J. Basic Microbiol. 32 (1992) 3, 193-200
(Institut fur Systemdynamik und Regelungstechnik 'der Universitat Stuttgart. W-7000 Stuttgart 80, Germany)
Effect of temperature and medium composition on mycelial growth of Streptowlyces tendae in submerged culture U. REICHL,R. KINGand E. D. GILLES (Received 14 June 199I/Accepted 12 Noiiember 1991) To investigate the influence of culture conditions on growth of filamentous bacteria in submerged culture, S. tendue was cultivated at various temperatures and in various media. For this purpose, a temperature-controlled growth chamber was constructed, which allows the cultivation of filamentous bacteria and fungi in oxygen saturated medium. To observe the development of mycelia emerging from spores, this growth chamber was mounted on a microscope stage and series of images were analyzed by an image processing system. Growth kinetics obtained in liquid culture were identical to those determined on solid media. Specific growth rate and apical extension rates of individual hyphae seemed to be higher than those observed on solid media. due to a better supply of medium in submerged culture. A two-fold increase in specific growth rate, mean apical extension rate and branching rate was observed when the temperature was increased from 27 "C to 37 "C, therefore. the length of the hyphal growth unit was not changed. Growth in synthetic media used here was considerably slower compared with complex medium. A decrease in glucose concentration from 40.0 g I - ' to 1.0 g 1resulted in an increase in branching rate and specific growth rate, while apical extension rate of individual hyphae was unchanged.
On solid media and in liquid culture, growth and branching of filamentous actinomycetes begins with the germination of spores. After a short period of exponential increase, the emerging germ tube(s) extends at a linear rate. Some time later, formation of branches leads to an exponential increase in total hyphal length (KRETSCHMER 1978, KRETSCHMER et al., 1981, ALLANand PROSSER 1983, PROSSER et al. 1988, REICHLct al. 1990). The overall kinetics of these apical growth and branching processes are comparatively well understood. Investigations concerning the influence of different culture conditions on extension or branching rates, however, are usually neglected, This fact is presumably due to the limited possibilities of controlling culture conditions on solid media, e.g. to change nutrient concentrations during growth or to achieve constant growth conditions without local concentration gradients or depletion of substrates. In continuous culture, growth takes place under steady state conditions with respect to substrates. Here, investigations are hampered by the fact that extension and branching rates are not determined directly (RIESENBERG and BERGTER 1979, RIESENBERG et al. 1979, KRETSCHMER et a/. 1981) but must be derived from the specific growth rate and the hyphal growth unit (BERGTER 1978). Further problems arise from the largely unknown influence of the stirrer on morphology. To obtain a better understanding of the influence of culture conditions on growth and morphology of filamentous microorganisms in submerged culture, a temperature-controlled growth chamber was used. To prevent the development of concentration gradients, the growth chamber was continuously supplied with fresh, oxygen saturated medium. This allowed the direct microscopic observation of the early growth and branching of Streptomyces tendae. Growth kinetics were determined from series of images analysed by an image processing system (REICHL et al. 1990). 14
J. Basic Microbiol. 32 (1992) 3
194
U. REICHLet ui.
Materials and methods Organism: Srreproniyces tericiae TU 901 used in this study produces a group of antifugal antibiotics called nikkomycins (DAHNct a/. 1976, LOHRet 01. 1990). Spores were obtained from frozen spore suspensions (approximately 1.9 . lo5 spores ml-', viable count), prepared from the same stock suspensions for all experiments. Before inoculation, the spore suspension was incubated at 37 'C for 2 h.
Media: Complex medium: soya peptone, 5 g I - ' (Oxoru. powder); yeast extract. 5 g I-' (OXOID, powder): glucose. 30 g 1- ' (MERCK).Synthetic mcdia with a glucose concentration of 40.0 and 1.0 g I - ' glucose (MERCK);dimethylglutaric acid. 8.0 g 1- '; K 2 H P 0 , . 3 H,O, 570 mg 1- '; NaCI, 1 g 1-': MgSO, 7 H,O. 1 g 1- ' ; FeSO, ' 7 H20, 20 mg I - ' ; ZnSOL 7 HzO. 10 mg 1- '; MnSO, . H,O, 7 mg I - ' ; Na,-citrate. 2 H,O, 230mgI-'; CaCI,. 2 H,O, 100rngl-'; (NH,),SO,, 3.3 g I-'; pH 7.0, by addition of 3 N NaOH; sterilized by filtering. Synthetic medium without glucose was prepared as described above but with glucose and dimethylglutaric acid omitted; pH 7.0. by addition of KH,PO,, 0.34 g 1 - ' ; sterilized by filtering. Preparation of growth chamber: For sterilization. the growth chamber was filled with ethanol (70%) and incubated at 100 'C until the ethanol had evaporated. After cooling to room temperature, the growth chamber was filled with 0.5 ml poly-d-lysine (SIGMA) to facilitate fixation of spores, afterwards, 1 ml of the spore suspension was added. Finally the growth chamber was mounted on a microscope stage and connected with tubes for temperature control and supply of media.
5
U
(b)
Fig. I A scale drawing of the growth chamber: (a) section through the centre; (b) seen from above;
T,o:
water channel for temperature control (Input, Output); hf,,o: input and output tube for substrate; A : aluminium base: U : upper top to seal down the upper slide; O r , L : upper and lower slide (microorganisms growing on the lower slide); S: screws to fix top; Q: rear channel for temperature control; C : central chamber.
195
Mycelial growth of S . toildue in submerged culture
Construction of growth chamber: The growth chamber mentioned earlier (REICHLet ul. 1990) was redesigned to allow submerged cultivation with a continuous supply of fresh medium. The 44 x 96 mm base was constructed from aluminium with a microscope slide ( 2 6 x 7 6 m m ) fixed at the bottom (Fig. 1). Most of the mycelia grow just above this slide. A second slide (25 x 25 mm) was used as top, which can be removed for cleaning the inner chamber. Sealing is obtained by small strips of parafilm (M, American National Can). The distance between both slides amounted to 3 mm. total volume was about 1.2 ml. For nutrient supply, the input tube (2 mm in diameter) was connected by silicon rubber tubing to a shake flask containing fresh, oxygen-saturated medium. The shake flask and the growth chamber were connected to a temperature-controlled water bath. All silicon rubber tubing was insulated. The decrease in temperature from water bath to growth chamber amounted to 2-3 "C. During germination and growth, the growth chamber was supplied with 36 ml medium h - ' . Image analysis: The growth chamber was mounted on a scanning table of an inverse microscope (ZEISS,IM35) equipped with phase contrast. A monochrome TV camera (BOSCH, chalnikon tube) connected to an image processing system (IPS, Kontron with DEC/VMS) was used to take series of images (every 15 min) of developing mycelia at a magnification of *200 - *400, depending on growth phase. Growth was characterized by the following measurements and derived parameters: length of individual hyphae ( L [pm]), total length of all hyphae (L,[pm]), number of branches ( N [-I), hyphal growth unit (HGU = L," [pm]), specific growth rate of the mycelium ( p [h-'1). specific branching rate (p [pm-' h-'I) and the extension rates of individual hyphae ( a [pm h-']) as described by REICHL r t al. (1990). Also. at the instant of the formation of a new apical branch, the distance between the apex and this first branch (L,,[pm]) and the distance between the first two apical branches ( L , [pm]) were calculated. All culture experiments were analysed for the first 4-7 h after germination as long as growth took place in a single plane.
Results Growth of a single tnyceliuni
As an example of the development from a spore to a mycelium with numerous branches, results of the analysis of a series of 26 images are shown in Fig. 2 and 3. The growth chamber was circulated with oxygen saturated complex medium at 31 "C. After a lag phase of approximately 3 h, a germ tube was formed which extended after a short period of acceleration at a linear rate. Due to the formation of new branches, the
600 500 400
300 200
Fig. 2 Linear ( + ) a n d logarithmic ( x )plots of the total hyphal length against time. Specific growth rate p is 0.82 h -
100
0 0
1
2
3
4
Time [ h ] 14*
5
6
7
196
U. REICHLet al. 150
/* ./
J '
120
E z -
,/" 90
r
+
0
60 _I
Fig. 3 Time course of length of three branches. Mean slope of regression lines E was 26.3 pm hK' 2
1
0
3
4
5
Time [ h ]
total hyphal length increased exponentially with a specific growth rate of 0.82 h - ' (Fig. 2). All branches examined grew only apically with a mean linear extension rate of 26.3 pm h - ' (Fig. 3). Standard errors of apical extension rates, calculated by regression analysis of length of individual hyphae against time, were always less than 1.5 pm h-'. The linear growth of these branches, like that of the germ tube, was preceded by a phase of exponential growth. Hyphal growth unit, calculated by linear regression analysis after the formation of the fourth branch was 28.1 pm. Hyphal growth unit (HGU, [pm]) and branching rate (p, [pm-' h-'I) calculated using the model of BERGTER (1978) were 32.2 pm and 0.025 pm h ', respectively.
+
~
~
2.00
1.75
1.50
-
-
f al
c
1.25 L
1.00
+
3
0 L
0.75 0.50
0.25 0.00 25
30
Temperature
35 [OC]
40
cn
.-
Fig. 4
Effect of temperature on specific growth rate p ( x ) and rates of cxtension of individual hyphae c( (+ J; mean v+e and standard deviation of 20 branches
197
Mycelial growth of S. teridae in submerged culture
Influence
01-temperature
Temperature was varied between 27 - 37 "C (complex medium) to investigate influences on growth and morphology (Fig. 4). Specific growth rates and branching rates were determined from three experiments at each temperature. For calculation of apical extension rates, growth of 20 branches was followed. Specific growth rates and linear apical extension rates increased by a factor of two (Fig. 4) within the temperature range of 10 "C.Consequently, the calculated specific branching rate (BERGTER 1978) p, also increased from 0.018 pm-' h-' to 0.033 pm-' h-' (mean values). At each temperature, apical extension rates of individual hyphae were described by a normal distribution with a standard deviation ranging from 3.5 pm h-' (27 "C) to 6.4 pm h-' (37 "C, Fig. 4). Thus, extension rates of single hyphae differ slightly at constant cultivation conditions. Mean hyphal growth unit calculated by regression analysis was constant at all temperatures investigated, being 30.8 4.5 pm, which was less than the mean value of model (BERGTER 1978). 34.2 2.1 pm calculated from BERGTER'S
Influence of medium
The influence of different media is given in Table 1. The use of a synthetic medium resulted in a considerable increase in the lag phase from 15 h to 20 h (complex medium 3 h to 4 h). The number of germinating spores was also reduced, but was not investigated in detail. All experiments were done at 37 "C. The composition of complex and synthetic media (glucose, 40.0 g I-') was selected according to the media usually used for fermentations of S . tendae in batch and continuous culture (LOHRet al. 1990). Three experiments were analysed with each medium and apical extension rates of individual hyphae were calculated from a minimum of 15 branches each; L, and L,, were calculated from a minimum of 10 measurements at each experiment. Table 1 Experimental data for growth of S. tendae in complex and synthetic media
complex medium synthetic, 40.0 g 1- Glc synthetic, 1.0 g 1- Glc synthetic, only citrate
L,,
[!Jm-' h-'I
L: [pml
0.035 0.016 0.033 0.0 14
14.6 8.7 11.9 15.0
52.6 35.8 24.7 66.7
B,
[!Jm h-'I
HGU, [WI
36.5 12.2 11.2 29.2
32.3 27.7 18.4 45.1
P
G(
P-'I 1.13 0.44 0.61 0.63
bml
p , specific growth rate of the mycelium; ii, mean extension rate of individual hyphae: HGU,, hyphal growth unit calculated according to BERGTER(1978); p,, specific branching rate (BERGTER1978); L,, length of the developing first segment and L,, distance between the apex and the first branch when developing this new apical branch
Due to shorter distances between branches which additionally often crossed, investigations in synthetic media were more complicated than in complex medium. In most cases, development could be analysed only up to the formation of 20 branches. This fact resulted in a higher variation within the single experiments. The use of the synthetic medium (glucose, 40.0 g 1-') resulted in a decrease in specific growth rate to 0.44 h- ', which was only 40% of the specific growth rate of the complex medium (Table 1).A decrease in glucose concentration from 40.0 g 1-' to 1.0 g 1-' resulted in an increase in specific growth rate and specific branching rate, while mean extension
198
U.REICHLet al.
rates of individual hyphae and the length of the developing first segment Lz did not change significantly. Corresponding to the increase in branching rate, mean length of the unbranched apical region L,. decreased (Table 1). Thus, new branches are formed earlier, while mean length of developing segments remained constant. Growth in synthetic medium without glucose was also improved and specific growth rate amounted to 0.63 h-'. In complex medium, mean apical extension rate was highest, being 3 times greater than that in synthetic medium with glucose (4O.Ogl-') and 1.3 times higher than that in synthetic medium without glucose. Hyphal growth unit increased from 18.4 pm (synthetic medium, 1.0 g 1- ' glucose) to 32.3 pm (complex medium) and 45.7 pm (synthetic medium without glucose).
Discussion The use of a temperature-controlled growth chamber continuously supplied with oxygen saturated medium allowed investigations of the early growth of S . tendae in liquid medium. In contrast to investigations presented earlier (REICHLer al. 1990) continuous cultivation led to well defined cultivation conditions during the whole growth phase. The use of poly-D-lySin facilitated fixation of spores and mycelia of S. tendae on the bottom of the growth chamber thus allowing an easy monitoring of the development of mycelia. Growth of single branches into the free medium o r of slowly drifting mycelial flocks can also be analysed and showed no significant differences in apical extension rates or specific growth rates (unpublished results). Additionally, modifications in medium and temperature or even the application of specific substances such as stains or antibiotics during growth are possible. Both bright field and phase contrast observations of developing mycelium were done up to a 400-fold magnification. The quality of the images to be analysed was, however, reduced by the depth of medium, which decreased illumination of the objects. For the magnifications used here the quality of images was little influenced. However, in combination with higher demands, the medium layer selected should be as small as possible and the upper microscope slide should be replaced by a cover glass. Furthermore, only media without solid particles should be used. Growth of S. tenclue in liquid culture was very similar to that of other streptomycetes grown on solid media (SCHUMANN rt al. 1976. KRETSCHMER 1982. ALLANand PROSSER 1983). but direct comparisons of growth parameters are not possible as growth was investigated at different temperatures as well as in different media. In comparison with growth of streptomycetes on solid media, it is noticed that specific growth rate and apical extension rate of individual hyphae are relatively high; though whether this depends on the selected strain or is influenced by the cultivation in liquid medium cannot be decided. For this purpose. investigations of various streptomycetes under the same culture conditions are necessary. However, a better growth in liquid culture may have occured as in contrast to investigations of ALLANand PROSSER (1983) and KRETSCHMER (1982) a cessation of growth of single hyphae was hardly detected. This is presumably due to a better supply of substrates to hyphae, which are always surrounded by fresh medium. In good agreement with the temperature dependence of many enzyme-catalyzed reactions (LAIDLER et t i l . 1958. BAILEY 1986) specific growth rate. branching rate and mean extension rate of individual hyphae increased by a factor of nearly two within a temperature range of 10 . C. Morphology characterized by the hyphal growth unit therefore remained constant. The increasing temperature in a range between 27-37 "C, which is usually used for fermentations, had no obvious influence on morphology of S. tendae but involves an acceleration of the overall growth process. In contrast. change in medium constituents clearly influenced morphology. The decrease of specific growth rate and apical extension
Mycelial growth of S. tendae in submerged culture
199
rate in synthetic medium corresponds well with results obtained for other streptomycetes grown in continuous cultivation in a fermenter (SCHUMANN et al. 1976, RIESENBERC et a/. 1979, KRETSCHMER et al. 1981, KRETSCHMER 1982). This can be explained by the additional metabolic activity of microorganisms necessary for growth in synthetic media. A decrease in glucose concentration increased branching rate by an earlier formation of new apical branches. Mean length of developing segments and apical extension rates however remained constant. These results indicate an inhibition of branching caused by high glucose concentrations, which leads to a reduced growth rate. Further investigations are in progress to investigate the influence of ammonia, phosphate, pH and dissolved oxygen concentration on growth and morphology of S. tendae. For this purpose the growth chamber provides an ideal tool to investigate growth in submerged culture, which is very similar to growth in a fermenter. This way, it is not only possible to develop or to validate kinetic expressions for important steps in the overall growth process but also to estimate kinetic parameters necessary for modelling growth of S. tendae in a fermenter. We think that this information will clarify the complex correlations between growth of filamentous bacteria and the respective cultivation conditions and help in developing fermentation models based on reliable information about the biological mechanisms of growth.
Acknowledgement This work was supported by the Bundesministerium fur Forschung und Technologie and the DECHEMA.
References ALLAN,E. J. and PROSSER,J. I., 1983. Mycelial growth and branching of Sfreptoniyces coelicolor a3(2) on solid medium. J. gen. Microbiol.. bf 129, 2029-2036. BAILEY, J. E. and OLLIS.D. F., 1986. Biochemical Engineering Fundamentals. McGraw-Hill, Chemical Engineering Series, New York. BERGTER, F., 1978. Kinetic model of mycelial growth. Z. allg. Mikrobiol.. 18, 143- 145. DAHN,U., HAGENMAIER, H., HOHNE,H., KONIG,W. A., WOLF,G. and ZAHNER,H.. 1976 Nikkomycin: Ein neuer Hemmstoff der Chitinsynthese bei Pilzen. Arch. Microbiol.. 107, 143- 160. KING,R., BUSCHULTE, T. K., YANG,H. and GILLES. E. D., 1990. Mathematical models of filamentously growing microorganisms. In: Dechema Biotechnology Conferences, Vol. 4 (Ed. : BEHRENS, D.). VCH Verlagsgesellschaft. Weinheim, 1990, 989-993. KRETSCHMER, S., 1978. Kinetics of vegetative growth of Thermoacrinoinyces z1ztlgaris. Z. allg. Mikrobiol.. 18, 701-711. S., RIESENBERG, D. and BERGTER, F.. 1981. Comparative analysis of mycelial growth. KRETSCHMER. G.). Gustav Fischer In: Actinomycetes, Zbl. Bakt. Suppl. 11 (Eds.: SCHAAL,K. P. and PULVERER, Verlag, Stuttgart, New York, 131 - 135. KRETSCHMER, S., 1982. Dependence of the mycelial growth pattern on the individually regulated cell cycle in Srreptomyces graimticolor. Z. allg. Mikrobiol., 22, 335 - 347. K., 1958. The Chemical Kinetics of Enzyme Action. The Clarendon Press, Oxford. LAIDLER, LOHR,D., BUSCHULTE, T. K. and GILLES, E. D., 1989. Continuous cultivation of Streptorrivces (endue in different media. Appl. Microbiol. and Biotechnol., 32, 274-279. PROSSER,J. I., GRAY.D. I. and GOODAY, G. W., 1988. Cellular mechanisms for growth and branch formation in streptomycetes. In: Biology of Actinomycetes (Eds.: OKAMI,Y., BEPPU, T. and OGAWARA, H.). Japan Scientific Societies Press, Tokyo, 316- 320. REICHL,U., BUSCHULTE, T. K. and GILLES,E. D., 1990. Study of the early growth and branching of Streptom,vees tmdae by means of an image processing system. J. Microsc.. 158. 55-62.
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RIESENBERG. D. and BERGTER.F.. 1979. Dependence of macromolecular composition and morphology of Sirc,pror)ij~cx~s /ij.groscopirirs on specific growth rate. 2 . allg. Mikrobiol.. 19. 41 5-430. RIESENBERG. D.. ERDMANN. A . and BERGTER.F., 1979. Distribution functions of variables characterizing the niycelial morphology of Strepptoriijws hygroscopicus grown in glucose-limited chemostat cultures. Z . allg. Mikrobiol.. 19. 481 -487. ScHuHhiAw. E. und BERGTER.F.. 1976. Mikroskopische Untersuchungen zur Wachstumskinetik von Srrep/onij,ccs hj~groscopic~us. Z. allg. Mikrobiol.. 16, 201 - 21 5 . Mailing address: Dr.-Ing. U . REICHL.Institut fiir Systemdynamik und Regelungstechnik, Pfaffenwaldring 9. W-7000 Stuttgart 80. Germany
(Institut fur Systemdynamik und Regelungstechnik 'der Universitat Stuttgart. W-7000 Stuttgart 80, Germany)
Effect of temperature and medium composition on mycelial growth of Streptowlyces tendae in submerged culture U. REICHL,R. KINGand E. D. GILLES (Received 14 June 199I/Accepted 12 Noiiember 1991) To investigate the influence of culture conditions on growth of filamentous bacteria in submerged culture, S. tendue was cultivated at various temperatures and in various media. For this purpose, a temperature-controlled growth chamber was constructed, which allows the cultivation of filamentous bacteria and fungi in oxygen saturated medium. To observe the development of mycelia emerging from spores, this growth chamber was mounted on a microscope stage and series of images were analyzed by an image processing system. Growth kinetics obtained in liquid culture were identical to those determined on solid media. Specific growth rate and apical extension rates of individual hyphae seemed to be higher than those observed on solid media. due to a better supply of medium in submerged culture. A two-fold increase in specific growth rate, mean apical extension rate and branching rate was observed when the temperature was increased from 27 "C to 37 "C, therefore. the length of the hyphal growth unit was not changed. Growth in synthetic media used here was considerably slower compared with complex medium. A decrease in glucose concentration from 40.0 g I - ' to 1.0 g 1resulted in an increase in branching rate and specific growth rate, while apical extension rate of individual hyphae was unchanged.
On solid media and in liquid culture, growth and branching of filamentous actinomycetes begins with the germination of spores. After a short period of exponential increase, the emerging germ tube(s) extends at a linear rate. Some time later, formation of branches leads to an exponential increase in total hyphal length (KRETSCHMER 1978, KRETSCHMER et al., 1981, ALLANand PROSSER 1983, PROSSER et al. 1988, REICHLct al. 1990). The overall kinetics of these apical growth and branching processes are comparatively well understood. Investigations concerning the influence of different culture conditions on extension or branching rates, however, are usually neglected, This fact is presumably due to the limited possibilities of controlling culture conditions on solid media, e.g. to change nutrient concentrations during growth or to achieve constant growth conditions without local concentration gradients or depletion of substrates. In continuous culture, growth takes place under steady state conditions with respect to substrates. Here, investigations are hampered by the fact that extension and branching rates are not determined directly (RIESENBERG and BERGTER 1979, RIESENBERG et al. 1979, KRETSCHMER et a/. 1981) but must be derived from the specific growth rate and the hyphal growth unit (BERGTER 1978). Further problems arise from the largely unknown influence of the stirrer on morphology. To obtain a better understanding of the influence of culture conditions on growth and morphology of filamentous microorganisms in submerged culture, a temperature-controlled growth chamber was used. To prevent the development of concentration gradients, the growth chamber was continuously supplied with fresh, oxygen saturated medium. This allowed the direct microscopic observation of the early growth and branching of Streptomyces tendae. Growth kinetics were determined from series of images analysed by an image processing system (REICHL et al. 1990). 14
J. Basic Microbiol. 32 (1992) 3
194
U. REICHLet ui.
Materials and methods Organism: Srreproniyces tericiae TU 901 used in this study produces a group of antifugal antibiotics called nikkomycins (DAHNct a/. 1976, LOHRet 01. 1990). Spores were obtained from frozen spore suspensions (approximately 1.9 . lo5 spores ml-', viable count), prepared from the same stock suspensions for all experiments. Before inoculation, the spore suspension was incubated at 37 'C for 2 h.
Media: Complex medium: soya peptone, 5 g I - ' (Oxoru. powder); yeast extract. 5 g I-' (OXOID, powder): glucose. 30 g 1- ' (MERCK).Synthetic mcdia with a glucose concentration of 40.0 and 1.0 g I - ' glucose (MERCK);dimethylglutaric acid. 8.0 g 1- '; K 2 H P 0 , . 3 H,O, 570 mg 1- '; NaCI, 1 g 1-': MgSO, 7 H,O. 1 g 1- ' ; FeSO, ' 7 H20, 20 mg I - ' ; ZnSOL 7 HzO. 10 mg 1- '; MnSO, . H,O, 7 mg I - ' ; Na,-citrate. 2 H,O, 230mgI-'; CaCI,. 2 H,O, 100rngl-'; (NH,),SO,, 3.3 g I-'; pH 7.0, by addition of 3 N NaOH; sterilized by filtering. Synthetic medium without glucose was prepared as described above but with glucose and dimethylglutaric acid omitted; pH 7.0. by addition of KH,PO,, 0.34 g 1 - ' ; sterilized by filtering. Preparation of growth chamber: For sterilization. the growth chamber was filled with ethanol (70%) and incubated at 100 'C until the ethanol had evaporated. After cooling to room temperature, the growth chamber was filled with 0.5 ml poly-d-lysine (SIGMA) to facilitate fixation of spores, afterwards, 1 ml of the spore suspension was added. Finally the growth chamber was mounted on a microscope stage and connected with tubes for temperature control and supply of media.
5
U
(b)
Fig. I A scale drawing of the growth chamber: (a) section through the centre; (b) seen from above;
T,o:
water channel for temperature control (Input, Output); hf,,o: input and output tube for substrate; A : aluminium base: U : upper top to seal down the upper slide; O r , L : upper and lower slide (microorganisms growing on the lower slide); S: screws to fix top; Q: rear channel for temperature control; C : central chamber.
195
Mycelial growth of S . toildue in submerged culture
Construction of growth chamber: The growth chamber mentioned earlier (REICHLet ul. 1990) was redesigned to allow submerged cultivation with a continuous supply of fresh medium. The 44 x 96 mm base was constructed from aluminium with a microscope slide ( 2 6 x 7 6 m m ) fixed at the bottom (Fig. 1). Most of the mycelia grow just above this slide. A second slide (25 x 25 mm) was used as top, which can be removed for cleaning the inner chamber. Sealing is obtained by small strips of parafilm (M, American National Can). The distance between both slides amounted to 3 mm. total volume was about 1.2 ml. For nutrient supply, the input tube (2 mm in diameter) was connected by silicon rubber tubing to a shake flask containing fresh, oxygen-saturated medium. The shake flask and the growth chamber were connected to a temperature-controlled water bath. All silicon rubber tubing was insulated. The decrease in temperature from water bath to growth chamber amounted to 2-3 "C. During germination and growth, the growth chamber was supplied with 36 ml medium h - ' . Image analysis: The growth chamber was mounted on a scanning table of an inverse microscope (ZEISS,IM35) equipped with phase contrast. A monochrome TV camera (BOSCH, chalnikon tube) connected to an image processing system (IPS, Kontron with DEC/VMS) was used to take series of images (every 15 min) of developing mycelia at a magnification of *200 - *400, depending on growth phase. Growth was characterized by the following measurements and derived parameters: length of individual hyphae ( L [pm]), total length of all hyphae (L,[pm]), number of branches ( N [-I), hyphal growth unit (HGU = L," [pm]), specific growth rate of the mycelium ( p [h-'1). specific branching rate (p [pm-' h-'I) and the extension rates of individual hyphae ( a [pm h-']) as described by REICHL r t al. (1990). Also. at the instant of the formation of a new apical branch, the distance between the apex and this first branch (L,,[pm]) and the distance between the first two apical branches ( L , [pm]) were calculated. All culture experiments were analysed for the first 4-7 h after germination as long as growth took place in a single plane.
Results Growth of a single tnyceliuni
As an example of the development from a spore to a mycelium with numerous branches, results of the analysis of a series of 26 images are shown in Fig. 2 and 3. The growth chamber was circulated with oxygen saturated complex medium at 31 "C. After a lag phase of approximately 3 h, a germ tube was formed which extended after a short period of acceleration at a linear rate. Due to the formation of new branches, the
600 500 400
300 200
Fig. 2 Linear ( + ) a n d logarithmic ( x )plots of the total hyphal length against time. Specific growth rate p is 0.82 h -
100
0 0
1
2
3
4
Time [ h ] 14*
5
6
7
196
U. REICHLet al. 150
/* ./
J '
120
E z -
,/" 90
r
+
0
60 _I
Fig. 3 Time course of length of three branches. Mean slope of regression lines E was 26.3 pm hK' 2
1
0
3
4
5
Time [ h ]
total hyphal length increased exponentially with a specific growth rate of 0.82 h - ' (Fig. 2). All branches examined grew only apically with a mean linear extension rate of 26.3 pm h - ' (Fig. 3). Standard errors of apical extension rates, calculated by regression analysis of length of individual hyphae against time, were always less than 1.5 pm h-'. The linear growth of these branches, like that of the germ tube, was preceded by a phase of exponential growth. Hyphal growth unit, calculated by linear regression analysis after the formation of the fourth branch was 28.1 pm. Hyphal growth unit (HGU, [pm]) and branching rate (p, [pm-' h-'I) calculated using the model of BERGTER (1978) were 32.2 pm and 0.025 pm h ', respectively.
+
~
~
2.00
1.75
1.50
-
-
f al
c
1.25 L
1.00
+
3
0 L
0.75 0.50
0.25 0.00 25
30
Temperature
35 [OC]
40
cn
.-
Fig. 4
Effect of temperature on specific growth rate p ( x ) and rates of cxtension of individual hyphae c( (+ J; mean v+e and standard deviation of 20 branches
197
Mycelial growth of S. teridae in submerged culture
Influence
01-temperature
Temperature was varied between 27 - 37 "C (complex medium) to investigate influences on growth and morphology (Fig. 4). Specific growth rates and branching rates were determined from three experiments at each temperature. For calculation of apical extension rates, growth of 20 branches was followed. Specific growth rates and linear apical extension rates increased by a factor of two (Fig. 4) within the temperature range of 10 "C.Consequently, the calculated specific branching rate (BERGTER 1978) p, also increased from 0.018 pm-' h-' to 0.033 pm-' h-' (mean values). At each temperature, apical extension rates of individual hyphae were described by a normal distribution with a standard deviation ranging from 3.5 pm h-' (27 "C) to 6.4 pm h-' (37 "C, Fig. 4). Thus, extension rates of single hyphae differ slightly at constant cultivation conditions. Mean hyphal growth unit calculated by regression analysis was constant at all temperatures investigated, being 30.8 4.5 pm, which was less than the mean value of model (BERGTER 1978). 34.2 2.1 pm calculated from BERGTER'S
Influence of medium
The influence of different media is given in Table 1. The use of a synthetic medium resulted in a considerable increase in the lag phase from 15 h to 20 h (complex medium 3 h to 4 h). The number of germinating spores was also reduced, but was not investigated in detail. All experiments were done at 37 "C. The composition of complex and synthetic media (glucose, 40.0 g I-') was selected according to the media usually used for fermentations of S . tendae in batch and continuous culture (LOHRet al. 1990). Three experiments were analysed with each medium and apical extension rates of individual hyphae were calculated from a minimum of 15 branches each; L, and L,, were calculated from a minimum of 10 measurements at each experiment. Table 1 Experimental data for growth of S. tendae in complex and synthetic media
complex medium synthetic, 40.0 g 1- Glc synthetic, 1.0 g 1- Glc synthetic, only citrate
L,,
[!Jm-' h-'I
L: [pml
0.035 0.016 0.033 0.0 14
14.6 8.7 11.9 15.0
52.6 35.8 24.7 66.7
B,
[!Jm h-'I
HGU, [WI
36.5 12.2 11.2 29.2
32.3 27.7 18.4 45.1
P
G(
P-'I 1.13 0.44 0.61 0.63
bml
p , specific growth rate of the mycelium; ii, mean extension rate of individual hyphae: HGU,, hyphal growth unit calculated according to BERGTER(1978); p,, specific branching rate (BERGTER1978); L,, length of the developing first segment and L,, distance between the apex and the first branch when developing this new apical branch
Due to shorter distances between branches which additionally often crossed, investigations in synthetic media were more complicated than in complex medium. In most cases, development could be analysed only up to the formation of 20 branches. This fact resulted in a higher variation within the single experiments. The use of the synthetic medium (glucose, 40.0 g 1-') resulted in a decrease in specific growth rate to 0.44 h- ', which was only 40% of the specific growth rate of the complex medium (Table 1).A decrease in glucose concentration from 40.0 g 1-' to 1.0 g 1-' resulted in an increase in specific growth rate and specific branching rate, while mean extension
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U.REICHLet al.
rates of individual hyphae and the length of the developing first segment Lz did not change significantly. Corresponding to the increase in branching rate, mean length of the unbranched apical region L,. decreased (Table 1). Thus, new branches are formed earlier, while mean length of developing segments remained constant. Growth in synthetic medium without glucose was also improved and specific growth rate amounted to 0.63 h-'. In complex medium, mean apical extension rate was highest, being 3 times greater than that in synthetic medium with glucose (4O.Ogl-') and 1.3 times higher than that in synthetic medium without glucose. Hyphal growth unit increased from 18.4 pm (synthetic medium, 1.0 g 1- ' glucose) to 32.3 pm (complex medium) and 45.7 pm (synthetic medium without glucose).
Discussion The use of a temperature-controlled growth chamber continuously supplied with oxygen saturated medium allowed investigations of the early growth of S . tendae in liquid medium. In contrast to investigations presented earlier (REICHLer al. 1990) continuous cultivation led to well defined cultivation conditions during the whole growth phase. The use of poly-D-lySin facilitated fixation of spores and mycelia of S. tendae on the bottom of the growth chamber thus allowing an easy monitoring of the development of mycelia. Growth of single branches into the free medium o r of slowly drifting mycelial flocks can also be analysed and showed no significant differences in apical extension rates or specific growth rates (unpublished results). Additionally, modifications in medium and temperature or even the application of specific substances such as stains or antibiotics during growth are possible. Both bright field and phase contrast observations of developing mycelium were done up to a 400-fold magnification. The quality of the images to be analysed was, however, reduced by the depth of medium, which decreased illumination of the objects. For the magnifications used here the quality of images was little influenced. However, in combination with higher demands, the medium layer selected should be as small as possible and the upper microscope slide should be replaced by a cover glass. Furthermore, only media without solid particles should be used. Growth of S. tenclue in liquid culture was very similar to that of other streptomycetes grown on solid media (SCHUMANN rt al. 1976. KRETSCHMER 1982. ALLANand PROSSER 1983). but direct comparisons of growth parameters are not possible as growth was investigated at different temperatures as well as in different media. In comparison with growth of streptomycetes on solid media, it is noticed that specific growth rate and apical extension rate of individual hyphae are relatively high; though whether this depends on the selected strain or is influenced by the cultivation in liquid medium cannot be decided. For this purpose. investigations of various streptomycetes under the same culture conditions are necessary. However, a better growth in liquid culture may have occured as in contrast to investigations of ALLANand PROSSER (1983) and KRETSCHMER (1982) a cessation of growth of single hyphae was hardly detected. This is presumably due to a better supply of substrates to hyphae, which are always surrounded by fresh medium. In good agreement with the temperature dependence of many enzyme-catalyzed reactions (LAIDLER et t i l . 1958. BAILEY 1986) specific growth rate. branching rate and mean extension rate of individual hyphae increased by a factor of nearly two within a temperature range of 10 . C. Morphology characterized by the hyphal growth unit therefore remained constant. The increasing temperature in a range between 27-37 "C, which is usually used for fermentations, had no obvious influence on morphology of S. tendae but involves an acceleration of the overall growth process. In contrast. change in medium constituents clearly influenced morphology. The decrease of specific growth rate and apical extension
Mycelial growth of S. tendae in submerged culture
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rate in synthetic medium corresponds well with results obtained for other streptomycetes grown in continuous cultivation in a fermenter (SCHUMANN et al. 1976, RIESENBERC et a/. 1979, KRETSCHMER et al. 1981, KRETSCHMER 1982). This can be explained by the additional metabolic activity of microorganisms necessary for growth in synthetic media. A decrease in glucose concentration increased branching rate by an earlier formation of new apical branches. Mean length of developing segments and apical extension rates however remained constant. These results indicate an inhibition of branching caused by high glucose concentrations, which leads to a reduced growth rate. Further investigations are in progress to investigate the influence of ammonia, phosphate, pH and dissolved oxygen concentration on growth and morphology of S. tendae. For this purpose the growth chamber provides an ideal tool to investigate growth in submerged culture, which is very similar to growth in a fermenter. This way, it is not only possible to develop or to validate kinetic expressions for important steps in the overall growth process but also to estimate kinetic parameters necessary for modelling growth of S. tendae in a fermenter. We think that this information will clarify the complex correlations between growth of filamentous bacteria and the respective cultivation conditions and help in developing fermentation models based on reliable information about the biological mechanisms of growth.
Acknowledgement This work was supported by the Bundesministerium fur Forschung und Technologie and the DECHEMA.
References ALLAN,E. J. and PROSSER,J. I., 1983. Mycelial growth and branching of Sfreptoniyces coelicolor a3(2) on solid medium. J. gen. Microbiol.. bf 129, 2029-2036. BAILEY, J. E. and OLLIS.D. F., 1986. Biochemical Engineering Fundamentals. McGraw-Hill, Chemical Engineering Series, New York. BERGTER, F., 1978. Kinetic model of mycelial growth. Z. allg. Mikrobiol.. 18, 143- 145. DAHN,U., HAGENMAIER, H., HOHNE,H., KONIG,W. A., WOLF,G. and ZAHNER,H.. 1976 Nikkomycin: Ein neuer Hemmstoff der Chitinsynthese bei Pilzen. Arch. Microbiol.. 107, 143- 160. KING,R., BUSCHULTE, T. K., YANG,H. and GILLES. E. D., 1990. Mathematical models of filamentously growing microorganisms. In: Dechema Biotechnology Conferences, Vol. 4 (Ed. : BEHRENS, D.). VCH Verlagsgesellschaft. Weinheim, 1990, 989-993. KRETSCHMER, S., 1978. Kinetics of vegetative growth of Thermoacrinoinyces z1ztlgaris. Z. allg. Mikrobiol.. 18, 701-711. S., RIESENBERG, D. and BERGTER, F.. 1981. Comparative analysis of mycelial growth. KRETSCHMER. G.). Gustav Fischer In: Actinomycetes, Zbl. Bakt. Suppl. 11 (Eds.: SCHAAL,K. P. and PULVERER, Verlag, Stuttgart, New York, 131 - 135. KRETSCHMER, S., 1982. Dependence of the mycelial growth pattern on the individually regulated cell cycle in Srreptomyces graimticolor. Z. allg. Mikrobiol., 22, 335 - 347. K., 1958. The Chemical Kinetics of Enzyme Action. The Clarendon Press, Oxford. LAIDLER, LOHR,D., BUSCHULTE, T. K. and GILLES, E. D., 1989. Continuous cultivation of Streptorrivces (endue in different media. Appl. Microbiol. and Biotechnol., 32, 274-279. PROSSER,J. I., GRAY.D. I. and GOODAY, G. W., 1988. Cellular mechanisms for growth and branch formation in streptomycetes. In: Biology of Actinomycetes (Eds.: OKAMI,Y., BEPPU, T. and OGAWARA, H.). Japan Scientific Societies Press, Tokyo, 316- 320. REICHL,U., BUSCHULTE, T. K. and GILLES,E. D., 1990. Study of the early growth and branching of Streptom,vees tmdae by means of an image processing system. J. Microsc.. 158. 55-62.
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RIESENBERG. D. and BERGTER.F.. 1979. Dependence of macromolecular composition and morphology of Sirc,pror)ij~cx~s /ij.groscopirirs on specific growth rate. 2 . allg. Mikrobiol.. 19. 41 5-430. RIESENBERG. D.. ERDMANN. A . and BERGTER.F., 1979. Distribution functions of variables characterizing the niycelial morphology of Strepptoriijws hygroscopicus grown in glucose-limited chemostat cultures. Z . allg. Mikrobiol.. 19. 481 -487. ScHuHhiAw. E. und BERGTER.F.. 1976. Mikroskopische Untersuchungen zur Wachstumskinetik von Srrep/onij,ccs hj~groscopic~us. Z. allg. Mikrobiol.. 16, 201 - 21 5 . Mailing address: Dr.-Ing. U . REICHL.Institut fiir Systemdynamik und Regelungstechnik, Pfaffenwaldring 9. W-7000 Stuttgart 80. Germany
