Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi1 Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
S. L. ROSENBERG Physical Chemistry Laboratory, General Electric Corporate Research and Development, Schenectady, New York 12301 Accepted June 25, 1975
ROSENBERG, S. L. 1975. Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi. Can. J. Microbiol. 21: 1535-1540. A glucose-containing mineral medium supplemented with 0.01% yeast extract is described upon which all the species ofthermophilic and thermotolerant fungi tested will grow. Thirteen of the 21 species do not require the yeast extract supplement for growth. Using this solid, supplemented mineral medium, the pH and termperature optima for growth of all strains were measured. No correlation was found between temperature optimum and pH optimum among members of the group tested. ROSENBERG, S. L. 1975. Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi. Can. J. Microbiol. 21: 1535-1540. On decrit un milieu mineral contenant du glucose et enrichi de 0.01% d'extrait de levure sur lequel toutes les especes de moisissures thermophiliques et thermotolerantes CtudiCes se developpent. Treize des 21 especes n'exigent pas I'extrait de levure pour leur croissance. En utilisant ce milieu mineral solide, enrichi, on a determine le pH et la temperature optimum pour la croissance de toutes les especes. I1 n'existe aucune corrtlation entre la temperature optimum et le pH optimum parmi les membres du groupe etudiks. [Traduit par le journal]
Introduction Because of the interest of this laboratory in the thermophilic biodegradation of cellulosic and lignocellulosic materials (2), we were interested in screening a number of thermophilic and thermotolerant (5) fungi for these degradative abilities. Before this program could be initiated, however, optimal growth conditions for the chosen organiims had to be established. Since the presence of large quantities of easily metabolizable amino acids and sugars found in most complex media might lead to the repression of synthesis or the inhibition of activity of the enzyme systems of interest, it was necessary to find a simple mineral medium upon which all strains could grow. In addition, since no single temperature or pH could be expected to accommodate good growth of all these diverse organisms and indeed might be expected to inhibit the growth of some, the optima of these two parameters were sought. Approximate temperature optima have been published for all the organisms to be discussed (see Table 1 for references). These optima have 'Received March 27, 1975.
been established by measuring the rate of increase in diameter of single fungal colonies growing on the surface of agar plates. Temperature intervals of 5 to 10 "C have generally been used, and the organisms have been cultivated on a complex medium at a single (usually neutral to slightly alkaline) pH. In this work we have attempted first to define the optimal pH for growth of each organism in our supplemented mineral medium working at or near the published temperature optimum. We then performed more precise temperature optimum studies at each organism's pH optimum using the supplemented mineral medium to be described.
Materials and Methods Organisms The organisms used in this study are listed in Table 1. Included are published temperature optima and references to this work. All strains were obtained from the American Type Culture Collection, Rockville, Maryland, U.S.A. Media The following solid complex media were used: yeast starch agar, YpSs (5); yeast glucose agar, YG (5); peptone-glucose agar, PG (8); malt extract agar, MX (1.5z malt extract 2 z agar in deionized water); and yeast extract -starch agar, YES (0.5z yeast extract, 1.5z wheat starch, 1.59, agar in deionized water).
+
CAN. J . MICROBIOL. VOL. 21, 1975
TABLE 1 Strains of thermophilic and thermotolerant fungi examined
Organism
ATCC No.
Approx. temp. optimum, "C
Temp. ref.
Storage medium
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
-
Allescheria terresrris Apinis Aspergillus fun~igatlrsFresenius Chaetomiun~thermophile var. coprophile Cooney et Emerson C. therr?~ophile var. dissitrrm Cooney et Emerson Cl~rysosporirrm pruinosum (Gilman et Abbott) Carmichael Hrrmicola grisea var. thermoidea Cooney et Emerson H. i?zsolet~s Cooney et Emerson H. lan~rgitzosa(Griffon et Maublanc) Bunce Synonym: Thermomyces lanuginosus Tsiklinsky Malbranchea pulchella var. sulfurea (Miehei) M~rcormiehei Cooney et Emerson M. plrsillrrs Lindt Myriococcurn albon~ycesCooney et Emerson Sporotrichum puloerulentutn Novobranova Synonyms: Clzrysosporium prrrit~osum(10) C. lignorun~(7) S . thermophile Apinis Stilbella thermophila Fergus Talaronzyces emersonii Stolk T. tl~ermophilrrsStolk Synonym : Talaromyces duponti Tilertt~oascrrsaorantiacos Miehei Tl~ertnotnycesstellatus (Bunce) Apinis Thielauia therniophila Fergus et Sinden Torrrla thermophila Cooney et Emerson All media were prepared with tap water or deionized water as required. Peptone, yeast extract, malt extract, and agar were products of Difco Laboratories, Detroit Michigan, U.S.A. For temperature and pH optimum studies a solid supplemented mineral medium (SM) of the following composition was used: (NH4),S04, 5.0 g; K H 2 P 0 4 , 6.04 g ; Na2HP04, 0.85 g ; yeast extract, 0.1 g ; "trace elements" solution, 10 ml; agar, 15 g; deionized water to 960 ml. The trace elements solution had the following composition: MgSO4,7H20, 5.0g; ZnSO4.7H2O,0.2 g; FeSO, 7 H Z 0 ,0.5 g; MnS04.4H20,0.5 g; CaCI,, 0.5 g; versenol, 5.0 g; deionized water to 250 ml. The pH of the medium was adjusted to 6.2 with a few drops of 1.0 N HCI or 1.0 N NaOH using a Leeds and Northrup No. 7403 pH meter and a Beckman No. 39502 combination electrode, and the medium was autoclaved. After autoclaving, 40 ml of a 25% (wlv) heat-sterilized glucose solution was added to the mineral medium, and the pH was further adjusted as required by adding measured amounts of heat-sterilized 1 N NaOH or membrane-filter-sterilized I N HCI (Millipore Corp., Bedford, Massachusetts, U.S.A.). Petri dishes, 90 x 15 mm, were filled with about 50 ml of medium and dried overnight at 40 "C before use. All plates held their preset pH to k 0 . 2 unit in growth-free areas during an experiment. Cotlditiotu of Incubation Inoculated plates were placed in foil-capped battery jars that contained water to prevent drying, and the
1 14 5 5 13 5 5 5
MX YG YG YG PG YpSs YpSs MX
5 5 5 5 13
PG PG YES YpSs YES
5 3 9 5
YG YES PG YpSs
jars were incubated at the appropriate temperature. A t temperatures of 40 "C and above, small fans were placed inside the incubators to eliminate thermal gradients. All incubators held their preset temperatures to k 0 . 5 " C . Growth Measurements Plates of S M were centrally inoculated with small blocks of mycelium and agar cut from the leading edge of colonies growing on solid medium. The inoculating blocks were about 2 mm square and 1 mm thick. This produced on each plate a single, circular colony whose radius was measured in four directions daily along two perpendicular colony diameters. The average deviation from the mean of these measurements was always less than 2 mm and generally less than 1 mm. Conditions of p H or temperature leading to the maximum increase in colony radius in a given period of time were taken as optimum for the growth of the organism (16, 17). A preliminary p H optimum study was conducted using SM and inocula grown on P G plates. Colonies which grew fastest in the preliminary series were used a s an inoculum source for a more refined pH optimum study, the results of which are shown in Fig. 1. Temperatures used in this study were set a t or near the published optimum or within the optimum range for each organism (see Fig. 1). Sporotrichum puluerulentum was an exception. It was incubated at 25 "C t o slow the growth rate so that pH-dependent differences could be measured more accurately. Temperature optimum studies were carried out in S M
ROSENBERG: TEMPERATURE AND pH OPTIMA O F FUNGI
TABLE 2
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
Summary of physiological and nutritional characteristics of thermophilic and thermotolerant fungi studied*
Organism
Optimum PH
Optimum Temp., "C
Yeast extract supplementation required
Allescheria terrestris Aspergillus fumigatus Chaetomium thermophile var. coprophile C. thermophile var. dissitum Chrysosporium pruinosum Humicola grisea var. thermoidea H. insolens H. lanuginosa Malbranchea pulchella var. sulfurea Mucor miehei M . pusillus Myriococcum albomyces Sporotrichum puluerrtlentum S . thermophile Stilbella thermophila Talaromyces emersonir T. thermophilus Thermoascus aurantiacus Thermomyces stellatus Thielavia thermophila Torula thermophila
4.0-6.1 5.9-6.8 6.8 5.5 4.1-4.9 7.3-7.6 7.6 6.8-7.3 7.2 5.5 6.1 7.6 3.9-5.2 6.1-6.8 7.9 3.4-5.4 7.2-8.1 6.8 5.4-5.9 6.8 7.3
42.5-47.5 3 5-40 50-52.5 47.5 37.5 47.5 45-47.5 47.5-52.5 47.5 42.5-47.5 40-45 45 40 42.5-45 45 47.5 47.5 52.5 37.5 40-45 42.5-47.5
Not No Yes Yes No Yes Yes No No No: No Yes No No Yes No No No Yes No Yes
*Temperature and p H optlma l ~ s t e d~ n c l u d eall d a t a polnts from Fig. 1 curves, whlch are >95Y, o f maxlmum value ?Absence of a yeast extract requirement means that the colony could spread from the ~ n o c u l a t ~ opolnt n and cover the plate. Presence of a requirement means that either n o growth occurred after 10 days of Incubation or that s l ~ g hgrowth t occurred on the nutrients present In the ~noculatlngblock followed by a 10-day p e r ~ o do f n o further growth (Increase In colony d ~ a m e t e r ) . $Growth much h e a v ~ e rw ~ t hadded yeast extract.
that some differences in temperature optima occurred from strain to strain in this species. The temperature optima obtained for M. Storage of Cultrires pulchella var. sulfurea, M. miehei, S. thermoCultures were stored at room temperature on slants phile, H. grisea var. thermoidea, T. thern~ophilus, of the complex medium, on which they grew well or best (Table 1). They were transferred to fresh homologous and H. insolens are higher than values recently reported by Chapman (4). They agree well, medium every 6 months. however, with data presented by Kane and Results and Discussion Mullins (11) and Crisan (6). These differences Figure 1 shows the relations between tempera- may be strain-specific. In earlier studies, summarized by Crisan (6), ture, pH, and growth for the 21 organisms examined. There appear to be no obvious cor- the temperature intervals used varied between 5 relations between temperature and pH optima and 10 "C. In this work we chose smaller 2.5 "C among members of the group, nor is there much intervals in the region of fastest growth to similarity between members of the same genus measure the peak or peak range more precisely. in this respect. Temperature preferences do seem In some cases, cf. Allescheria terrestris and to be similar at the level of the genus, but more Thielavia thermophila, a 2.5 "C change in incubation temperature can lead to a 50% decline in extensive studies must be done to confirm this. The temperature optima obtained (summa- apparent growth rate. Using 5 or 10 "C steps in rized in Table 2) generally fall within or overlap incubation temperature would obscure the the ranges reported previously for these species observation of the sensitivity of such organisms (6, 10, 11, 13) with the exception of Thermoascus to small temperature changes. For future nutritional and physiological aurantiacus. Cooney and Emerson (5) noted at or very near each organism's optimum pH. The inocula were grown as described above at their p H optima.
CAN. J . MICROBIOL. VOL. 21, 1975 ALLESCHERIA T E R R E S T R I S A S P E R G I L L U SFUYIGATUS - p H 5.2, 3 DAYS' - - p H 6.7, 3 D A Y S
---
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
40
40°,
---
4 DAYS
40°,
3 DAYS
r
-
C H A E T O Y I U Y THERMOPHILE VAR. COPROPHILE
p p
- p H 6.7,
-- ---
45',
2 DAYS 4 DAYS
.
-
25 20 I5 10
VAR. D l S S l T U Y
I - p H 5.2,
.-
u
45',
4 DAYS
CHRYSOSPORIUY PRUINOSUY H U M I C O L A GRISEA VAR. p H 4.3, 1 D A Y rHERYOlDEA 40°, I DAY 4-pH7.0,3DAYS
-
-
4 ---
4
4 DAYS
?\
HUMICOLA INSOLENS --
40
I
pH 7 7 , 2 DAYS 40°,
3 DAYS
---
HUMICOLA LANUGINOSA -
-
-
- pH 7 0 , 3 DAYS - - - 4 5 ' , 3 DAYS
--
MALBRANCHEA PULCHELLA VAR S U L F U R E A p H 71, 7 DAYS -4 5 O , 5 DAYS
-
,_*'
10
7' --
1
4
- pH 6 7,
4 0 6 , 1 DAY
4
---
I
TEMP
MUCOR P U S I L L U S
pH 5 2 , 1 DAY
40°,
l OAY 1 DAY
MYRIOCOCCUM A L B O Y Y C E S pH 7 6 , 2 DAYS 40",
3 DAYS
-
ROSENBERG: TEMPERATURE AND pH OPTIMA O F FUNGI SPOROTRICHUM THERMOPHILE
SPOROTRICHUM PULVERULENTUM
- pH 4 . 8 , ---
40
-
,-..-'i
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
35-
/
30 25E
- ---
2 DAYS
25',
- pH 6.1,
1 DAY
45',
STILEELLA THERMOPHILA
- pH 7.9, 5 D A Y S - --- 4 S 0 , 4 D A Y S
3 DAYS
3 DAYS
'L
1
z:rrA;{//, I 20-
E
\
1
---
U 0 LL
pH 4 3 , 4S0,
- - pH 7.6,
4 DAYS
---
3 DAYS
-
4 DAYS
p H 6.7,
---
4 S 0 , 5 DAYS
40°,
1 DAY
4 DAYS
--
--
20 15
I
'.
10 5 0
TEMP
'C
1
35
45
55
25
THERMOMYCES S T E L L A T U S -- p H 5 2, I I DAYS
---
E
L
p
I
H
40°,
I
I
2
I
4
I
6
l
35
45
55
I
25
---
g
8
l
I
4S0,
I
2
---
3 DAYS
3 DAYS
I
4
I
I
6
35
45
55
TORULA T H E R M O P H I L A
T H l E L A V l A THERMOPHILA
- PH 6 7 .
13 DAYS
I
\
L
\.
25
/..'
I
I
1
8
;
l
2
p H 7 0 , 3 DAYS 4 0 ° , 3 DAYS
I
I
4
I
6
I
I
l
~
l
8
FIG.1 (Continued). Average radius of fungal colonies at one time point as a function of temperature -or pH---.
studies we also wished to describe the pH optima for growth of these organisms. To our knowledge, this is the first published study of p H requirements for growth of thermophilic fungi. These optima varied widely from a low of 3.4-5.4 for Talaromyces emersonii to a high of 7.2-8.1 for T. thermophilus. Originally, we had hoped to be able to use a n unsupplemented mineral medium for this work. This proved impossible, however, so the yeast
extract (0.01%) was included. Since nothing was then known about the p H requirements of these organisms, the possibility existed that the supplementation was needed to fill a growth requirement created by the imposition of an extreme pH, relative to the organism's optimum (12). After the p H and temperature optima of all strains had been determined, the need for 0.01% yeast extract supplementation of the solid
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
1540
CAN. J. MICROBIOL. VOL. 21. 1975
mineral medium was reexamined using the established optimum conditions. These results are shown in the last column of Table 2. These are the same results that were obtained initially working a t the temperatures shown in the legends of Fig. 1 and a fixed p H of 7.5. This p H was chosen for the initial studies since it was known that all the organisms could grow on complex media of this pH. The p H and temperature optima abstracted from the data in Fig. 1 are also shown in Table 2. Preliminary experiments (Rosenberg, unpublished results) indicate that those organisms which require the yeast extract supplement use the glucose in the medium as the main growth substrate.
Acknowledgment The author thanks R. E. Brooks. E. V. Crisan, and R. Emerson for helpful discussions and thoughtful criticism. 1. APINIS. A. E. 1963. Occurrence of thermophilous microfungi in certain alluvial soils near Nottingham. Nova Hedwigia Z. Kryptogamenkd. 5: 57-78. W. D. 1974. Single cell proteins from cel2. BELLAMY. lulosic wastes. Biotechnol. Bioeng. 16: 869-880. 3. BUNCE.M. E. 1961. Hrrtnicoln slellalrrs sp. nov., a thermophilic mold from hay. Trans. Br. Mycol. Soc. 44: 372-376. 4. C H A P M A NE. , S. 1974. Effect of temperature on growth rate of seven thermophilic fungi. Mycologia, 66: 542-546. 5. COONEY. D. G., andR. EXIERSON. 1964. Thermophilic fungi. W. H. Freeman and Co., San Francisco.
6. CRISAN,E . V. 1973. Current concepts of thermorphilism and the thermophilic fungi. Mycologia, 65: 1171-1 198. 7. ERIKSSON, K. E.,and W. RZEDOWSKI. 1969. Extracellular enzyme system utilized by the fungus Chrysosporitrm lignor~tm for the breakdown of cellulose. l . Studies on the enzyme production. Arch. Biochem. Biophys. 129: 683-688. 8. FERGUS, C. L. 1964. Thermophilic and thermotolerant molds and actinomycetes of mushroom compost during peak heating. Mycologia, 56: 267-284. 9. FERGUS. C. L., and J. W. SINDEN.1969. A new thermophilic fungus from mushroom compost: T l ~ i e l a v i a ~ l ~ e r m o p h i lspec. n nov. Can. J. Bot. 47: 1635-1637. 10. HOFSTEN,B. v., A N D A. v. HOFSTEN.1974. Ultrastructure of a thermotolerant basidiornycete possibly suitable for production of food protein. Appl. Microbiol. 27: 1142-1 148. 1 I. KANE,B. E . , and J. T . MULLINS. 1973. Thermophilic fungi in a municipal waste compost system. Mycologia, 65: 1087-1 100. 12. KOSER.S. A. 1968. Vitamin reauirements of bacteria and yeasts. Charles C. ~ h o m a s Springfield, , Ill. p. 505 ff. 13. NILSSON,T . 1965. Mikroorganismer i flisstackar. Sven. Papperstidn. 68: 495-499. 14. OFOSU-ASIEDU, A., and R. S. S M I T H 1973. . Some factors affecting wood degradation by thermophilic and thermotolerant fungi. Mycologia, 65: 87-98. 15. STOLK,A. C. 1965. Thermophilic species of Tolarotnyces Benjamin and Tl~ermonscrrsMiehei. Antonie van Leeuwenhoek J. Microbiol. Serol. 31: 262-276. 16. T R I N C IA.. P. J . 1969. A kinetic study ofthegrowthof Aspergillrrs nid~ilonsand other fungi. J. Gen. Microbiol. 57: 1 1-24. 17. T R I N C IA, . P. J. 1971. Influence of the width of the peripheral growth zone on the radial growth rate of fungal colonies on solid media. J. Gen. Microbiol. 67: 325-344.
S. L. ROSENBERG Physical Chemistry Laboratory, General Electric Corporate Research and Development, Schenectady, New York 12301 Accepted June 25, 1975
ROSENBERG, S. L. 1975. Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi. Can. J. Microbiol. 21: 1535-1540. A glucose-containing mineral medium supplemented with 0.01% yeast extract is described upon which all the species ofthermophilic and thermotolerant fungi tested will grow. Thirteen of the 21 species do not require the yeast extract supplement for growth. Using this solid, supplemented mineral medium, the pH and termperature optima for growth of all strains were measured. No correlation was found between temperature optimum and pH optimum among members of the group tested. ROSENBERG, S. L. 1975. Temperature and pH optima for 21 species of thermophilic and thermotolerant fungi. Can. J. Microbiol. 21: 1535-1540. On decrit un milieu mineral contenant du glucose et enrichi de 0.01% d'extrait de levure sur lequel toutes les especes de moisissures thermophiliques et thermotolerantes CtudiCes se developpent. Treize des 21 especes n'exigent pas I'extrait de levure pour leur croissance. En utilisant ce milieu mineral solide, enrichi, on a determine le pH et la temperature optimum pour la croissance de toutes les especes. I1 n'existe aucune corrtlation entre la temperature optimum et le pH optimum parmi les membres du groupe etudiks. [Traduit par le journal]
Introduction Because of the interest of this laboratory in the thermophilic biodegradation of cellulosic and lignocellulosic materials (2), we were interested in screening a number of thermophilic and thermotolerant (5) fungi for these degradative abilities. Before this program could be initiated, however, optimal growth conditions for the chosen organiims had to be established. Since the presence of large quantities of easily metabolizable amino acids and sugars found in most complex media might lead to the repression of synthesis or the inhibition of activity of the enzyme systems of interest, it was necessary to find a simple mineral medium upon which all strains could grow. In addition, since no single temperature or pH could be expected to accommodate good growth of all these diverse organisms and indeed might be expected to inhibit the growth of some, the optima of these two parameters were sought. Approximate temperature optima have been published for all the organisms to be discussed (see Table 1 for references). These optima have 'Received March 27, 1975.
been established by measuring the rate of increase in diameter of single fungal colonies growing on the surface of agar plates. Temperature intervals of 5 to 10 "C have generally been used, and the organisms have been cultivated on a complex medium at a single (usually neutral to slightly alkaline) pH. In this work we have attempted first to define the optimal pH for growth of each organism in our supplemented mineral medium working at or near the published temperature optimum. We then performed more precise temperature optimum studies at each organism's pH optimum using the supplemented mineral medium to be described.
Materials and Methods Organisms The organisms used in this study are listed in Table 1. Included are published temperature optima and references to this work. All strains were obtained from the American Type Culture Collection, Rockville, Maryland, U.S.A. Media The following solid complex media were used: yeast starch agar, YpSs (5); yeast glucose agar, YG (5); peptone-glucose agar, PG (8); malt extract agar, MX (1.5z malt extract 2 z agar in deionized water); and yeast extract -starch agar, YES (0.5z yeast extract, 1.5z wheat starch, 1.59, agar in deionized water).
+
CAN. J . MICROBIOL. VOL. 21, 1975
TABLE 1 Strains of thermophilic and thermotolerant fungi examined
Organism
ATCC No.
Approx. temp. optimum, "C
Temp. ref.
Storage medium
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
-
Allescheria terresrris Apinis Aspergillus fun~igatlrsFresenius Chaetomiun~thermophile var. coprophile Cooney et Emerson C. therr?~ophile var. dissitrrm Cooney et Emerson Cl~rysosporirrm pruinosum (Gilman et Abbott) Carmichael Hrrmicola grisea var. thermoidea Cooney et Emerson H. i?zsolet~s Cooney et Emerson H. lan~rgitzosa(Griffon et Maublanc) Bunce Synonym: Thermomyces lanuginosus Tsiklinsky Malbranchea pulchella var. sulfurea (Miehei) M~rcormiehei Cooney et Emerson M. plrsillrrs Lindt Myriococcurn albon~ycesCooney et Emerson Sporotrichum puloerulentutn Novobranova Synonyms: Clzrysosporium prrrit~osum(10) C. lignorun~(7) S . thermophile Apinis Stilbella thermophila Fergus Talaronzyces emersonii Stolk T. tl~ermophilrrsStolk Synonym : Talaromyces duponti Tilertt~oascrrsaorantiacos Miehei Tl~ertnotnycesstellatus (Bunce) Apinis Thielauia therniophila Fergus et Sinden Torrrla thermophila Cooney et Emerson All media were prepared with tap water or deionized water as required. Peptone, yeast extract, malt extract, and agar were products of Difco Laboratories, Detroit Michigan, U.S.A. For temperature and pH optimum studies a solid supplemented mineral medium (SM) of the following composition was used: (NH4),S04, 5.0 g; K H 2 P 0 4 , 6.04 g ; Na2HP04, 0.85 g ; yeast extract, 0.1 g ; "trace elements" solution, 10 ml; agar, 15 g; deionized water to 960 ml. The trace elements solution had the following composition: MgSO4,7H20, 5.0g; ZnSO4.7H2O,0.2 g; FeSO, 7 H Z 0 ,0.5 g; MnS04.4H20,0.5 g; CaCI,, 0.5 g; versenol, 5.0 g; deionized water to 250 ml. The pH of the medium was adjusted to 6.2 with a few drops of 1.0 N HCI or 1.0 N NaOH using a Leeds and Northrup No. 7403 pH meter and a Beckman No. 39502 combination electrode, and the medium was autoclaved. After autoclaving, 40 ml of a 25% (wlv) heat-sterilized glucose solution was added to the mineral medium, and the pH was further adjusted as required by adding measured amounts of heat-sterilized 1 N NaOH or membrane-filter-sterilized I N HCI (Millipore Corp., Bedford, Massachusetts, U.S.A.). Petri dishes, 90 x 15 mm, were filled with about 50 ml of medium and dried overnight at 40 "C before use. All plates held their preset pH to k 0 . 2 unit in growth-free areas during an experiment. Cotlditiotu of Incubation Inoculated plates were placed in foil-capped battery jars that contained water to prevent drying, and the
1 14 5 5 13 5 5 5
MX YG YG YG PG YpSs YpSs MX
5 5 5 5 13
PG PG YES YpSs YES
5 3 9 5
YG YES PG YpSs
jars were incubated at the appropriate temperature. A t temperatures of 40 "C and above, small fans were placed inside the incubators to eliminate thermal gradients. All incubators held their preset temperatures to k 0 . 5 " C . Growth Measurements Plates of S M were centrally inoculated with small blocks of mycelium and agar cut from the leading edge of colonies growing on solid medium. The inoculating blocks were about 2 mm square and 1 mm thick. This produced on each plate a single, circular colony whose radius was measured in four directions daily along two perpendicular colony diameters. The average deviation from the mean of these measurements was always less than 2 mm and generally less than 1 mm. Conditions of p H or temperature leading to the maximum increase in colony radius in a given period of time were taken as optimum for the growth of the organism (16, 17). A preliminary p H optimum study was conducted using SM and inocula grown on P G plates. Colonies which grew fastest in the preliminary series were used a s an inoculum source for a more refined pH optimum study, the results of which are shown in Fig. 1. Temperatures used in this study were set a t or near the published optimum or within the optimum range for each organism (see Fig. 1). Sporotrichum puluerulentum was an exception. It was incubated at 25 "C t o slow the growth rate so that pH-dependent differences could be measured more accurately. Temperature optimum studies were carried out in S M
ROSENBERG: TEMPERATURE AND pH OPTIMA O F FUNGI
TABLE 2
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
Summary of physiological and nutritional characteristics of thermophilic and thermotolerant fungi studied*
Organism
Optimum PH
Optimum Temp., "C
Yeast extract supplementation required
Allescheria terrestris Aspergillus fumigatus Chaetomium thermophile var. coprophile C. thermophile var. dissitum Chrysosporium pruinosum Humicola grisea var. thermoidea H. insolens H. lanuginosa Malbranchea pulchella var. sulfurea Mucor miehei M . pusillus Myriococcum albomyces Sporotrichum puluerrtlentum S . thermophile Stilbella thermophila Talaromyces emersonir T. thermophilus Thermoascus aurantiacus Thermomyces stellatus Thielavia thermophila Torula thermophila
4.0-6.1 5.9-6.8 6.8 5.5 4.1-4.9 7.3-7.6 7.6 6.8-7.3 7.2 5.5 6.1 7.6 3.9-5.2 6.1-6.8 7.9 3.4-5.4 7.2-8.1 6.8 5.4-5.9 6.8 7.3
42.5-47.5 3 5-40 50-52.5 47.5 37.5 47.5 45-47.5 47.5-52.5 47.5 42.5-47.5 40-45 45 40 42.5-45 45 47.5 47.5 52.5 37.5 40-45 42.5-47.5
Not No Yes Yes No Yes Yes No No No: No Yes No No Yes No No No Yes No Yes
*Temperature and p H optlma l ~ s t e d~ n c l u d eall d a t a polnts from Fig. 1 curves, whlch are >95Y, o f maxlmum value ?Absence of a yeast extract requirement means that the colony could spread from the ~ n o c u l a t ~ opolnt n and cover the plate. Presence of a requirement means that either n o growth occurred after 10 days of Incubation or that s l ~ g hgrowth t occurred on the nutrients present In the ~noculatlngblock followed by a 10-day p e r ~ o do f n o further growth (Increase In colony d ~ a m e t e r ) . $Growth much h e a v ~ e rw ~ t hadded yeast extract.
that some differences in temperature optima occurred from strain to strain in this species. The temperature optima obtained for M. Storage of Cultrires pulchella var. sulfurea, M. miehei, S. thermoCultures were stored at room temperature on slants phile, H. grisea var. thermoidea, T. thern~ophilus, of the complex medium, on which they grew well or best (Table 1). They were transferred to fresh homologous and H. insolens are higher than values recently reported by Chapman (4). They agree well, medium every 6 months. however, with data presented by Kane and Results and Discussion Mullins (11) and Crisan (6). These differences Figure 1 shows the relations between tempera- may be strain-specific. In earlier studies, summarized by Crisan (6), ture, pH, and growth for the 21 organisms examined. There appear to be no obvious cor- the temperature intervals used varied between 5 relations between temperature and pH optima and 10 "C. In this work we chose smaller 2.5 "C among members of the group, nor is there much intervals in the region of fastest growth to similarity between members of the same genus measure the peak or peak range more precisely. in this respect. Temperature preferences do seem In some cases, cf. Allescheria terrestris and to be similar at the level of the genus, but more Thielavia thermophila, a 2.5 "C change in incubation temperature can lead to a 50% decline in extensive studies must be done to confirm this. The temperature optima obtained (summa- apparent growth rate. Using 5 or 10 "C steps in rized in Table 2) generally fall within or overlap incubation temperature would obscure the the ranges reported previously for these species observation of the sensitivity of such organisms (6, 10, 11, 13) with the exception of Thermoascus to small temperature changes. For future nutritional and physiological aurantiacus. Cooney and Emerson (5) noted at or very near each organism's optimum pH. The inocula were grown as described above at their p H optima.
CAN. J . MICROBIOL. VOL. 21, 1975 ALLESCHERIA T E R R E S T R I S A S P E R G I L L U SFUYIGATUS - p H 5.2, 3 DAYS' - - p H 6.7, 3 D A Y S
---
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
40
40°,
---
4 DAYS
40°,
3 DAYS
r
-
C H A E T O Y I U Y THERMOPHILE VAR. COPROPHILE
p p
- p H 6.7,
-- ---
45',
2 DAYS 4 DAYS
.
-
25 20 I5 10
VAR. D l S S l T U Y
I - p H 5.2,
.-
u
45',
4 DAYS
CHRYSOSPORIUY PRUINOSUY H U M I C O L A GRISEA VAR. p H 4.3, 1 D A Y rHERYOlDEA 40°, I DAY 4-pH7.0,3DAYS
-
-
4 ---
4
4 DAYS
?\
HUMICOLA INSOLENS --
40
I
pH 7 7 , 2 DAYS 40°,
3 DAYS
---
HUMICOLA LANUGINOSA -
-
-
- pH 7 0 , 3 DAYS - - - 4 5 ' , 3 DAYS
--
MALBRANCHEA PULCHELLA VAR S U L F U R E A p H 71, 7 DAYS -4 5 O , 5 DAYS
-
,_*'
10
7' --
1
4
- pH 6 7,
4 0 6 , 1 DAY
4
---
I
TEMP
MUCOR P U S I L L U S
pH 5 2 , 1 DAY
40°,
l OAY 1 DAY
MYRIOCOCCUM A L B O Y Y C E S pH 7 6 , 2 DAYS 40",
3 DAYS
-
ROSENBERG: TEMPERATURE AND pH OPTIMA O F FUNGI SPOROTRICHUM THERMOPHILE
SPOROTRICHUM PULVERULENTUM
- pH 4 . 8 , ---
40
-
,-..-'i
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
35-
/
30 25E
- ---
2 DAYS
25',
- pH 6.1,
1 DAY
45',
STILEELLA THERMOPHILA
- pH 7.9, 5 D A Y S - --- 4 S 0 , 4 D A Y S
3 DAYS
3 DAYS
'L
1
z:rrA;{//, I 20-
E
\
1
---
U 0 LL
pH 4 3 , 4S0,
- - pH 7.6,
4 DAYS
---
3 DAYS
-
4 DAYS
p H 6.7,
---
4 S 0 , 5 DAYS
40°,
1 DAY
4 DAYS
--
--
20 15
I
'.
10 5 0
TEMP
'C
1
35
45
55
25
THERMOMYCES S T E L L A T U S -- p H 5 2, I I DAYS
---
E
L
p
I
H
40°,
I
I
2
I
4
I
6
l
35
45
55
I
25
---
g
8
l
I
4S0,
I
2
---
3 DAYS
3 DAYS
I
4
I
I
6
35
45
55
TORULA T H E R M O P H I L A
T H l E L A V l A THERMOPHILA
- PH 6 7 .
13 DAYS
I
\
L
\.
25
/..'
I
I
1
8
;
l
2
p H 7 0 , 3 DAYS 4 0 ° , 3 DAYS
I
I
4
I
6
I
I
l
~
l
8
FIG.1 (Continued). Average radius of fungal colonies at one time point as a function of temperature -or pH---.
studies we also wished to describe the pH optima for growth of these organisms. To our knowledge, this is the first published study of p H requirements for growth of thermophilic fungi. These optima varied widely from a low of 3.4-5.4 for Talaromyces emersonii to a high of 7.2-8.1 for T. thermophilus. Originally, we had hoped to be able to use a n unsupplemented mineral medium for this work. This proved impossible, however, so the yeast
extract (0.01%) was included. Since nothing was then known about the p H requirements of these organisms, the possibility existed that the supplementation was needed to fill a growth requirement created by the imposition of an extreme pH, relative to the organism's optimum (12). After the p H and temperature optima of all strains had been determined, the need for 0.01% yeast extract supplementation of the solid
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of Melbourne on 06/06/13 For personal use only.
1540
CAN. J. MICROBIOL. VOL. 21. 1975
mineral medium was reexamined using the established optimum conditions. These results are shown in the last column of Table 2. These are the same results that were obtained initially working a t the temperatures shown in the legends of Fig. 1 and a fixed p H of 7.5. This p H was chosen for the initial studies since it was known that all the organisms could grow on complex media of this pH. The p H and temperature optima abstracted from the data in Fig. 1 are also shown in Table 2. Preliminary experiments (Rosenberg, unpublished results) indicate that those organisms which require the yeast extract supplement use the glucose in the medium as the main growth substrate.
Acknowledgment The author thanks R. E. Brooks. E. V. Crisan, and R. Emerson for helpful discussions and thoughtful criticism. 1. APINIS. A. E. 1963. Occurrence of thermophilous microfungi in certain alluvial soils near Nottingham. Nova Hedwigia Z. Kryptogamenkd. 5: 57-78. W. D. 1974. Single cell proteins from cel2. BELLAMY. lulosic wastes. Biotechnol. Bioeng. 16: 869-880. 3. BUNCE.M. E. 1961. Hrrtnicoln slellalrrs sp. nov., a thermophilic mold from hay. Trans. Br. Mycol. Soc. 44: 372-376. 4. C H A P M A NE. , S. 1974. Effect of temperature on growth rate of seven thermophilic fungi. Mycologia, 66: 542-546. 5. COONEY. D. G., andR. EXIERSON. 1964. Thermophilic fungi. W. H. Freeman and Co., San Francisco.
6. CRISAN,E . V. 1973. Current concepts of thermorphilism and the thermophilic fungi. Mycologia, 65: 1171-1 198. 7. ERIKSSON, K. E.,and W. RZEDOWSKI. 1969. Extracellular enzyme system utilized by the fungus Chrysosporitrm lignor~tm for the breakdown of cellulose. l . Studies on the enzyme production. Arch. Biochem. Biophys. 129: 683-688. 8. FERGUS, C. L. 1964. Thermophilic and thermotolerant molds and actinomycetes of mushroom compost during peak heating. Mycologia, 56: 267-284. 9. FERGUS. C. L., and J. W. SINDEN.1969. A new thermophilic fungus from mushroom compost: T l ~ i e l a v i a ~ l ~ e r m o p h i lspec. n nov. Can. J. Bot. 47: 1635-1637. 10. HOFSTEN,B. v., A N D A. v. HOFSTEN.1974. Ultrastructure of a thermotolerant basidiornycete possibly suitable for production of food protein. Appl. Microbiol. 27: 1142-1 148. 1 I. KANE,B. E . , and J. T . MULLINS. 1973. Thermophilic fungi in a municipal waste compost system. Mycologia, 65: 1087-1 100. 12. KOSER.S. A. 1968. Vitamin reauirements of bacteria and yeasts. Charles C. ~ h o m a s Springfield, , Ill. p. 505 ff. 13. NILSSON,T . 1965. Mikroorganismer i flisstackar. Sven. Papperstidn. 68: 495-499. 14. OFOSU-ASIEDU, A., and R. S. S M I T H 1973. . Some factors affecting wood degradation by thermophilic and thermotolerant fungi. Mycologia, 65: 87-98. 15. STOLK,A. C. 1965. Thermophilic species of Tolarotnyces Benjamin and Tl~ermonscrrsMiehei. Antonie van Leeuwenhoek J. Microbiol. Serol. 31: 262-276. 16. T R I N C IA.. P. J . 1969. A kinetic study ofthegrowthof Aspergillrrs nid~ilonsand other fungi. J. Gen. Microbiol. 57: 1 1-24. 17. T R I N C IA, . P. J. 1971. Influence of the width of the peripheral growth zone on the radial growth rate of fungal colonies on solid media. J. Gen. Microbiol. 67: 325-344.
