Balla, P

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COXTKHHJTIOXS TO THE KNOWLEDGE OF THERMOPHILIC ACTIXOMYCETES OCCURRING !N CHAMPIGNON COMPOSTbyP. BALLADepartment of Microbiotogy of the Eötvös Lóránt! University. Budapest Received on September 13 th, 1966I t  is only from the period o f the last 30 years that we have information about comprehensive and detailed examinations o f the thermophilic actinomy­cètes. H a i n e s  (1932) has grouped the common saprophytic actinomycètes according to their growth temperature. K n o r r  (1933) has found such soil sample in which, when exposed to a temperature o f 180 — 200° C, not all o f the actinomycètes were destroyed. J a g  n o w (1957) has examined the effect o f temperature and humidity in the proportions o f spores and mycelia o f various actinomycètes. Thermophilic actinomycètes have been isolated by G i 1 b e r t (1904) from soils, by M i e h e (1907) from the decomposing masses o f plant and by S c h ü t z e  (1908) from decomposing clover hay. W a k s m a n ,  C o r d o n  and H u i p o i  (1939) have examined in detail the outstanding role played by thermophilic actinomycètes in decomposition processes taking place in thermophilic composts. H e n s s e n (1957) has recognised that cer­tain species o f and Æocardm are found abundantly in thermophiliccomposts and systematized them mainly according to their morphological properties. The chapter on thermophilic actinomycètes in W a k s m a n's (1961) actinomycètes system includes also the species isolated and identified by H e n s s e n .  The systems o f K r a s s i l n i k o v  (1949) and B e r g e  y (1957) in respect o f actinomycètes, arc also important in the investigation o f thermophilic actinomycètes.K r a  s i l n i k o v  and A g r e (1965) have described a new species by the name o f cAro?Ho<ye?m. K o s m a t c h e v  (1959) argued thatthe thermophilic properties are not sufficient to establish independent genera, and recommended that they should be classified in the same genera as the mesophilic forms.B l o n d e a u  (1959) has demonstrated that the presence o f thermophilic actinomycètes acts favourably on the development o f the champignon. In the present paper this interaction will be analyzed and some preliminary results will be reported.Materia! and Method1. Description q/ ?/;<? ê.sl ?HrPer/o/Com])ost made o f horse manó re and coming from the Kőbánya establish- ment o f the Mushroom Crowing Enterprise was used as test matériái. The preparation o f compost includes three main phases:a j  the preparative phase (moistening, spreading, etc.)& the phase o f controlled fermentation the phase o f heat treatment.Due to the activity o f microorganisms the temperature o f the compost gradualiy rises during fermentation; after the 12th— 13th day, when tempera­ture is as high as 40 — 45° C, the powdery colonies characteristic o f actinomy- cetes appear in great masses.I t  is important that the water content o f the compost should not be less than 60 — 65% by the end o f fermentation, since this is the optimum level o f  humidity for microorganisms.In the course o f heat treatment the compost is treated with direct steam o f 50 — 60° C in order to destroy the harmful microorganisms. This ^pasteuri­zation" takes four days.The samples examined were taken from various phases o f compost prepa­ration :fresh manure before compost-making (marked 1.); fermented compost before heat treatment ( I I  ); compost treated for 24, 48, 72, 06 hours ( I I I — VI.); compost prior to champignon inoculation (V II.); compost after champignon plucking (VM I.).2. Zso/oDon q/' oc/inowycchs s/ro/ns a/a/ raa/g/m/ q  ̂yerc/.sA 1:10 suspension o f compost samples and hot water was made with the shaking technique. Due to the action o f hot water the microorganisms attached to the plant parts are more easily detached ( H 1 o n d e a u 1950).A dilution series was made o f this suspension, and 0,5 ml aliquots of the various concentrations were plated on different culture media in Petri dishes. The inoculated culture media were incubated in the thermostat for 3 — 6 days. Bv means o f further plating the pure culture o f the strains was obtained.The following culture media were employed for isolation (W  a k s m a n 1061):n j glycerol-glycine agar (P  1 o t h o)6̂1 glucose-asparagine agar (asparagine agar) rp ep ton e-bee f extract or nutrient agar (pepton agar) r/J sucrose-nitrate agar +0,6 g yeast extract (Czapek-yeast agar) Supplemented with actidion, penicillin and nystatin, the same culture media were used for counting the germs o f actinomycetes ( W i l l i a m s  — D a n e s, 1065).3. TVs/ o/ criMose dccoM/Msd/onThe pure actinomycetes strains isolated were examined in two different ways for their capacity o f decomposing cellulose:2S P. B Á LLÁaj in V i n o g r a d s k y ' s standard silica gel solution, with filter-paper in Petri dishes,& in deep culture in 50ml flasks, in a culture medium containing only inorganic substances and a cellulose preparation (H u n g a t e 1950) as carbon source. As a control glucose was used as carbon source.4. (bimrpAo/ogu'caTJ mef/iodThe microscopic (morphological) examination of active cellulose decom­posing strains was made with the hanging-drop method. A culture medium with liquid starch and glucose-yeast was used for preparing a hanging-drop culture. In order to prevent the cultures from running dry, sterile water was poured into the Petri dish under the microscopic slides.Results and ConclusionsThe samples were examined on four different selective culture media, with six series for each sample, under identical conditions. The average numbers of germs after an incubation period of 144 hours, with the various samples and culture media are listed in Table 1.T'nMc 7.Average number ot germs nt tbcrmophi'ic actinomycètes in I  —VHf.  compost sampies, on tour cuiture mediaTHERMOPHILIC ACTINOMYCETES' 29From the results obtained the following conclusions can be drawn:1. On all of the four culture media there is a difference in the order of magnitude of the germ number obtained with fresh manure (1) and with com­post gained by fermentation (II), respectively. This can be explained by the fact that temperature is rising during compost making as a consequence of the decomposing processes, and this has a beneficial effect on the multiplication of thermophilic microorganisms.Samp'?.Nutrient mediaPepton Czapek Asp. ldotho1 4 - l ( ) 3 2 - 1 0 3 3 - 1 0 2 3 - 1 0 211 2 - 1 0 4 3 - 1 0 4 5 - 1 0 3 3 - 1 0 3I I I 9 - l f ) S 7 -1 0 3 6 - 1 0 3 4 - 1 0 3I V 4 - l ( ) 3 5 - 1 0 3 5 - 1 0 3 4 - 1 0 2V 2 - 1()3 3 - 1 0 3 2 - 1 0 2 3 - 1 0 2V I 3 - l ( ) 3 0 - 1 0 3 3 -1 02 3 - 1 0 2V I I 2 - l t d 3 - 1 0 4 2 - 1 0 3 3 - 1 0 3V I I I 3 1 Q 3 3 -1 0 3 7 - 1 0 2 3 - 1 02. In the samples froni the heat treatment phase ( I I I —VI.), the germ number decreases on all o f the four culture media. This may be due to the inhibited development o f thermophilic actinomycètes by radical heat treat­ment.3. In the period after heat treatment and prior to inoculation with cham­pignon spores, the compost is undisturbed. As shown by the results of sample V I1.. this is probably advantageous for the development o f actinomycètes.4. 'Die vigorous growth o f the champignon as well as the changed tempera­ture conditions may account for the further decrease o f germ number by an order o f magnitude, as observed in the sample taken after chamniynon nluckiu-r (V III .).5. As shown by the experimental results, thermophilic actinomycètes are growing best on pepton agar and C'zapek-veast agar, whereas their development is less vigorous on the two other media.In the second part o f experiments the pure strains o f thermophilic acti­nomycètes were examined from the point o f view o f cellulose decomposition. The silica gel method was applied as a qualitative test. A  distinction between the activity o f the different strains in the liquid culture was made on the basis o f growth rate. Combining the results o f the two methods, the strains could be grouped on the basis o f their cellulose-decomposing capacity as follows:30 P. B A LLAThe quantitative examination o f the decomposing capacity o f strains developing at a medium and a vigorous rate, respectively, on cellulose media is part o f my future work.On the basis o f microscopical examinations and with the help o f H e n s- sen' s  system ( H e n s s e n ,  1957) the strains o f vigorously decomposing capacity could be identified according to their microscopical ¡Morphological properties.d / ic ro .s io p / ro/  e/anw/er/.s/h'.s ¡ y  g/ra/?; 7T1,„. ¿n /Ac Aa  " y o a /  d ro p .'The vegetative mycelia are non septate, they are branched (Fig. 1.) and twisted in an ol 1 culture (Fig. 2.), the aerial hyphae are branched (Fig. 3.), the spores are born one by one on simple and sometimes branched sporophores (Fig. 4.), the spores are round (Fig. 5.) and 1,6 /< in size. All that proves that the said strain belongs to the species yAcrwowoaogpara cacra/a H e n s s e n 1957.A / ¿ c ro g ro p a a /  rAnrnc/er/g/fcg a/' g/ra/a 7 '.4.,,.'The vegetative mycelia are mainly straight, non-septate (Fig. 6.), aerial mycelia are 0,8 — 1,2 p thick, 40 — 50 p long and branched (Fig. 7.), the sporopho­res arc spirally curved and formed one by one or in groups from side branches o f the aerial mycelia, the round spores are produced in chains o f 5— 10, which remain together (Figs. 8., 9.. 10.). The data show that this strain is TVieriMopo- /pgpara po/ygpora H e n s s e n  1957.THERMOPHILIC ACTINOMYCETES 31Fig. 1. Vegetative myceiia of TArrmomo- no.spora cnrtafa. M =  100 : 1Fig. 2. Vegetative myceiia in an oi<i cutture ofî'AcfinmMonosporacMrtwfn. Al =  272: 1Fig. 3. Aerial myceiia of TAerntowono^poracnrtafa. M — 272 : 1Fig. 4. Sporophoric form ot 7'Aermomo- no.spora cart-a/a. M =  200 : 132 P. B Á L L ÁFig. 5. Spores of TAerwo'Monospora cMrtfata. M — 200 : 1Fig. 6. Vegetatíve myceiia of TAe; wo- po^pora poiysporr;. M =  200 : 1Fig. 7. Aerial myceiia of T/tcrmopoíp^pompoiy.spora. ) í  =  200 : iFig. 8. Sporophoric form of TAermo- po^yapora po/y^pora. M — 200 : 1THERMOPHILIC ACTINOM YCETES 33Fig. 9. Spore chains of TZ;ermopoZyspora poZyspora ramaining together, M =  200 : )Fig. 10. Spores of TAermopoZyspora poZyspora. M =  200 : 1Fig. 11. Vegetative mycelia of iSZrepZawiyCM ZAerworaZyarM. M = [00 : 1Fig. 12. Aerial myceiia of -SZrepZowyceaZAermoHaZyarfs. M =  100 : 13 A NN ALES — Sectio Biotogica — Tomus 9- 10.34 P. B Á L L ÁJMicroscopMM? c&ira^enWcg of Â<? r̂o/M.s ^The vegetative mycelia are not septate (Fig. 11.), the aeriai hyphae are branched (Fig. 12.). Spiral sporophores, 16-24  p in length, are produced late­rally (Fig. 13.), the spores develop in spirals (Fig. 14.) they are oval-shapedFig. 13. Spiral sporophores of <Sh*epio?nyceg iAerMMa-Mlyarfs. M — 200 : 1Fig. 14. Spore chains of ¿Rreplonu/cea i/icrniora/yarfs. M = 200 : 1Fig. 15. Spores of ¿^replomycas MerniorMlyarfs. M = 200 : 1(Fig. 15.) and 0,8 — 1,6 ¡a in size. These correspond to <S7r<?y%o?nyce.? /AerwMWMZyd- r?'g H e n s s e n 1957.For their final identification the determination o f their physiological properties is needed.S u m m a r yExperiments were made to establish on four different culture media the number o f germs o f thermophilic actinomycètes in various stages o f compost applied to champignon. Some correlations were found between the development o f actinomycètes and that o f the champignon. The multiplication o f thermo­philic actinomycètes was found to be affected by changing temperature condi­tions during the processes o f compost making and heat treatment, as well as in a subsequent phase, by the vigorous growth o f champignon. Among the pure strains isolated, 6 proved to be o f vigorous activity as far as cellulose decompo­sition is concerned, and these were identified on the basis o f their morphologi­cal characteristics.REFERENCES1. B e  r g e y 's  manual of determinative bacteriology Ed. 7. 1957. London.2. B t o n d e a u , H. 1959: Study on some Actinomyces developing during „pasteurization" of manure. Effect of Actinomyces on the subsequent development of the champignon. Mushroom Science IV . Copenhagen. 153 — 175.3. G i l b e r t ,  A. 1904: Über Actinomyces thermophilus und andere Actinomyceten. Z. Hyg. Infektionskrankh. 47. 383 — 406.4. H a i n e s, R. 1932: The influence of temperature on the rate of growth of saprophytic Actinomyces. J. Exptl. Biol. 9. 45 — 60.5. H e n s s e n ,  A. 1957: Beiträge zur Morphologie und Systematik der thermophilen Acti­nomyceten. Arch. Microbiol. 26. 373 — 414.6. H e n s s e n ,  A. 1957: Über die Bedeutung der thermophilen Mikroorganismen für die Zersetzung des Stallmistes. Arch. Microbiol. 27. 63 — 81.7. H u n g a t e .  R. E. 1950: The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14. 1-49.s. J a g n o w, G. 1957: Beiträge zur Ökologie derStreptomyceten. Arch. Microbiot. 26. 175 — )91.!'. K n o r r ,  AL 1933: Über die wiederstandsfähigen Keime in Sterilizationsproben. (Akti- nomyzeten.) Centr. Bakteriol. Parasiten!:. Abt. I. 127. 269 — 276.10. K r a s s i l n i k o v ,  N. A. 1949: Opredelitel' bakterii i aktinomitsetov. IzdatelstvoAka- demii Nauk, Moscow.) ! .  K r a s s i l n i k o v ,  N.  A . —A g r e ,  N. S. 1965: The brown group of Actinobifidachro- mogena n. sp. Microbiologija, 34. 284 — 291.12. K o s m a t c h e  v, A. E. 1959: Mikrobiologija 28. 938 —943.13. M i e h e ,  H. 1907: DieSelbsterhitzungdesHeues.G. Fischer, Jena.] 4. S c h ü t z  e, H. 1908: Beiträge zur Kenntnis der thermophilen Actinomyceten und ihrer Sporenbildung. Arch. Hyg. u. Bacteriol. 67. 35 — 57.15. W a k s m a n ,  S. A. 1961: Theaetinom ycetes.Vol.il. Baltimore.)6. W a k s m a n ,  S. A. — C o r d o n ,  T. C. 1939: Thermophilic decomposition of plant residues in composts by pure and mixed cultures of microorganisms. Soil. Sei. 47. 217 — 225.17. W a k s m a n ,  S. A., C o r d o n ,  T. C. and H u l p o i ,  N. 1939: Influence of tempera­ture upon the microbiological population and decomposition processes in composts of stable manure. Soil. Sei. 47. 83— 113.18. IV i 11 i a m s, S. T. — D a n e s, F. L. 1965: Use of antibiotics for selective isolation anti enumeration of Actinomycetales in soi). J. Gen. Microbiol. 38. (2) 251 — 262.THERMOPHILIC ACTINOM YCETES 353*

Contributions to the knowledge of thermophitic actinomycètes occurring in champignon compost

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Contributions to the knowledge of thermophitic actinomycètes occurring in champignon compost

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