ifluence of co2 concentration on mycelium growth

8/13/2019 Ifluence of CO2 Concentration on Mycelium Growth

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6t3European J.Appl.Microbiol. 1 , 3 2 7 - 3 3 5 ( 1 9 7 5 )bySpringer-Verlag1975

I n f l u e n c e of C O 2 o n c e n t r a t i o no n the M y c e l iu m G r o w t h of T h r e e P l eu r o tu s S p e c i e sF. ZADR AZILResearch InstitutevonSengbusch, Hamburg

Received August 14, 1 9 7 4 ;Final Version July 2, 1975

Summary1 . IncreasedCO2concentrationin the air has apositive effecton

mycelium growthin thecultivationofPleurotus ostreatus, P. floridaandP.eryngii (Agar cultures).

2 . Between 16 and 2 2 CO2 in the air is theoptimumfor thegrowthofthe 3above-mentioned Pleurotus species.

3 . Concentrationsof 36vol. CO2inhibited growthof all 3species.4 . Both sterileandpaste urize d straw substrata showed 2 0 - 2 5 (of vol.)CO2 3days after inoculation. Mycelium growthwasnormal.CO2concen

tration standsinrelationto theamountofinoculation material, temperature, compositionofsubstratum,andother environmental factors.

5 . Thestimulating effectof CO2 on thegrowthof thePleurotus speciesstudiedandtheir hig h tole ranceto CO2havea special importanceinPleurotus farming.

6 . Under semianaerobic conditions with highCO2concentrationin thegaseous phaseofsubstratiim,thecompetitive microorgan ismsareeliminatedandPleurotus myceliumcangrow we llin thenon-steriles\ib-stratum.Carbon dioxide belongs to the most important ecological

factors. The quantity of CO2in the ecosystem determines biological activity and the quantity of micro-organism (Ahrens &Thalmann, 1970; Zadrazil, 1971, e t c . ) . On the contrary onecan consider the differing concentrations of CO2in the ecosystem as a selective factor in the range of species ofmicro-organisms (MacFadyen, 1973; Zadrazil, 1973a).

The CO2requirement of bacteria, yeasts and moulds has longbeen recognized (Valley & Rettger, 1927; Hermann, 1963; Bronn& Luca, 1973). Removal of CO2from the ecosystem can causedecrease of growth (La-fferty, 1963).The optimum CO2concentration varies for each type of microorganism.

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In 1971, Walsh & Stewart proved exchanges between oxygen,carbon dioxide and the cellulolytical activity in some kindsof fungi. In 1974, Schan^l found a direct relationship between the CO2concentration and the exoenzymatic activity ofwood-destroying fungi.

The influence of CO2on the mycelium growth and the fruitbody building of the basidiomycetes was mainly studied withthe Agaricus bisporus (white m u s h r o o m ) . In 1958, 1959 and 1972,Tschierpe determined that at 2 CO2and above, the myceliumof Agaricus bisporuswas inhibited in growth; at a concentrationabove 32 vol. , the growth stopped.

Rast & Bachofen (1965, 1967a, b) determined that the mycelium of Agaricus bisporusis able to fixate CO2. San Antonio &Thomas (1972, 1973) specified the optimum concentration fortheA.bisporus as between 0.1 and 0.5 CO2.

In 1954 and 1956, Plunkett also recognized CO2as the mostimportant factor of the fruit body building of Collybia velutipesand in 1963 Niederpruem studied the same with the SchlzophyHumcommune.

The influence of CO2on the growth of wood-destroying fungiin connection with wood protection was studied by Lambert(1933), Zycha ( 1 9 3 7 ) ,Plunkett (1954, 1 9 5 6 ) , Gundersen (1961),Rypacek ( 1 9 6 6 ) ,Jensen (1967) and Schanel ( 1 9 7 0 ) .As in theabove-mentioned papers little information is given about theinfluence of CO2on the Pleurotus species, we studied in thefollowing the effect of different concentrations of carbondioxide under various conditions as a basis for the biotechnology of Pleurotus production.

METHODS

Cultures of fungi:Pleurotus florida (FovoSe) - selected culture Pi own breeding

of Pleurotus florida (Eger, 1965)Pleurotus ostreatus(Jacq. ex Fr.) Kummer - provenance Barn, culture P4

Pleurotus eryngii(DC.ex Fr.) - own isolation - Spain, culture P5

Experiment N0.IGrowthofMycelium onAgar-Plates with Different Conc entrations ofC02Cultures in petri dishes were cultivated at 250c in incubators with different contents of CO2.Normal air (0.03 CO2was used as control.

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One half of the petri dishes inoculated in the center witha round inoculum of 7 mm diameter each were placed into anormal incubator at 25oc; the other half of the petri disheswas put into an incubator (Heraeus KB 600 K - C O 2 ) under acontrollable and constant CO2concentration.

At the following concentrations of CO2in the air, thePleurotus species were incubated: 5.0, 10.0, 16.6, 22.0, 28.8and 37.5 vol. . During the period of the experiment, thedifference of the CO2concentration was approximately 0.5 .Analysis of theExperiments. The radial mycelium growth wasmeasured in regular time intervals. The velocity of the mycelium growth (diameter of culture and time) was discovered asa linear correlation for 5 days. For each CO2 concentrationwe made 3 to 5 experiments, repeating each one four times.The number of measurements in Table 1 are shown under n.The Composition ofNutrient Media. Malt extract 5 g, soy beanflour 10 g, pepton 1 g, K H 2 P O 4 0.5 g, MgS04.7H20 0.5g, FeCl3(1% solution) 1 ml, yeast extract 0.1 g, agar 15 g, 1 1 H2O,pH 5.2.

Experiment No.2Submerged Culture. Submerged culture on the shake apparatus(500 ml Erlenmeyer flask with 200 ml nutrient media) wasinoculated with Pleurotus mycelium (3 agar mycelium piecesof 7 mm 0). For gas addition, we used the following concentration of CO2:5, 10 and 15 vol. of CO2in air). The mycelium growth was finished after 5 or 9 days and the dry weightand the pH value of nutrient media were determined.

Experiment No.3Production Experiment. In order to determine the CO2productionin sterile and pasteurized substratum (90^0, 1 h) we mademycelium growth experiments. In 1 1 jars 0.5 kg of straw substratum was sterilized (Zadrazil, 1973b) and inoculated with3 agar pieces of mycelium 07 mm). The CO2concentration inthe substratum was measured with the Drager-Gasspiirgerat type19/31.

RESULTS

The results showingf the effect of CO2on the growth of mycelium (Experiment N0.I) are shown in Table 1 and Fig.1.

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T a b l e 1E f f e c t o f c a r b o n d i o x i d e o n t h e r a d i a l m y c e l i io m g r o w t h (mm i n 5 d a y s ) o fP, ostreatus, P . florida and P . eryngii( n = n u m b e r o f m e a s u r e m e n t s )V o l , CO2 i n t h e a i r P . ostreatus P . florida P . eryngii

i n mm n i n mm n i n mm n

5 6 1 . 9 2 0 5 6 . 6 2 0 3 9 . 3 3 0C o n t r o l ( 0 . 0 3 ) 5 8 . 4 2 0 4 8 . 1 3 0 3 4 . 9 4 810% 5 5 . 7 4 6 5 5 . 4 5 2 3 3 . 4 8 4C o n t r o l ( 0 . 0 3 ) 4 8 . 5 5 2 5 1 . 4 5 2 2 9 . 4 8 81 6 . 6 6 1 . 5 8 8 6 0 . 4 8 0 3 3 . 3 1 0 4C o n t r o l ( 0 . 0 3 ) 4 9 . 1 1 0 4 5 2 . 2 1 1 2 3 0 . 3 1 0 42 2 . 0 5 6 . 7 1 5 0 5 4 . 5 1 5 0 2 5 . 4 2 2 0C o n t r o l ( 0 . 0 3 ) 5 3 . 0 1 7 0 4 9 . 9 1 8 0 1 9 . 4 2 2 02 8 . 8 5 7 . 2 1 3 6 5 9 . 2 1 4 4 2 8 . 7 3 0 4C o n t r o l ( 0 . 0 3 ) 5 4 . 3 1 6 0 5 5 . 4 1 5 2 3 0 . 9 2 883 7 . 5 2 8 . 7 1 0 4 3 1 . 3 2 3 2 1 7 . 3 1 3 6C o n t r o l ( 0 . 0 3 ) 4 4 . 9 1 6 0 5 1 . 5 1 2 8 2 9 . 8 2 4 8

M y c e l i u m g r o w t h130-

9 08 07 06 05 0AO3 02 01 00

P l e u r o t u s o s t r e a t u sR e u r o t u s F l o r i d aPleurotus eryngi i

CO2 con cen tra t ion (Vol 7o)1 0 1 5 2 0 2 5 3 0 3 5 4 0

Fig.lE f f e c t o f c a r b o n d i o x i d e o n t h e m y c e l i i i mg r o w t h o f Pleurotusostreatus, P . floridaand P . eryngii ( c o n t r o l-0.03% CO2 = 100 %M y c e l i u m g r o w t h )

It may be obse rved that a C O 2 con cen tra tio n in the air ofup to 28 vol .% vol. stimu lates the growth of Pleurotus ostreatusand P . florida. P.eryngii is the exc ept ion wi th a limit forstimulat ion of about 2 2% , by 28% C O 2myce lium is inhibited.The highe st con centra tion during our studies of 37.5 vol .%reduced myceli um g rowth in all three Pleur otus species byabou t 4 0% as compared wi th a con tro l at 0.0 3% C O 2 (see Table1 and Fig. 1)

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T a b l e 2E f f e c t o f CO2 c o n c e n t r a t i o n on the mycel i iam growth (6 r e p e a t s / e x p e r i m e n t s )in submerged cu l t ur e (P.florida s t r a i n PI)

5-day growth pH o f cu l t u r e 9 -day g row thmycel ium a t end o fw e i g h t i n mg e x p e r i me n t mg pH

Gas wi th ,air 576.4 5.3c o n t r o l 100.0 0.21A i r + 5 CO2 299. 3 5.9 67 3.0 5.8

80.0 0.08 80.0 0.14A i r + 10 CO2 333 .5 5.7 77 8. 9 5.8

50.0 0.01 150.0 0.15A i r + 15 CO2 349.8 5.6 766.9 5.8

40.0 0. 08 15 0.0 0 . 13

Similar results witha stimulating effectof CO2on thesubmerged cultures. ExperimentNo.2 (Table 2 ,were noticed.

The influenceof CO2onmycelium growth remained in thesame sequencein all theexperiments. With increasingCO2concentration in the air,alsothemycelium weightof thevarious cultures increased.'But theaverage differencesbetweentherepetitions were bigger thanthedifference betweenthe variantsof theexperiments.Inthis case,one canonlyspeakof atendency.The pHchangesby theadditionof gastothesubmerged culture show quiteaninsecure correlationtothemycelium growth.

In ordertoprove thatthePleurotus mycelium grows underpractical conditionsathigh concentrationsof CO2/theCO2content ininoculated substrata (sterileandpasteurized)wasmeasured.The air wasanalyzedatregular intervals,theresults being shownasF i g . 2 .Inordertotesttheactivityof various mycelium masses,twoquantitiesof inoculationmaterial were introduced intothesubstratum. Three daysafter inoculation,the CO2concentration inboth caseshadalready risentoover20 . Asexcepted,thecase with10inoculum showedagreater concentration thanthecase with5 .4 and 6days respectively after inoculation, CO2concen-trationhadreached amaximum, then decreased, finallyshowingnomaterial alteration fromthe 13th to the 31st day.After 13daysthesubstratumwaswell permeated.

Inatemperature experiment (mycelium growthat 25 and 30^C)with 1 oinoculation material,the CO2concentration climbedslowlyandcontinuously, reachinga maximum after20days,at 25 c thiswas 30 and at30OC,35 CO2(Fig.3). After this

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CO.

1 0 7 S p a w n

P F l o r i d a

0 2 4 6 8 10 12 14 16 18 2 0 22 24 26 28 30- d a y s

Fig.2Effect of spawn quantity{Pleurotus florida onCO2 concentration inwheat straw during themycelium growth.A. 10% spawnB. 5% spawn

0 2 4 6 8 10 12 14 16 18 20 22 24d a y s

Fig.3.Effect of temperature onCO2 concentration in wheatstraw substratum during themycelium growth (l ospawn)A. Pleurotus florida 30^CB. Pleurotus florida 25^0C.Pleurotus eryngii 30^CD . Pleurotus eryngii 25^0

time, the C O 2conc entr atio n dec line d. In the 30^C system thedecl ine was mor e rapid than in the 25^0 . Similar tempera tureeff ects wer e noted wi th p.eryngii, but wit h lower C O 2 concentration and slower growth. Here the C O 2did not r eac h theoptimum concentration for growth.

The high tolerance of Pleurotus species to C O 2is dem onstrated by this example, and the results could be reproducedin differ ently organized exp eri men ts. Carbon dioxi de prod uction is never thele ss depe nden t on the ingredien ts in the substratum (unpublished).

DISCUSSIONFrom the results the stimulating effect of C O 2on the myc eli umgrow th of 3 Ple uro tus s peci es is evi den t. Wit h an i ncre aseof the C O 2conce ntrat ion from 0.03 % to 16 %, the myc eliu mgro wth of both P.ostreatus and P.florida was stimula ted.P.eryngii still showed a posi tive result at 22. 0% C O 2 anda growth decrease at 28% C O 2. P.ostreatus and P. floridawere inhibited at a highe r conc entr atio n.

By repeating various experiments, the results were repro-duce able and in agree ment wit h those of Schanel (1970). He

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determined that p.ostreatus shows the bes t grow th at 10 vo l. %C O 2 (120.4 %), only 10 8% at 20 % C O 2 ,and at 3 0% C O 2a de pres sion of 83% against the control level (100%).

Under differ ent conditio ns in submerged culture similarincre ases of mycel ium gro wth wer e shown. 5% , 10% and 15% C O 2in the air incr ease the yie ld of myc eli um. On aera tion wit hnormal air the results were partly positive, partly negativecompared to nonaer ated culture s and those supplied wit h C O 2 /res pec tiv ely . The res ult s of the dail y grow th (mg/day) didnot corr esp ond as muc h as in the case of agar cult ure s. Thediffe rent res ults can be explained with the special beha viorof Basid iomyc etes in submerged c ultu re. The myce lium growthdepends on the special conditions of cultivation. We obtaineddif fer ent types of grow th (secondary spore pro duc tio n) andrepro duct ion rates in an exper iment with Pleur otus and otherBasid iomyc etes under similar con dit ions . The germi nation andgrowth of secondary spores have most proba bly inf luenced theyield of mycel ium in the submerged cult ure.

We could also prove the special tolerance of the Pleurotusmycelium toward high C O 2con cen tra tio ns bo th in steri le andpasteu rized straw substratum. 3 days after the inoculati onthere was a C O 2conce ntrat ion of more than 20 % ; the my celiumdev elo ped qui ckly and nor mal ly. In a closed s ystem, the C O 2concentration reached 35%. The high resistance of Pleuro tusand the stimulating effect of C O 2on the growth of Ple uro tusmyce lium are the most important bio -tec hnic al fa ctors inindustrial Pleurotus producti on.

The myce lium growth develops under semian aerobi c conditi onswi th a high con tent of C O 2in the gaseou s part of the s ubst ratum. This fact does not exclude the neces sity of oxy gen forthe myce lium gr owth. In 1974, Zadrazil determine d the relation between mycelium growth and the decrease of oxygen inthe nutrient media. The high concentration of C O 2in thesubstratum not only has a stimulating effect on the myceliumgrow th of the Ple uro tus s peci es but also an inhi biti ve effe cton oth er species in the non -st eri le str aw subs trat um (seeZadrazil, 1973).

Pleu rotus cultures (pasteurized substr atum whic h havegrown under semianaerobic conditions were free from otheraerobic organism. On the other hand, those cultures whichhave grown under the same condit ion s, but fully aerobic , we re,apart from Pleur otus myceli um, occupi ed by weed fungi .

In this case the C O 2is the mos t im porta nt fact or in Pleu rotus cul ture . With a high concen trati on of C O 2 / the abilit yof rep rod uct ion of the pro duc tio n is secure d and saves thehigh expendi ture of work of the sterile techn ique .

In mas s producti on of myceli um on solid media , the con tents

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of CO2could be regulated from the beginning of cultivationby adding and circulating C02-enriched air. Thus the temperature of CO2and O2could be regulated and kept at an optimum level during the cultivation.

The fruit body building of Pleurotus species in comparisonto the period of mycelium growth is a real aerobic process.The decomposition of the cellulose-lignin-complex is directlyrelated to the exchange of air (Zadrazil, 1974). Under aerobicconditions, the substratum was decomposed more rapidly thanunder semianaerobic conditions. In 1974, Zadrazil summarizedthe complex of cultivating conditions of Pleurotus production.

The experiments regarding CO2fixation of Pleurotusspecies will be continued.

Acknowledgement. Wewould liketoofferourthanksto theDeutsche Forschungsgemeinschaftfortheir supportof ourstudies.Wefurther wishtothank Mrs. Marianne Schneidereitfor herassistancein these experiments.

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Bronn,W.K., Luc,S.: (1973). 3.Symposiiam Technische Mikrobiologie, BerlinEger,G.: (1965).Arch.Mikrobiol.50,343-356MacFadyen,A.: (1973). Pedobiologia13,140-149Gundersen,K.: (1961).Nature190, 649Hermann,D.: (1963).Beij.Arch.Mikrobiol.45 373-397Jensen,K.F.: (1967). Forest Sei.13,384-389Lafferty,R.M.: (1963). Arch.Mikrobiol.44,373-405Lambert,E.B.: (1933).J.Agr.Res.47,599-608Niederpruem,D.J.: (1963).J.Bact.85,1300-1308Plunkett,B.E.: (1954).VIII Congr.Int.Bot., Paris, Rapp.andComm.Sect.18-20.p. 101-102Plunkett,B.E.: (1956).Ann .Bo t. (London)N.S. 20,563-586Rast,D., Bachofen,R.: (1965).VerhandlungenderSchwe iz. Na turforsehend en

Gesellschaft,p.125-129Rast,D., Bachofen,R.: (1967a). Arch.Mikrobiol.57,392-405Rast,D., Bachofen,R.: (1967b). Arch.Mikrobiol.58,339-356Rypcek,V.: (1966). Biologie holzzerstrender Pilze. Jena:VEB. G.FischerSan Antonio,J.P., Thomas,R.: (1972).Mushr oom Scien ce 8.. 623-629San Antonio,J.P., Thomas,R.: (1973). Mushroom News,Vol. 21, Nr. 12

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Tschierpe,H.J.: (1959). Mushroom Scie nce IV, 211-221 K openha genTschierpe,H.J.: (1972).Mushroom Science VIII , 553-591 LondonTschierpe,H.J.: (1958). Die Bedeutung des Kohlendioxyds fr den Kultur

champignon. Diss., Techn. Universitt BerlinValley,G., Rettger,L.F.: (1927).J.Bact. 14, 101-137Walsh,J.H., Stewart,C.S.: (1971). Trans.Br.Mycol.Soc. 57, 75-84Zadrazil,F.: (1971). Der Einflu von langjhrigen Dngungsmanahmen mit

Stroh- und Grndngung sowie Stickstoffdngung auf einige physikalische, chemische und biologische Bodeneigenschaften. Diss., UniversittGieen, Landw. Fakultt

Zadrazil,F., Schneidereit,M.: (1972). Der Champignon 135, 25-32Zadrazil,F.: (1973a).Berich t der D FG. (unpublished)Zadrazil,F.: (1973b).D er Champignon 139, 3-4Zadra zil,F .: (1974 inprint). Mushroom Science IX, TokioZycha, H.: (1937). Zbl. Bakt. II Abt. 9 7, 222-224

D r. Frantisek Zadrazil, Forschungsstelle von Sengbusch GmbH,D -200 0 Hamburg 67 , Waldredder 4 , Federal Republic o f Germany

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ifluence of co2 concentration on mycelium growth

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