FEMS Microhk)logyLetters 93 119t~2)279-284 ~ 1992 Federation of European Micrt~biological S~cieties (B78-1(197/92/$05.(10 Published by Elsevier
279
FEMSLE 114911
Description of conidia from submerged cultivation of Thermomyces ianuginosus for use as a uniform inoculum Susanne Havn Eriksen, Iben Haasum, Bo Jensen and J0rgen Olsen Depaanlent of Gent'ral Micr~bioh~g),, Univer,~ityof Cop~'nhagvn, Copf~lhag~'n, Denmark Received 21) March 1992 Accepted 23 March t992
Key words: Thermophilic fungus; Morphology; Conidia; Germination: Thermom~'ces ianuginosus
1. SUMMARY Conidia produced by submerged cultivation of the thermophilic fungus Therrnomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45°C more than 90% germination was achieved at 50°C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia. 2. INTRODUCTION The
imperfect,
thermophilic fungus Therreproduces asexually by
momyces ianuginosus
Corre.~pondence to: S.H. Eriksen, Deparlment of General Microbiology, University of Copenhagen. Stalvgade 83H. DK1307, Copenhagen K. Denmark.
forming aleurioconidia [1,2]. Descriptions of conidia from this fungus regarding morphology [ 1.3-5] and physiology [4,6] have until now referred to conidia derived from surface cultltres grown on agar plates. These conidia, when mature, are dark brown, globose with a diameter in the range of 5.5-12 #m, and the thick outer conidial wall is characteristically wrinkled [1,3-5]. Immature conidia have been described as colourless and smooth walled [1]. Physiological studies of conidia from "1".lanuginosu.~ are sparse. Conidia from 1". lanuginosus germinate welt in a complex medium containing yeast extract, whereas germination is poor in synthetic medium [6]. Germination began with a swelling of the spore, the spore volume increased by 49% in 230 min and the tip of the germ tube was observed emerging through a crack in the spore wall observed after 195 rain [4]. T. lal~ttginost~s however, also conidiates in submerged cultivation in liquid medium. In this study, conidia from surface and submerged cultivation have been compared with the aim of obtaining a homogeneous and reproducible inoculum.
28~1
3. MATERIALS AND METHODS
3.1. Microorganism Thermoon,ces kmughzosus (single spore isolate from strain no. 1457, The Royal VetcrinaJ3, and Agricultural University, Copenhagen, Denm~rk) was used. Stock cultures of the fungus were produccd as previously described [7] exccpt for temperature which was 45°C. After 10 days at 45°C, the stock cultures were kept at ambient temperaturc.
3.2. Medium The medium used was modified from that previously reported [7] and contained (in g/l): soluble starch (Merck, 1252), 15.1); yeasl extract (Difco, Bactok 4.0; K2HPO4"3H20, 1.O; MgSOa • 7H20, 0.5; and 0.1 ml Vogel's trace element solution [8]. All components were dissolved in distilled water. The pH was adjusted to 6.5. For surface cultivation the medium was gelled with 20 g/I agar.
3.3. Prodt~ction of conidia Surface conidia were obtained by suspending conidia from two 9-cm agar plate cultures in 50 ml of a salt solution comprising (g/I): K2HPO 4 • 3H20, 1.0; MgSO 4 • 7H 20, 0.5; and Triton X-100, 0.(15% (v/v) and filtcring through four layers of sterile gauze.
Submerged conidia were produced in 300-ml Erlenmeyer flasks with 50 ml of medium. Each flask was inoculated with surface conidia to a final concentration of approx. 1 × l0 r' per ml and incubated on a rotary shaker in a constant-temperature incubator for 3 days at 220 rpm and 45°C, unless otherwise stated. The conidia were separated from the mycelium by filtering through four layers of sterile gauze. The filtrate was centrifuged at l l 0 0 × g (Sorvali GSA-rotor)for 10 rain at 5°C. The pellet was washed once and resuspended in the salt solution described .4bore to a concentration of approx. 2.5 X 10 7 conidia per ml.
3. 4. Germination e.~periments Germination experiments were performed in 3(}0-ml Erlenmeyer flasks with 50 ml medium, preheated to 50°C. The flasks were inoculated to a final concentration of 1 × 10~' conidia per ml and incubated at 50~C with shaking at 220 rpm. Samples of 1 ml were taken hourly and examined immediately or stored at 5°C until examined. Conidial germination was determined microscopically (× 400 magnification) in a Thoma haemocytometer (0.0025 mm 2 × 0.05 ram) by counting the number of conidia with germ-tubes of a length equal to or longer than the spore width [9]. By this criterion a prolonged lag phase was inevitably introduced, as germination could often be recog-
Fig. 1. Light micrographs of eonidia produced by submerged (A) or surface cultivation (B) of T. ktm~ghuJx,,s. The fungus was grown in a yeast ex|ract/stareh medium al 45"C in shake flasks at 220 tom or on agar pl~iles. Bar = 10 ,urn.
2SI
nized at an earlier stage. At least 2(1t) conidia were investigated from each of three replicates and percentage germination calculated.
+3
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ZS. Diameter The conidial diameters were estimated microscopically (× 400 magnification) with a measurement ocular (Olympus WFl0XMicro). 200 conidia of each type were measured.
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4. RESULTS AND DISCUSSION
4. I. Comparison of conidia Thermomyces Ionugioo&us produced two main types of conidia. During submerged cultivation in shake flasks relatively small, coiourless conidia with a smooth outer-wall layer were developed (Fig. IA); these conidia lacked the pedicel characteristic of surface conidia [1]. Conidia produced by surface cultivation on agar plates (Fig. I B) were for the major part dark brown larger, had a sculptured wall structure, and a pedicel attached.
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Fig, 2, Size distribution of conidia produced by submerged {A) or surface cul~i~alion (B) of T. lantigino,s~s. The fungus was grown in a yeasl extract/starch medium at 45+(.7 in shake flasks at 22[I rpm or on agar plates.
Fig. 3. Gerrr4nalion of conidia from T, htnt+~,,ino.~t+.i" prl)duced in a yeast extract/xtarch medium at 45~C by submerged cultivation in shake flasks at 220 rpm (o) or surface cultivation on agar plates (iJ, The c~midia v-ere ~crminated in the yeast extract/starch medium ul 51Y'C in ~hak~ fla,&~ at 22(1 rpm.
These conidia showed a strong tendency to agglutinate. In addition to the typical surface conidia, some smaller conidia with a smooth surface, resembling the submerged conidia, were also present. This type was in a previous description [1] defined as 'immature" conidia. "(he mean diameter of the conidia derived from submerged cultivation was 5.1 ~m which was significantly smaller than the mean value of 6.1~ p.m for thc surface conidia tr:i,,,_.o.2A and 2B). Thc surfacc conidia also :+howed a greater variation in size (Fig. 2B) and morphology (Fig. 1); from 3.3 to 5.0/xm these were all colourle~s and smooth walled, while from 5.3 to 7.5 #m the fraction of smooth-wailed conidia gradually decreased, and finally from 7.8 #.m all [he conidia wcrc dark brown and rough walled. The variation in size of the surface conidia was rather large compared to earlier estimation [!,3~5] in which the sizes of conidia from surface cultivations were tbund to be 6-10 /.tm, 7-12 ~m, or 5.5-10/~m respectively. This may indicate that in these previous studies the smooth-walled conidia were not accounted for, probably because they were considered to be immature. The time course of germination of the two conidial types was compared (Fig. 3). The conidia produced by submerged cultivation germinated faster than the surface conidia, as 90% of the
1
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submerged eonidia had germinated aftcr 3 h of incubation, whereas the surface conidia took 5 h to reach the same level. This delay in germination was due not only to a longer lag period (which accounted for 1 h at the most), but also to a lesser degree of synchronization in the ~erminalion. The time course of germination of the surface conidia is comparable to earlier results [4], although in that study only 50% of the conidia were able to germinate. In the present study 90% of the conidia from surface cultivation germinated and the smaller, smooth-walled, un. coloured conidia germinated as efficiently as the larger, rough-walled, dark-coloured conidia. 4.Z Formation and germination af conidia produced during submerged cuhiration
When conidia were produced by submerged cultivation at different temperatures in the range from 37 to 55°C, the highest number of conidia was formed a~ 45°C, and the optimum temperature for germination was 50-53°C (data not shown). The temperature of formation of submerged conidia was shown to influence the germination rate (Fig. 4). The optimum temperature for conidial formation was 40-45°C since 90% of these conidia had germinated after 3 h whereas it took conidia from 48 and 50°C 4 h to reach this level,
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Fig. 4. Effect of temperature of formation on Ihe germination of conidia from Z ianuginosus. The temperature of incubation for conidial formation was 37 ( ,x 1, 40 ( 0 ), 45 10), 48 ( & ), 50 (I), and 55°C (o}. The eunidia wele. germinated in the yeast exlraet/stareh medium at 50°C in shake flasks at 220 rpm.
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Time sin(e inocu~olion (hours)
Fig. 5. Effect of the conidial age at the lime of harvesting on the g¢:millation of conidia from T. lmmginosl~s. Conidia were produced by submerged cultivation in a yeast extract/starch medium at 45°C in shake flasks at 22(1 rpm for 2 ( 13 ). 3 (o). 4 (,~), or 5 days (o). Conidia from day 2 were reincubated for
another 24 h in the cullure ~'iltrate (IlL The conidia were germinaled in the yeast extract/starch medium at 50°C in shake flasksat 220 rpm. and those prt~dueed at 55°C needed 5 h. The almost parallel course of the germination curves indicates that the time needed to obtain complete germination is primarily a result of the length of the lag phase, differing according to the temperature of formation. However, the conidia produced at 37°C also showed reduced germination having reached a steady level of only 65% after 4 h of incubation. A similar relationship between temperature of formation and germination rate has been shown for surface conidia from T. lanuginosus, although maximal germination was obtained with conidia produced at 55°C [4]. In order to investigate whether the culture age at the time of harvesting of the conidia had any influence on germination, conidia harvested at days 2, 3, 4, and 5 of cultivation were germinated (Fig. 5). The conidia from days 3, 4, and 5 germinated equally well, whereas conidia from day 2 showed a reduction in the capacity of germination as a level of only 45% was reached afteJ 4 h of incubation. However, when these conidia were re-incubated for another 24 h in the spent culture filtrate, the germination percentage increased to 80% after 4 h of incubation. Consequently it seems as ;.f the eonidia, after release from the myceliura, demand some kind of maturation to achieve full capacity for germination.
283
For experimentai and industrial applications with fungi it is important that the inoculurn is homogeneous and reproducible. The results presented indicate that for 7". lanuginosus the conidia produced in submerged cultivation may be a good choice for an inoculum due to a homogeneous morphology, little agglutination, and a fast and synchronous germination.
REFERENCES [ I] Cooney. D,G, and Emerson, R. (1964)Thermophilic Fungi. W,H. Freeman and Company, San Francisco.
[2} Cole. G.T. and Samson. R.A. (~979) Patterns of Development in Conidi:,l Fungi. Pitman, London. [3} Pugh, G.LF,, B$akeman. J.P. and Morgan-Jonc.~, G. t 19f-~) Trans. Brit. Mycol. Soc. 47. [|5-121. [41 Nakaji. M.K. (1978) Ultrastructure and Pkysiolo~ of the Thermt,philic Fungus lhtmicola lanuginosa. Ph.D. Thesis, University of Kentucky, L0ui.wille, K¥. [5] Ellis, D.H. (t98l)Tran~. Brit. Mycol. Soc. 77, 22~-241. [6] Haasum. I.. Eriksen. S.H.. Jenscn, B. and Olsen, J. (|gO1) Appl. Microhiol. Bioteehnol. 34. 656-660. [71 Jen~n. B.. O].'~n. J. and Allermann. K. (i(~t]8) Can..!. microbiol. 34. 2]8-223. [8] Vogel, H.J. { 1964) Am, NaL 98, 435-446. [9] Manners, J.G. (19fi6) In: The fungus spore (M~delin, M.F., Ed.L pp. 1fi5-t74. Butlerworths, London.
279
FEMSLE 114911
Description of conidia from submerged cultivation of Thermomyces ianuginosus for use as a uniform inoculum Susanne Havn Eriksen, Iben Haasum, Bo Jensen and J0rgen Olsen Depaanlent of Gent'ral Micr~bioh~g),, Univer,~ityof Cop~'nhagvn, Copf~lhag~'n, Denmark Received 21) March 1992 Accepted 23 March t992
Key words: Thermophilic fungus; Morphology; Conidia; Germination: Thermom~'ces ianuginosus
1. SUMMARY Conidia produced by submerged cultivation of the thermophilic fungus Therrnomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45°C more than 90% germination was achieved at 50°C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia. 2. INTRODUCTION The
imperfect,
thermophilic fungus Therreproduces asexually by
momyces ianuginosus
Corre.~pondence to: S.H. Eriksen, Deparlment of General Microbiology, University of Copenhagen. Stalvgade 83H. DK1307, Copenhagen K. Denmark.
forming aleurioconidia [1,2]. Descriptions of conidia from this fungus regarding morphology [ 1.3-5] and physiology [4,6] have until now referred to conidia derived from surface cultltres grown on agar plates. These conidia, when mature, are dark brown, globose with a diameter in the range of 5.5-12 #m, and the thick outer conidial wall is characteristically wrinkled [1,3-5]. Immature conidia have been described as colourless and smooth walled [1]. Physiological studies of conidia from "1".lanuginosu.~ are sparse. Conidia from 1". lanuginosus germinate welt in a complex medium containing yeast extract, whereas germination is poor in synthetic medium [6]. Germination began with a swelling of the spore, the spore volume increased by 49% in 230 min and the tip of the germ tube was observed emerging through a crack in the spore wall observed after 195 rain [4]. T. lal~ttginost~s however, also conidiates in submerged cultivation in liquid medium. In this study, conidia from surface and submerged cultivation have been compared with the aim of obtaining a homogeneous and reproducible inoculum.
28~1
3. MATERIALS AND METHODS
3.1. Microorganism Thermoon,ces kmughzosus (single spore isolate from strain no. 1457, The Royal VetcrinaJ3, and Agricultural University, Copenhagen, Denm~rk) was used. Stock cultures of the fungus were produccd as previously described [7] exccpt for temperature which was 45°C. After 10 days at 45°C, the stock cultures were kept at ambient temperaturc.
3.2. Medium The medium used was modified from that previously reported [7] and contained (in g/l): soluble starch (Merck, 1252), 15.1); yeasl extract (Difco, Bactok 4.0; K2HPO4"3H20, 1.O; MgSOa • 7H20, 0.5; and 0.1 ml Vogel's trace element solution [8]. All components were dissolved in distilled water. The pH was adjusted to 6.5. For surface cultivation the medium was gelled with 20 g/I agar.
3.3. Prodt~ction of conidia Surface conidia were obtained by suspending conidia from two 9-cm agar plate cultures in 50 ml of a salt solution comprising (g/I): K2HPO 4 • 3H20, 1.0; MgSO 4 • 7H 20, 0.5; and Triton X-100, 0.(15% (v/v) and filtcring through four layers of sterile gauze.
Submerged conidia were produced in 300-ml Erlenmeyer flasks with 50 ml of medium. Each flask was inoculated with surface conidia to a final concentration of approx. 1 × l0 r' per ml and incubated on a rotary shaker in a constant-temperature incubator for 3 days at 220 rpm and 45°C, unless otherwise stated. The conidia were separated from the mycelium by filtering through four layers of sterile gauze. The filtrate was centrifuged at l l 0 0 × g (Sorvali GSA-rotor)for 10 rain at 5°C. The pellet was washed once and resuspended in the salt solution described .4bore to a concentration of approx. 2.5 X 10 7 conidia per ml.
3. 4. Germination e.~periments Germination experiments were performed in 3(}0-ml Erlenmeyer flasks with 50 ml medium, preheated to 50°C. The flasks were inoculated to a final concentration of 1 × 10~' conidia per ml and incubated at 50~C with shaking at 220 rpm. Samples of 1 ml were taken hourly and examined immediately or stored at 5°C until examined. Conidial germination was determined microscopically (× 400 magnification) in a Thoma haemocytometer (0.0025 mm 2 × 0.05 ram) by counting the number of conidia with germ-tubes of a length equal to or longer than the spore width [9]. By this criterion a prolonged lag phase was inevitably introduced, as germination could often be recog-
Fig. 1. Light micrographs of eonidia produced by submerged (A) or surface cultivation (B) of T. ktm~ghuJx,,s. The fungus was grown in a yeast ex|ract/stareh medium al 45"C in shake flasks at 220 tom or on agar pl~iles. Bar = 10 ,urn.
2SI
nized at an earlier stage. At least 2(1t) conidia were investigated from each of three replicates and percentage germination calculated.
+3
p. ....
~
.-
~-i
ZS. Diameter The conidial diameters were estimated microscopically (× 400 magnification) with a measurement ocular (Olympus WFl0XMicro). 200 conidia of each type were measured.
•
~, ,..,
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~i.~
?0 ,
i
':
3
4
-,
4. RESULTS AND DISCUSSION
4. I. Comparison of conidia Thermomyces Ionugioo&us produced two main types of conidia. During submerged cultivation in shake flasks relatively small, coiourless conidia with a smooth outer-wall layer were developed (Fig. IA); these conidia lacked the pedicel characteristic of surface conidia [1]. Conidia produced by surface cultivation on agar plates (Fig. I B) were for the major part dark brown larger, had a sculptured wall structure, and a pedicel attached.
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Conidiol
7
dle.meter
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.. 9
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Fig, 2, Size distribution of conidia produced by submerged {A) or surface cul~i~alion (B) of T. lantigino,s~s. The fungus was grown in a yeasl extract/starch medium at 45+(.7 in shake flasks at 22[I rpm or on agar plates.
Fig. 3. Gerrr4nalion of conidia from T, htnt+~,,ino.~t+.i" prl)duced in a yeast extract/xtarch medium at 45~C by submerged cultivation in shake flasks at 220 rpm (o) or surface cultivation on agar plates (iJ, The c~midia v-ere ~crminated in the yeast extract/starch medium ul 51Y'C in ~hak~ fla,&~ at 22(1 rpm.
These conidia showed a strong tendency to agglutinate. In addition to the typical surface conidia, some smaller conidia with a smooth surface, resembling the submerged conidia, were also present. This type was in a previous description [1] defined as 'immature" conidia. "(he mean diameter of the conidia derived from submerged cultivation was 5.1 ~m which was significantly smaller than the mean value of 6.1~ p.m for thc surface conidia tr:i,,,_.o.2A and 2B). Thc surfacc conidia also :+howed a greater variation in size (Fig. 2B) and morphology (Fig. 1); from 3.3 to 5.0/xm these were all colourle~s and smooth walled, while from 5.3 to 7.5 #m the fraction of smooth-wailed conidia gradually decreased, and finally from 7.8 #.m all [he conidia wcrc dark brown and rough walled. The variation in size of the surface conidia was rather large compared to earlier estimation [!,3~5] in which the sizes of conidia from surface cultivations were tbund to be 6-10 /.tm, 7-12 ~m, or 5.5-10/~m respectively. This may indicate that in these previous studies the smooth-walled conidia were not accounted for, probably because they were considered to be immature. The time course of germination of the two conidial types was compared (Fig. 3). The conidia produced by submerged cultivation germinated faster than the surface conidia, as 90% of the
1
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submerged eonidia had germinated aftcr 3 h of incubation, whereas the surface conidia took 5 h to reach the same level. This delay in germination was due not only to a longer lag period (which accounted for 1 h at the most), but also to a lesser degree of synchronization in the ~erminalion. The time course of germination of the surface conidia is comparable to earlier results [4], although in that study only 50% of the conidia were able to germinate. In the present study 90% of the conidia from surface cultivation germinated and the smaller, smooth-walled, un. coloured conidia germinated as efficiently as the larger, rough-walled, dark-coloured conidia. 4.Z Formation and germination af conidia produced during submerged cuhiration
When conidia were produced by submerged cultivation at different temperatures in the range from 37 to 55°C, the highest number of conidia was formed a~ 45°C, and the optimum temperature for germination was 50-53°C (data not shown). The temperature of formation of submerged conidia was shown to influence the germination rate (Fig. 4). The optimum temperature for conidial formation was 40-45°C since 90% of these conidia had germinated after 3 h whereas it took conidia from 48 and 50°C 4 h to reach this level,
TCC. r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Fig. 4. Effect of temperature of formation on Ihe germination of conidia from Z ianuginosus. The temperature of incubation for conidial formation was 37 ( ,x 1, 40 ( 0 ), 45 10), 48 ( & ), 50 (I), and 55°C (o}. The eunidia wele. germinated in the yeast exlraet/stareh medium at 50°C in shake flasks at 220 rpm.
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Time sin(e inocu~olion (hours)
Fig. 5. Effect of the conidial age at the lime of harvesting on the g¢:millation of conidia from T. lmmginosl~s. Conidia were produced by submerged cultivation in a yeast extract/starch medium at 45°C in shake flasks at 22(1 rpm for 2 ( 13 ). 3 (o). 4 (,~), or 5 days (o). Conidia from day 2 were reincubated for
another 24 h in the cullure ~'iltrate (IlL The conidia were germinaled in the yeast extract/starch medium at 50°C in shake flasksat 220 rpm. and those prt~dueed at 55°C needed 5 h. The almost parallel course of the germination curves indicates that the time needed to obtain complete germination is primarily a result of the length of the lag phase, differing according to the temperature of formation. However, the conidia produced at 37°C also showed reduced germination having reached a steady level of only 65% after 4 h of incubation. A similar relationship between temperature of formation and germination rate has been shown for surface conidia from T. lanuginosus, although maximal germination was obtained with conidia produced at 55°C [4]. In order to investigate whether the culture age at the time of harvesting of the conidia had any influence on germination, conidia harvested at days 2, 3, 4, and 5 of cultivation were germinated (Fig. 5). The conidia from days 3, 4, and 5 germinated equally well, whereas conidia from day 2 showed a reduction in the capacity of germination as a level of only 45% was reached afteJ 4 h of incubation. However, when these conidia were re-incubated for another 24 h in the spent culture filtrate, the germination percentage increased to 80% after 4 h of incubation. Consequently it seems as ;.f the eonidia, after release from the myceliura, demand some kind of maturation to achieve full capacity for germination.
283
For experimentai and industrial applications with fungi it is important that the inoculurn is homogeneous and reproducible. The results presented indicate that for 7". lanuginosus the conidia produced in submerged cultivation may be a good choice for an inoculum due to a homogeneous morphology, little agglutination, and a fast and synchronous germination.
REFERENCES [ I] Cooney. D,G, and Emerson, R. (1964)Thermophilic Fungi. W,H. Freeman and Company, San Francisco.
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