Acetic acid tolerance in wood- and litter-decomposing Hymenomycetes Veikko Hintikka Finnish Forest Research Institute Introduction Wood, whether living or de.composing, forms a complicated and changmg system, in which the various chemical and biotic factors which affect the fungus mycelium within the wood can only be elucidated with difficulty. Among these factors, the acidity of the wood depends to a considerable degree on the presence of volatile .acids, the mo~t important of which are acetic and forrmc acids. According to PACKMAN ( 1960), the content of total free acids, mostly acetic acid, in fresh oak wood may be up to 0.45 o/o of the dry weight, in birch wood 0.09 o/o, in beech 0.10 %, and in Douglas fir 0.11 o/o . It is these acids that are responsible for the cr>rrcsive action of the wood of certain trees, especially oak and sweet chestnut, on metals, p<~rticulay lead, in which a chain reacti<;n from acetic acid to lead carbonate sets m (fARMER 1967). Owing to the presence of free acids, the pH of fresh wood may often be quite low. According to the data of GRAY ( 1958 ), the acidity of hardwoods varies between pH 2. 75 and 6.8, and among European species the most acid are the oaks (pH 3.35 ). Fraxinus excelsior (pH 3.55) , and Castanea sativa (pH 4.0), while values between pH 4.0 and 5.0 are common. The pH of softwoods varies between 2. 7 (Pinus strobus) and 8.8, the acidity of the wood of Picea excelsa being 4. 75 and that of Pinus silvestris 4. 75-5.25 (GRAY 1958). When wood is hydrolyzed in boiling dilute sulfuric acid, acetic acid is liberated in considerable amounts from the acetyl groups of the wood, and it is one of the major products of the dry distillation of wood. In conifer wood, acetyl groups are mainly associated with galactoglucomannans and in hardwoods with 4-0 methylglucuranoxylan, the yield of acetic acid in the former being 0.5-2 %, and in hardwoods considerably higher, 2-6 % (KLINGSTEDT 193 7, HAGGLUND 1951) According to PACKMAN ( 1960), acetic acid develops in wood, when kept under damp conditions at 48°G, and a corresponding amount of acetyl groups is lost from the wood. In his experiments the amount of total free acid, mostly acetic acid, increased during two years' storage in birch wood from 0.9 o/o to 2.4 %, in oak wood from 0.4 % to 6.5 o/o, and in Sitka spruce from 0.02 %, to 1.6 o/o, if the samples were not contaminated with micro-organisms. Correspondingly, the pH of the birch wood decreased from 4.6 to 3.32 in 126 days. STEWART et al. (1961) have found evidence that acid hydrolysis occurs within the living tree, probably owing to the formation of acetic acid from acetyl groups. To begin with, this autocatalytic process takes place very slowly, but the acetic acid liberated causes additional hydrolysis and formation of more acid over long periods of time. There is a parallel lowering in the pH of the wood, e.g. in Eucalyptus from pH 4.6 in 5-year-old sapwood to pH 3.3 in 420-year-old heartwood. 0 177 Similarly, young sapwood of Larix occidentalis contains only a small amount of acetic acid (0.039 %) , but this causes slow additional liberation of acetic acid by hydrolysis resulting in a content of 0.14 % acetic acid in heartwood 320 years old (CoTE et a!. 1961). Fungi and other micro organisms growing within wood may inhibit this accumulation, probably by consuming the acid (PAcKMAN 1960) . On the other hand, acetic acid is a common metabolite in fungus cultures and numerous species are known to produce it (CocHRANE 1958); among the wood-decomposing fungi it is produced by Polyporus species from carbohydrates (PERLMAN 1949, 1950) and by Fames anna-sus. In addition, many bacteria produce acetic acid (WAKSMAN 1931 ) e.g. from cellulose (Sru 1951) . In thermophilic bacterial attack on wood components the main products are acids, such as acetic, butyric, and lactic acids (VIRTANEN & HUKKI 1946). Adequate information is not available to allow a comparison of the occurrence of acetic acid in other types of natural substrates. According to ScHWARTZ et a!. ( 1954) , acetic acid may be present in podzol soils in quantities of 0. 73 to 1.08 rneq per 100 g soil, the content being thus lower than in wood. In flooded soils the content of acetic acid may be to 3 x 10- 3 rneq/rnl (HoLLIS & RoDRIQUEZ 1967) and this acid evidently plays a role in the microbiology of flooded soils (ALEXANDER 1961). If acetate is added to the soil, it is metabolized rather rapidly by the soil microbial population (STEVENSON & K '\ TZNELSON 1958). Thus, on the basis of the literature, it seems that acetic acid may accumulate in wood and affect the mycelia growing within it. In the present investigation, the tolerance of certain wood-decomposing and litter-inhabiting basidiomycetes to acetic acid was studied. Methods Fungus strains were cultivated on Hagem agar, the composition of which is: glucose 5 g, malt extract 5 g, KIII2P04 0.5 g, Mg:S04 · 7 H20 0.5 g, NH4Cl 0.5 g, FeCls ( 1% sci!.) 1 ml, agar 15 g and distilled water 1 l. After this mixture had been autoclaved for 20 min. at 120°C, measured amounts of sterile acetic acid were added with a pipet, and the agar poured in to 10 em petri 178 dishes. 1ihe radial growth was measured after a variable number of days (7-10) and thus the differences in the growth rates between the s·p ecies are not comparable. The strains used in thi·s investigation were isolated in the years 1964-67, and preserved at the laboratory of the Forest Biology Department, Finnish Forest Research Institute, at soc with transfers about twice a year. As the content of phosphate was rather high, it buffered the changes in pH brought about by the acid to some degree. The pH of the different concentrations were : 0% : 4:8, 0.01 %: 4.7, 0.05 % : 4;0, ).'1 %: 3.8, 0.2 %: 3.6, and 0.3 % : 3.4. These pH values agree fairly well with the values given a:bove for natural wood containing acetic acid. Results The soil hyrnenornycetes investigated proved to be rather sensitive to acetic acid (Table 1 and Fig. 1). The growth of typical litter-decomposing as well as mycorrhizal species ( Clitocybe, C ollybia, Suillus) was totally inhibited by concentrations of 0.01 %· More tolerant were Clavaria fistulosa, M arasmius androsaceus, and species of Pholiota, many of which in nature show a distinct preference for twigs, small pieces of wood or bark heaps, although they do not as a rule grow directly on thick logs. In general, fungi growing on conifer wood were considerably more tolerant to acetic acid, some of them growing at 0.1 %, namely Fomitopsis pinicola, Gloeophyllum sepiarium, Phaeolus schweinizii, Abortiporus borealis, Anisomyces odoratus, and Stereum sanguinolentum. These species are characteristic of thick logs of stumps or living sterns of conifers. Stereum s.anguinolentum occurs as a heart-rot parasite in living spruces, although it does not form besidiocarps on this substrate. Species growing on old decayed wood of conifers, such as X eromphalina campanella and Flammula penetrans, showed a markedly lower tolerance of acetic acid, the maximum being approximately the same as that of the most tolerant species of the litter-decomposing group. In species growing in nature on deciduous wood the tolerance was fairly similar to that of the conifer wood species, or even higher. Especially it should be noted that the species occurring on thick trunks of oak ( Daedalea quercina, Laetiporus sulphureus) or elm (Tytomyces fissilis) still grew fairly well at 0.2 %, a concentration at which all the other species investigated failed to grow. Oak wood especially is known to contain considerable amounts of acetic acid (FARMER 1967) . Discussion That acetic acid may have an ecological significance in the biology of wood-decomposing fungi is indicated by the experiments of SuoLAHTI (1951 ), who found that it exerts a stimulatory effect on the aerial mycelium of Stereum sanguinolentum. As acetic acid is one of the central metabolites in fungal metabolism, e.g. in the TCA cycle and in the synthesis of many compounds, it would be interesting to investigate to what extent the enzyme systems of lignicolous fungi differ form those of the litter-decomposing species. The present experiments suggest a distinct difference in this respect between the two ecological groups. When the values given for concentrations of acetic acid in wood and in soil are compared with the above values in the agar substrate, it should be noted that the former are based on the dry weight, and the actual concentration of the cid in the liquid phase may be different, depending of the moisture content, although exact determinations seem to be lacking. In any case the present experiments indicate that the tolerance, especially of species attacking oak wood, may be of considerable ecological importance. Wood is denser in structure than soil, and the .soluble and volatile substances within it are not exposed to leaching with rain water or evaporation to a same extent as in soil. Forest soils, especially, are comparatively well ventilated and, for instance, the carbon dioxide concentration does not as a rule exceed 1- 2 %, although in wood it often rises to 10- 15 % (THACKER & GooD 1952). Thus it i.s possible, although not conclusively proved, that acetic acid accumulates in wood in greater quantities than in soil, and may thus, like many other chemical factors, exert a selective effect on the invading microbial population. In connection with the present experiments some attempts were made to isolate lignicolous fungi from forest soils with 0.3 o/o acetic acid-Hagem-agar dilution plates. There were great differences in the fungus flora between the plates containing acid and those without acid. In Hagem agar many species of Mucor as well as other Phycomycetes species were present, but in acetic agar species of Penicillium totally dominated the plates and inhibited any mycelia of lignicolous basidiomycetes possibly present. Summary On the basis of a literature review, acetic acid may occur in wood from three different origins: 1) as free acid, especially in oak wood, 2 ) liberated through hydrolysis from acetyl groups of wood, and 3 ) metabolic products of micro-organisms. The content of acetic acid in the wood of living trees may exceed 0.4 % per unit of dry weight. The tolerance of 125 species of basidiomycetes was determined by adding sterile acetic acid to Hagem agar after autoclaving, and the radial growth of mycelia was measured after 7-10 days. The pH of the acid agar agreed fairly well with the values given for wood containing acetic acid . The typical litter-decomposing species were found to tolerate 0.01 % acetic acid or below, while species which in nature occur on small branches or twigs grew in slightly higher concentrations. Species growing on conifer wood tolerated as much as 0.1 %, the highest tolerance being observed in species which grow in thick logs or within living tree stems. Among species attacking deciduous wood, the highest tolerance (0.2-0.3 %) was found in the fungi which occur in living oaks and elms (Daedalea quercina, Laetiporus sulphureus) . The ecological significance of this difference is discussed and the possible accumulation of acetic acid and other metabolic products in wood due to its compact structure compared with easily leachable forest humus and litter is emphasized. Acknowledgments The aut•hor expresses his thanks to Mr. LALLI LAINE, M .Sc., for allowing the use of 15 strains of polypores detevmined a;nd isolated by him. Financial support by U .S. Public Law 480 grant Fg-Fi-132 is gratefully acknowledged. Received 30. 5. 1969. 179 Table 1. The effect of acetic acid on the radial growth (mm) of some hymeuomycetes. Soil fungi Species Agaricus sp . Agrocyge praecox 4scobolus sp . Clitocybe clavipes C. nebularis C. odora C. phyllophila Clitopilus prunulus Cla;varia fistulosa Collybia asema C. butyracea C. confluens C. dryophila C. peronata C. putilla Cystoderma amianthina C. granulosa Galerina pa,l udosa Hygrophoropsis aurantiaca L epiota clypeolaria Lepista nuda Lycoperdon pyriforme M acrolepiota rh acodes Mara-smius androsaceus M. epiphyllus M. oreades M. prasiosmus M. rotula M . scorodonius Micromphale perforans Mycena epipterygia M . galopus M . samguinolenta M . viscosa Pamaeolus sp. Phaeolepiota aurea Pholiota carbona;ria P. lenta · P. lubrica P. spumosa Psathyrella gracilis (?) Rhizopogon sp. Stropharia aeruginosa S . hornemannii S . semiglobata Suillus bovinus S. elegans S . variegatus 180 9 0.01 0 10 10 4 11 13 2 5 4 5 3 5 2 0 13 10 18 12 fi 8 19 10 11 20 12 15 8 1 7 0.005 10 12 13 7 10 19 11 11 2 1 3 4 11 11 10 1 4 7 0 0 8 0 8 10 1 0 0 2 11 6 11 9 10 ·6 24 21 18 5 5 3 10 10 25 6 10 10 5 6 22 27 16 14 7 11 26 25 15 13 14 5 9 12 5 6 5 3 41 16 23 15 5 8 12 3 11 15 7 5 0 7 5 3 4 40 16 25 13 10 16 3 10 3 6 19 7 42 15 23 o/r acetic acid O.D25 0.05 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 13 0 0 0 0 0 0 0 7 0 15 0 8 0 0 6 1 0 0 0 0 0 2 0 0 0 36 12 13 7 23 11 0 0 0 13 13 7 0 0 0 12 2 15 1 8 0 0 0 0 0 0 7 0 0 0 0 0 0 0.075 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 1 (cont.). Fungi growing on deciduous wood Species Aporpium semisupinum Armillaria mellea « « Bjerka:ndera adusta C oriolus hirsutus Daeda,lea quercina Daedaleopsis confragosa v. tricolor Flammula alnico/a F. sp . Fla ;.lmulina velutipes Fames fomentarius F omit apsis pinicola « « « « F. odoratissima Ganoderma applamatum G . lucidum Gloeoporus dichrous H apalopilus nidulans H ohenbuehelia serotina Hydnum septentrionale Hym enochaete rubiginosa Hypholoma fasciculare H . sublateritium I no notus obliquus I . radiatus I. rheades Lentinus bisus L . vulpinus (?) Lentinus sp. L enzites betulina La:etiporus sulphureus Lyophyllum ulmarium Pa:nellus stypticus Panus conchatus Phellinus conchatus P. ferruginosus P. igniarius P. pomaceus P. puncta·tus P. robustus v. hippophaes P. tremulae Pholiota aurivella P. mutabilis P. squarrosa Phyllotopsis nidulans Piptoporus betulinus Plcurotus ostreatus Polypilus frondosus Polyporus brumalis P. squamosus P. varius Pycnoporus cinnabarinus Stereum hirsutum S . purpureum Trametes suaveolens Tyromyces /issilis isolated from Betula Quercus Salix frag . Betula Betula Quercus 0 21 5 5 29 40 18 Betula Betula Betula Populus Betula Betula Tilia A . incana Salix 12 20 18 45 34 29 34 27 3 8 10 A. glutinosa Betula Betula Betula A. glutinosa Quercus w 7 41 6 4 10 Betula 22 Betula 19 A. glutinosa 11 Populus 11 Betula 25 Betula 25 Betula 18 Betula 37 Quercus 16 Acer platanoides 19 Quercus 14 Betula 13 S alix caprea 26 Corylus 21 Betula 20 Prunus ceraceus 20 Salix caprea Hippophae rhamnoides Populus Acer Betula decid. tree Betula Betula Aesculus Quercus Ulmus Tilia Sorbus Betula Betula Sa.Zix Ulmus 0.005 0.01 29 6 5 32 38 19 0.025 20 5 6 20 38 19 20 19 45 35 12 18 10 33 18 19 4 13 9 9 9 9 4 21 21 22 40 11 30 18 22 23 9 7 3 18 23 19 22 26 45 26 28 10 19 22 26 13 40 25 7 4 23 24 27 34 29 10 20 4 10 40 8 5 9 21 22 16 24 25 20 40 17 13 13 26 13 22 22 7 7 7 19 19 19 22 34 38 37 30 10 18 29 40 26 35 26 2 10 5 15 8 34 23 3 9 20 14 15 17 24 24 15 39 38 9 13 12 23 9 19 20 6 2 5 19 16 18 25 33 31 40 0.5 12 21 27 11 25 25 0.05 0.075 4 0 0 4 0 5 12 8 30 22 18 0.1 0 0 0 8 14 14 0.2 0 0 0 0 11 0.3 0 0 0 0 0 1 0 15 16 6 10 20 23 22 0 1 4 6 1 28 18 0 4 0 1 4 13 7 13 5 14 37 3 0 10 19 0 0 0 0 0 9 10 0 0 17 17 16 0 0 0.5 2 0 21 4 0 0 0 0 0 5 0.5 4 0 4 33 0 0 1 15 0 0 0 0 0 6 8 0 0 15 16 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 17 0 15 15 10 7 26 21 0 0 16 14 7 13 20 0 0 10 0 1 8 0 0 14 0 0 11 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 9 0 0 0 8 9 0 0 7 0 0 0 14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 1 0 12 25 0 0 0 11 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 181 Table 1 (cont.). Pinus Species Abortiporus borealis A nisomyces odoratus Coriolellus heteromorphus C. serialis Coriolus vaporarius Flammula penetrans Fomitopsis annosa F. pinicola « F. rosea Gloeophyllum abietinum G. sepiarium Hirschioporus abietinus Hypholoma ca'pnoides Ischnoderma resmosum Mycena luteoalcalin M. rubromarginata Phaeolus schweinitzii Phellinus pini Pholiota flammans Pleurotus mitis Polystictus circinatus v. triqueter Stereum sanguinolentum Tyromyces cinerascens T . fra:gilis Xeromphalina campanella = Fungi growing on conifer wood Pinus silvestris, Picea isolated from Pice a Pice a Picea Picea Picea Pinus Pinus Picea Pinus Pinus Pinus Picea Picea Picea Pinus stump Picea Pinus Pinus Abies balsamea Picea 0 12 4 27 13 20 5 43 35 40 13 10 25 18 6 46 9 11 19 10 3 14 19 Pinus Pinus Pinus stump 5 30 12 8 0.005 18 10 26 = Picea excelsa. 37 0.01 0.025 15 23 9 16 27 22 16 25 19 11 7 40 30 37 30 0.05 19 14 12 15 0 0 15 26 15 15 13 15 13 8 20 25 8 7 50 8 22 7 8 44 6 28 10 16 23 18 27 15 8 35 18 8 7 14 12 20 14 8 40 0 29 12 3 17 17 8 32 7 8 O.o75 16 6 0.1 11 8 0 0 0 20 0 6 0 0.5 0 0 0 9 10 0 5 2 5 34 0 0 27 4 0 8 6 17 0 0 23 0 0 27 0 0 0 0 4 0 0 0 0 6 20 0 4 3 9 0 0 5 0 20 0 0 0 0 0 0 0 0.2 0 0 0 0 0 0 0 0.5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Fig. 1. The effect of acetic acid on the radial growth of lignicolous ( 1- 4, 710, 13-14) and soilinhabiting (5- 6, 11- 12, 15) fungi. 1: Fomitopsis amnosa, 2 : H ohenbuehelia serotina, 3: Coriolus hirstus, 4 : Pycnoporus cinnabarinus, 5: Collybia dryophila, 6: Collybia asema, 7 : Daedalea quercina, 8 : Hirschioporus abietinus, 9 : Pan us conchatus, 10: Trametes serial is, 11: Mycena sanguinolenta, 12: Marasmius oreades, 13 : Stereum sanguinolentum, 14: Hypholoma capnoides, 15 : Suillus variegatus . 40 20 0.01 182 0.05 0.1 0.2 0.3% REFERENCES ALEXANDER, M ., 1961: Introduction to soil microbiology. - 472 pp. New York & London. CocHRANE, V. W., 1958: Physiology af fungi. 524 prp. New York - London. CoTE, W. A. Jr., B. W . SIMSON & T . E. TIMELL, 1967: Studies on larch arabinogalactan II. - Holzforsohung 21, 85-88. FARMER, R. H., 1967: Chemi&try in the utilization of wood. 193 pp. Oxford. GRAY, V . R ., 1958: Tlhe acidity of wood. Journ. Inst. Wood Sc. 195·8, 58-64. HoLLIS, ] . P . & R . 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