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

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1 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 constitution and microbiology of acid soils in the neighbourhood of Shefiield. Other papers will deal not only with general microbiological problems of these soils but also with the metabolic activity in pure culture of some of the organisms isolated. LOCATION AND VEGETATION The soil was obtained from a small wood at Ringinglow, 350 m. above sea-level, on the eastern slope of tbe moor wbich, stretching westward from this point to the Derwent valley, reaches a maximum height of 450 m. The tree cover consists of Pinus svhestris with Betula pubescens, and the field layer is dominated by Deschampsia flexitosa with Galiuni saxatile also present. O\er considerable areas Deschampsia is the only plant present, and its roots form a thick mat some 5 cm. thick overlying a dark brown humus layer in which partially decayed plant remains are clearly visible. This humus layer is 8-10 cm. in depth and the organic matter is mixed with sand from the under-lying layer. The soil used in the microbiological investigation was removed from this layer, each sample e.xtending from the top to the bottom of the layer and being thoroughly mixed before use. Sharply divided from this layer is the underlying sandy zone, light brown in colour and granular in appearance and about 14 cm. deep. The content of organic matter in this zone is small and decreases rapidly with increasing depth. Samples from this layer were examined solely from the chemical aspect. CHEMICAL ANALYSES Table i gives information about tbe soil layers, the upper being the humus zone, the middle and lower forming the underlying sandy zone. The upper layer was examined in greater detail, and the values recorded in Table 2 are from 100 g. organic matter. The total acidity of one sample of the upper layer was 97-8 m.equiv./ioo g. dry wt., of which 22-8 m.equiv. were from substances removable by successive extraction with benzene, alcohol and water. i8-2 m.equiv. were from substances insoluble in these solvents hut soluble in i ",, NaOH cold; the residual 56-8 m.equiv. were due to substances in the lignocellulose complex, since it was clear that the residual inorganic matter contributed nothing to the acidity of this fraction. Using the ether-extraction technique on soil acidified with 2A^H2SO,, to about/)h i, the total ether-extractable acidity was 6-i m.equiv./loog. dry wt. This may be regarded as the acidity due to a well-recognized group of biologically important organic acids, and of the total 0-9 m.equiv. was due to 'oxalic acid'.

2 The microbiology of acid soils 219 The titration curves showed that the soil contained both weak acids and bases; the almost straight-line form of the curves between ^H 10 and 4 suggested that a number of acids and bases contributed. 5 for undisturbed samples over the period May-September 1943 showed little variation from the average value of 423 mv. Nitrate was never present. 5 is the estimated potential atph 5, obtained by increasing the observed value by 58 mv. for every decrease in pa of one unit. Table I Upper Middle Lower Depth below root layer (cm.) pn Moisture content (^t,) Organic content as "0 dry wt. Acidity (m.equiv.)/ioo g. dry wt. Acidity (m.equiv.), ioo g. organic matter Residue following extraction with benzene, alcohol, water and i % NaOH Acidity (m.equiv.)/ioo g. dry wt. Acidity (m.equiv.)/ioo g. organic matter Table 2 Soluble in benzene Soluble in 95 % alcohol Soluble in water Soluble in i " NaOH, cold Cellulose Lignin Total nitrogen Amino nitrogen-alcohol soluble o-oi MICROBIOLOGY The values recorded below are per gram of soil in the natural state with regard to water content. The bacterial counts were made on glucose-peptone agar. Only isolated records were made of the total counts, and it is not possible to give a picture of seasonal changes. The following values from a sample taken on 26 November 1944 indicate the order of magnitude of the number of organisms which grow on this type of plate: 326xIO^ 394x10*', 331x10'', 338x10". While it is recognized that similar counts of fungi have no great significance except possibly in the case of the yeasts, fungal counts were made using 2",, malt agar. The following values were obtained from a sample taken on 12 February 1945 and indicate the order of magnitude of the counts: 3 04XI0-^ 2-7Oxio-\ 3-36x10-^ 3-30x10^, 3-12x10-', 2-92 X lo-'. Of the fungi present yeasts were the most numerous on the malt agar, accounting for between 40 and 70 % of the total colonies; of the remainder Penicillium was most abundant. 15-2

22O J. G. BOSWELL AND D. J. GOVER The most prominent colonies were those of the dark brown Dematium pullulans which was always present to the extent of 2-3%, and a Cephalosporium species in about the same numbers.

3 22O J. G. BOSWELL AND D. J. GOVER The most prominent colonies were those of the dark brown Dematium pullulans which was always present to the extent of 2-3%, and a Cephalosporium species in about the same numbers. Using the medium acidified to/)h 4 these two fungi became much more abundant. Species of Mucor, Rhisopus, Aspergillus, Actinomyces and Botrytis rarely developed on plates prepared from soil extracts, but they developed rapidly and showed abundant growth when particles of soil were spread on malt-agar plates followed by incubation at 22 C. It is probable therefore that these fungi were present in soil in an active vegetative form. Microscopic examination of the soil revealed abundant hyphae. The effect of the constitution of the medium on the growth of the fungi is most clearly shown by the great increase in the number of yeast colonies which occurs when gelatin is added to the malt agar plates to give a concentration of 2%. The number of yeast colonies is approximately doubled on the malt gelatin in comparison with the malt medium. While the culture media provide evidence of the fungal spores which are present in the soil the extent to which these organisms can multiply at the natural acidity of the soil is suggested by the following results: ph No./g. soil % yeasts X 10' X 10' X 10* 30 3'5 o8x 0 The failure of yeasts to develop at p\i 35 suggests that they have little part in soil metabolism under natural conditions, although apparently present in considerable numbers. The number present may be due to the existence of colonies in isolated pockets in which owing to the constitution of the neighbouring organic matter the ph in the vicinity of the organisms approaches neutrality. The fungi which develop at the lower ph values are species of Penicilliiim and Actinomyces, in particular the number of the latter which occur is increased and accounts largely for the actual increase in the numher of non-yeast fungi which appear at ph 5-5. With media at ph 2-5 only occasionally did fungal colonies appear, and in all cases these were Penicillium spp. The bacteria which develop on the glucose-peptone plates are naturally confined to one physiological group, those which can satisfy their nitrogen requirements by decomposing peptone. A wide range of morphological forms was observed, chiefiy rods of variable dimensions and only rarely cocci. Spore formers and non-sporing types were present and among the sporing rods (Bacillaceae) were cells with a pair of polar spores and others with only a single central spore producing cell distension. In some cultures the cells joined to form filaments up to 12 units in length, while in one the cells aggregated into small groups surrounded by mucilage. Metabolically some were obligate and others facultative aerobes. This group of micro-organisms is under more detailed examination. Cellulose decomposition Strips of filter paper were partially immersed in a suitable inorganic culture solution and inoculated with a water extract of soil i ml. from 10 g. soil in 500 ml. water. After some weeks the submerged cellulose and the medium turned dark brown in colour, the latter developed a well-marked acidity, the cellulose at the air-water interface disintegrated and pink gelatinous masses appeared in the zone of decomposition. A small rodshaped organism appeared to be the active agent. Little or no decomposition occurred

4 The microbiology of acid soils 221 under anaerobic conditions. It is difficult to determine the importance of this organism in the process of cellulose decomposition in the soil, since its activity in pure culture is greatly depressed by increases in acidity. At/)H 6'5 it decomposes cellulose actively, at 5-5 less actively, and at 4-5 and below no sign of cellulose decomposition occurred after I month. The possibility must be considered that since these organisms only decompose cellulose when in actual contact with the fibres, and since cellulose behaves as a weak acid (Rabinov & Heymann, I94r), it may be that these bacteria exist in a microclimate less acid than the reaction of the whole soil and are therefore more active in the decomposition of the cellulose in the soil than would be estimated from their activity in aqueous media at different ph values. When sheets of filter paper moistened with a suitable culture solution were inoculated with a little soil after i week at 25 C., species oi Penicilliuni and Aspergillus were strongly developed and Ftisarium had frequently appeared. When the culture medium was adjusted topu values as acid as 3-5, Trichoderma sp. developed. Actinomyces appeared occasionally, irrespective of the ph of the culture solution. Of all the fungi which appeared species of Aspergtllus produced the most extensive decomposition after culture for 2 weeks. Protein decomposition The protein-decomposing organisms isolated from the soil were very active in decomposing a 5 % gelatin: r % peptone medium. The decomposition resulted in the formation of amino acids and, following deamination, of free ammonia and organic acids. Mixed cultures of the organisms were incubated at 30 C. for i week with a protein-peptone medium. It was found that the amino acid/organic acid ratio varied between 1-25 and r'93 with an average value of r-55. The cultures used were obtained from soil samples taken over a period of 6 months. From the mixed cultures a very active proteindecomposing rod form was isolated which under the same experimental conditions gave the amino acid/organic acid ratio as r-40 aerobically; under anaerobic conditions the organism was equally active but with a ratio of r-6i. Sugar fermentation The inoculation of a i % sucrose solution contained in Pasteur tubes with mixed culture from a soil suspension resulted in rapid fermentation with the production of acid, gas and alcohol. The alcohol was probably the product of yeast fermentation, since colonies of this fungus appeared on the surface of the solution after a few days. The gas produced consisted in part of CO2, the percentage varying between 25 and 53, being least in cultures prepared from soil during May and greatest in September. Nitrifying bacteria The presence and activity of organisms capable of oxidizing ammonia and nitrite in the absence of organic matter were investigated by the inoculation of suitable media, in some cases with particles of soil and in other cases with soil extracts. With certain soil samples no oxidation of either ammonia or of nitrite occurred, and with one sample nitrite was reduced to ammonia. With a few samples oxidation of the substrates was observed, but this occurred at a very slow rate, and after incubation for i week nitrite was present in a concentration of about ro~* and nitrate about io~^ in solutions which initially contained

5 222 J. G. BoSWELL AND D. J. GoVER only ammonia and nitrite respectively. The identity of the organisms was not further investigated, but it is not possible to assume that they were Nitrosomonas and Nitrobacter, since H. Winogradsky (1935) has noted the existence of certain slow-acting nitrifiers, a nitrite-former, Nitrospira, and a nitrate-former, Bactoderma alba, and the presence of capsuled or Nitrocystis strains in forest soils. Nitrogen fixation Two media were employed, one containing mannitol as the sole carbon source and the other containing glucose; the first was used for the culture of aerobic organisms and the second for the culture of anaerobes. Cultures were inoculated either with particles of soil or with soil extracts. Active fermentation occurred with the formation of large numbers of bacteria, the aerobes formed a pellicle on the surface of the solution and the anaerobes gelatinous masses on the particles of calcium carbonate at the bottom of the solution. The aerobic organism had the characteristic form of Azotobacter, while the anaerobic genus was Clostridiiim; both appeared to be pure cultures. Both the organisms were active in repeated subcultures. While no nitrogen determinations were made nitrogen fixation was assumed following the identification of the organisms, and since the original media contained no nitrogen and there occurred abundant bacterial growth with active acid and gas fermentation. Asa measure of bacterial activity aliquots of each medium were acidified with H.^SOj, the volatile acids distilled and titrated against A7100 NaOH. The fermentation during Way and June was greater than at any other time with both Azotobacter and Clostridimn. Using the distillation technique the fermentation activities of both organisms in relation to the acidity of the medium were investigated. Both organisms showed reduced rates of fermentation at/)h 4-5 as compared with those at 5-5, but while with Clostridimn the reduction was 5o';"o with Azotobacter the reduction was 9o o. At pw 3-5 fermentation by Clostridimn was reduced to 8,, of that at 5-5, while Azotobacter was completely inactive. DISCUSSION The values recorded in Table i for the percentage of water and organic matter in the samples emphasize the relationship between high humus content of soil and the amount of retained moisture. The ph values bear no relationship to the total acidity of the soil samples, the total acidity decreasing rapidly with depth while the pw is lowest in the cm. zone. The /)H value is of course the resultant of the mixture of the strong and weak acids which are present as the result of the partial oxidation of the organic matter by the micro-organisms. Ecologically the maximum acidity of the soil is probably more important than the total amount of acid present, since, in general, nutritional studies show that it is the degree of acidity rather than the total acidity which is a controlling factor in plant growth. However, in examining the microbiological processes which occur in soil it is from values of the total acidity that most useful information can be derived. While the total acidity of the soil decreases with depth, the acidity/100 g. of organic matter increases rapidly, but the residual acidity (acidity/100 g. of organic matter after extraction of the soil successively with benzene, alcohol, water and i % NaOH) decreases sharply. It may therefore be concluded that the microbe activity results in oxidations leading to the production of COOH groups and to molecular degradations resulting in the formation of more soluble

6 The microbiology of acid soils 223 substances. It is clear that not only are cellulose, hemicelluloses and pectins decomposed but also the lignin complex, and further investigations are being directed to the isolation of organisms which not only attack lignin but also open the benzene ring. In the upper layer the acidity of the ligno-cellulose complex (residual acidity) is a considerable part of the total acidity, but in the middle and lower layers the acidity of this fraction is unimportant, and since the^h values vary little in these three layers it appears probable that the ph values of the samples are determined largely by the composition of the more soluble oxidation products. These soluble oxidation products are chemically illdefined, but it is clear from the extended analysis of the upper layer that the ether-soluble organic acids are a relatively small fraction of the whole. The 5 value of 423 mv. places the soil in the lower range of the oxidizing group. Since it is not possible to analyse the redox systems in detail at present and to determine precisely the reaction or reactions to which the potential is related, its significance can only be considered in general terms. The oxidation of the organic matter in the soil by micro-organisms may be regarded as a series of linked redox systems catalysed by bacterial and fungal enzymes with molecular oxygen as the final hydrogen acceptor. The rate of oxidation will be determined by the rate at which oxidizable substrates are produced and the availability of oxygen, the latter being controlled in large measure by the diffusion resistances of the soil. At any one time, therefore, the rate of formation of oxidizable substrate and the rate at which this undergoes oxidation will determine the concentration of reduced and oxidized substances within the soil, and the ratio of the concentration of these two groups determines the soil potential. The relatively low value of E^ suggests that, for this soil, oxygen availability is the factor limiting oxidation; nevertheless, the soil potential cannot be regarded as a direct measure of the rate of oxidation since it is certain that changes in the availability of oxygen affect the metabolic activity of the micro-organisms in a much more general manner than simply by increasing the oxygen available as the final hydrogen acceptor. In particular, the concentration of oxygen within the soil not only controls in some degree the constitution of the microbe flora but may also determine the nature of the enzyme system through which oxygen is utilized as the final hydrogen acceptor, quite apart from any stimulation of the growth and activity of those organisms which are more particularly concerned with the pre-oxidation stages of the organic breakdown. While in general, aeration of a soil (availability of o.xygen), rate of oxidation and soil potential march together, these are only the outward manifestations of the complex metabolic processes of the soil micro-organisms. While from the E^ value it might be expected that the soil would contain nitrate this radical has never been detected. This places the soil in that group of oxidizing soils with a ph below 3-8, deficient in nitrates described by Pearsall (1938). Microbiological experiments reveal the reason for the absence of nitrate, since nitrite- and ammoniaoxidizing organisms are almost completely absent. This is probably the result of soil acidity, since earlier records state that the nitrifying bacteria are only present above pu O. Further, since their activity does not confine them to a solid substrate as in the case of the cellulose-decomposing organisms, there is no reason to expect that they will occur in pockets isolated from the general acidity of the soil. The aeration of the soil is apparently not sufficient to promote the autoxidation of nitrite to nitrate which Corbet (1935) has mentioned as playing a more important part in the formation of nitrate in acid soils below ph 5 than the biological activity of Nitrobacter.

7 224 J- G. BoSWELL AND D. J. GoVER An interesting feature of this acid soil is the presence of micro-organisms which in pure culture do not multiply and are not metabolically active at ^H values near to that of an aqueous suspension of the soil. The most important examples are a bacterium which decomposes cellulose and the nitrogen-fixing bacteria. While the view that cellulose decomposition in acid soils is chieffy brought about by fungal attack is supported by observations made on this soil, it is of interest to isolate a bacterium capable of decomposing cellulose in aqueous media only at acidities much less than that of the whole soil. The degree of metabolic activity of the organism in the soil is as yet unknown, but it must be sufficient to maintain at least a small number of active colonies. Since chemical analysis has shown that oxidation, molecular degradation and increased acidity march together, it may well be that the ph at the surface of the cellulose fibre is higher than that in the general body of the soil. These organisms, therefore, whose substrate is a solid fibre may exist in isolated pockets and be little affected by the acidity of the whole soil. Such cellulose-decomposing micro-organisms may be widespread in acid soils, since a similar organism has been isolated from the Eriophorum peat located on the higher parts of the moor. Nitrogen-fixing bacteria have been isolated from this soil, although the /)H is much below that at which they can be grown in synthetic media lacking fixed nitrogen. There is, however, no evidence that they do fix nitrogen in the soil, and in view of the known ^H limits of their capacity for nitrogen fixation in pure culture it would seem probable that they grow in acid soil by absorbing fixed nitrogen from the medium and by the fermentation of the carbohydrate present. SUMMARY 1. A ' mor' bearing Pinus sylvestris, Betula pubescens and Deschampsia flexuosa was examined from the chemical and biological aspects. 2. Certain observations were made on the total acidity of the soil, the distribution of the acidity among the fractions of the organic matter in the humus layer, and of the acidity in relation to the depths of the samples from the surface. 3. A survey of microbiological activity revealed the presence of large numbers of bacteria and fungi. All the major groups of soil micro-organisms were well represented with the exception of the nitrifiers. These last were present in very small numbers, and this explained the failure to obtain positive reactions to tests for the nitrate ion. 4. In spite of the low ph a cellulose-decomposing bacterium and nitrogen-fixing bacteria were isolated. REFERENCES CORBET, A. S. (1935). Microbiological oxidation. Biochem. J. 29, PEARSALL, W. H. (1938). The soil complex in relation to plant communities. J. Ecol. 26, 194. RABINOV, G. & HEYMANN, E. (1941). The acid nature of cellulose. J. Phys. Cliem. 45, WiNOGRADSKv, H. (1935). The number and variety of nitrifying organisms. Proc. Int. Soc. Soil Sci. Commission, 3, 138.

THE MICROBIOLOGY OF ACID SOILS

[ 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