably give some measure of the production of spores, assuming that the vegetative form of the fungus in the soil is filamentous. Table 4 sets out some of the results obtained. The results reveal clearly a relationship between the number of colonies which can be grown from a unit weight of the peat and the moisture content of the bog. Table 4 Date 9 July 26 July 12 Aug. 29 Sept. 29 Nov. Weather notes { Dry, the surface layers of the j bog dried steadily V Fall of rain a few days previously Very wet month A second sample was air-dried for 14 days No. of colonies/g. peat ( X 10') 4-7S In addition to the genera mentioned above, species of Trichoderma, Aspergiltus, Botrytis, Penicillium, Gliomastix and Saccharomyces occurred occasionally. It was of interest to note that the last-named genus did not occupy the dominant position reported for the fungus in a 'mor' soil bearing Deschampsia flexuosa (Boswell & Gover, 1946). Extracts of the peat were used to test the activity of the microbial population in respect of cellulose, protein and sucrose decomposition. CELLULOSE DECOMPOSITION Three groups of cultures containing cellulose were set up, one with strips of cellulose partially immersed in a mineral salt solution, a second with the cellulose completely immersed and under anaerobic conditions and tbe third using sheets of cellulose moistened with the salt solution. In all cases the added nitrogen was in the form of nitrate. The anaerobic cultures gave no obvious signs of cellulose decomposition, but there was a slight browning of the paper at the surface of the medium. The moistened sheets of paper were the more successful aerobic culture media and colonies of Cephalosporium spp., Penicillium spp., Aspergillus sp. and Trichoderma sp. were isolated after i week. Of these fungi Cephalosporium was by far the most active. When grown on partially immersed strips of filter-paper active growth appeared just above the surface of the liquid, and within 10 days a translucent zone of decomposition was clearly marked. About one-half of the cellulose was decomposed in 21 days at room temperature; thereafter decomposition was slow and little more was decomposed in twice the time. The cellulose was estimated as that fraction of the solid substrate which was insoluble in cold i % NaOH. When the fungus was

5 176 J. G. BOSWELL AND J. SHELDON grown on cellulose dextrin agar clear zones developed round the colonies in 7 days. There was no evidence that the enzymes involved operated at any great distance from the mycelium. The fungus did not attack the cellulose under anaerobic conditions. As the aerobic decomposition of the cellulose proceeds the ph of the medium rises steadily. This is unlike other fungal decompositions of cellulose in which considerable amounts of acid are produced; Merulius lacrymans, for example, produces considerable amounts of acetic acid. The decrease in the acidity of the medium suggests either the reduction of the nitrate to ammonia or the preferential utilization by the fungus of the acidic radicals such as phosphate and nitrate. The Nessler reagent gave only the slightest positive reaction for ammonia, and thereby ruled out ammonia formation as the cause of the increased ph value. Further, the substitution in the medium of ammonium sulphate for sodium nitrate inhibited cellulose decomposition. Reduction of the ph value of the medium as far as 3-5 did not greatly affect the rate of cellulose decomposition. NITROGEN METABOLISM The examination of protein decomposition was carried out by inoculating known amounts of a protein-peptone medium with equal amounts of peat washings and incubating under both aerobic and anaerobic conditions. Active decomposition set in rapidly, and after 7 days the contents of the tubes were titrated to estimate both the free and the amino acidities. The increase in the content of free acid was small, rarely reaching o-i m-equiv./io ml. medium, but the increase in the amino-acid content was between i and 2 m-equiv./io ml. medium. Under anaerobic conditions the values for the free acidity were slightly higher and those for the amino acidity slightly lower than under aerobic conditions. Microscopic examination of the microbial populations of the cultures showed that they were wholly bacterial. Nevertheless, experiments with pure cultures of species of Cephalosporitim, Pullularia and Cladosporium showed that these fungi would all grow on a protein medium. Titration of the medium showed that in all cases the ph rose towards 8-5 and that the increase in the amino-acid content was very small, less than 0-2 m-equiv./io ml. medium. It would appear that the fungi hydrolyse t^he medium and absorb the products at about the same rates, and that the partial oxidation products of the amino-acids are not liberated into the medium, a form of metabolic activity quite different from that of the bacteria described above. The nitrogen relationships of the peat were examined with regard to possible nitrogen fixation and to the activity of nitrifying bacteria. The inoculation of suitable media with particles of peat failed to produce any fermentation which might be ascribed to the activity of a Clostridium sp. or any ammonia or nitrite transformations showing the activity of nitrifying bacteria. SUCROSE FERMENTATION Inoculation of a i % sucrose solution in Pasteur tubes with a little peat suspension produced fermentation in a few days. The products were acid but included no gas. A certain amount of ethyl alcohol was also formed; plating out of the fermenting medium revealed a high percentage of yeasts. Using pure cultures of Pullularia pullulans, Cephalosporium spp. and Cladosporium herbarum it was found that they fermented sucrose actively with the production of acid but no gas.

$Gover (1946). Extraction with a range of solvents shows that the organic matter in both these 'soils' fractionates to the same pattern.$

6 The microbiology of acid soils 177 DISCUSSION It is proposed to compare the peat formed by the partial decomposition of Eriophorum vaginatum with the raw humus ('mor' soil) described by Boswell & Gover (1946). Extraction with a range of solvents shows that the organic matter in both these 'soils' fractionates to the same pattern. However, the titratable acidities differ to a substantial extent. While the ph value of the 'mor' at 3-28 is less than that for the Eriophorum peat ^t 375! the titratable acidity of the former, calculated either on the basis of dry weight or of organic matter content, is smaller than that of the latter. The distribution of the acidity among the fractions also shows considerable diflferences. In the 'mor' the bulk of the titratable acidity is located in the ligno-cellulose complex remaining after extraction with o-25n-naoh, while in the peat from E. vaginatum only 25 % of the total acidity is located in this fraction, in spite of the fact that the sodium hydroxide extraction was much milder, the concentration being approximately o-o22n-naoh. Examination of the results obtained by alkaline extractions of the Eriophorum peat with different concentrations of sodium hydroxide, both in terms of the amount of material extracted and of the form of the titration curves of the residues, indicates the heterogeneous character and the weak acidities of the alkaline soluble fractions. Further, it is clear that water-soluble sulphur acids, either from industrial contamination or from the activity of sulphur oxidizing bacteria, are not major contributors to the total titratable acidity. The microbiological examination of the Eriophorum peat showed that the bacterial population by a direct counting technique was between 10^ and io^ /g- f''^sh weight, this value being of the magnitude found for other acid soils. The outstanding feature of the examination of the fungi was the ubiquitous appearance of species of three genera, Cephalosporium, Pullularia and Cladosporium, and the dominant position of the first as determined by plate counts. Six other genera have been identified as present on a limited number of the plates and it is of interest to note that no member of the Mucorales appeared. The limitations of the isolation technique used are clearly recognized, and further studies using other methods are essential before it can be established that the fungal population in this peat is limited to the genera mentioned. It must of course be recognized that this 'soil' is waterlogged for many months of the year, and during the winter is actually submerged under a shallow layer of water and that the microbiological population is probably specialized. The work of Conway (1949) has shown the very slow rate at which the plant remains in the bog are decomposed. The observations which have been made on the decomposition of cellulose by the organisms present at or near the bog surface have established the important position of Cephalosporium spp. in this degradation. Since metabolic studies show that this fungus only operates under aerobic conditions and in the presence of nitrate, it is clear that in a 'soil' which does not give a positive reaction for nitrate and which owing to waterlogging must be little aerated, it is unlikely that cellulose decomposition will proceed actively. Such decomposition which occurs in the lower layers must be through the activity of anaerobic organisms. The nitrogen relations of the peat were examined in detail. It was shown that protein decomposition could be carried on both by bacteria and by fungi. The former broke down the protein very rapidly, amino-acids and organic acids accumulating in the culture medium, while in fungal breakdown the products did not accumulate. As was to be

7 178 J. G. BoSWELL AND J. SHELDON expected from the absence of nitrate, tests for the presence of nitrifying bacteria were always negative. Nor was it possible to isolate a Clostridium capable of growth in a medium deficient in added nitrogen, although this has been achieved with many other acid soils rich in organic matter, including the 'mor' soil. Indeed no micro-organisms multiplied when this medium was inoculated with particles of the Eriophorum peat. SUMMARY 1. A peat formed by the partial decomposition of Eriophorum vaginatum, which dominates the existing surface vegetation, was examined both chemically and microbiologically. 2. Certain observations were made on the total acidity of the peat and on the distribution of the acidity among the fractions of the organic matter. 3. The microbiological survey revealed the presence of a large number of bacteria. The number of genera of fungi was limited, species of Cephalosporium, Pullularia and Cladosporium being dominant. The first of these genera was active in cellulose decomposition, while protein decomposition was effected by both bacteria and fungi. 4. The nitrifying bacteria and those capable of fixing gaseous nitrogen were absent. REFERENCES BoswELL, J. G. & GovER, D. J. (1946). The microbiology of acid soils. I. Neiv Phytol. 45, 218. CONWAY, V. M. (1949). Ringinglow Bog, near Sheffield. II. J. Fcol. 37, 148. JONES, P. C. T. & MOLLISON, J. E. (1948). A technique for the quantitative estimation of soil microorganisms, y. gen. Microbiol. 2, 54. THORNTON, H. G. & GRAY, P. H. H. (1934). The numbers of bacterial cells in field soils, as estimated by the ratio method. Proc. Roy. Soc. B, 115, 522.

THE MICROBIOLOGY OF ACID SOILS I. 'MOR' BEARING PINUS SYLVESTRIS AND BETULA PUBESCENS

THE MICROBIOLOGY OF ACID SOILS I. 'MOR' BEARING PINUS SYLVESTRIS AND BETULA PUBESCENS BY J. G. BOSWELL AND D. J. GOVER The University, Sheffield This is the first of a series of publications on the chemical