GROWTH CHARACTERISTICS OF FUNGI AND ACTINOMYCETES'

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1 GROWTH CHARACTERISTICS OF FUNGI AND ACTINOMYCETES' K. C. MARSHALL AND M. ALEXANDER Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York Received for publication February 17, 1960 For studies of the development of saprophytic and pathogenic fungi in liquid culture and in sterile soils, a method for comparing growth rates under varying conditions is lacking. Growth rates of microorganisms which divide by binary fission are readily determined, since the increase in cell numbers is exponential. Thus, a plot of the logarithm of bacterial numbers or activity as a function of time is linear, the slope of the line being a measure of the growth rate. Such estimates can be made by measuring the increase in population, disappearance of substrate, formation of metabolic products, turbidimetrically, or by manometric methods. In the fungi and actinomycetes, the type of vegetative development is of an entirely different character. Proliferation of such filamentous microorganisms is frequently estimated by radial development on an agar surface (Ryan, Beadle, and Tatum, 1943) or by the linear extension of individual hyphae (Smith, 1924; Stadler, 1952). But these methods often do not give a valid comparison of growth rates of different fungi or of the same fungus under varying nutrient conditions because they fail to account for differences in the density of the mycelial mats. The weight of mycelium produced in liquid culture gives a better comparative estimate of proliferation. Since hyphal extension in liquid medium is essentially unrestricted, Emerson (1950) has suggested that the growth of Neurospora crassa in such a medium is three dimensional, and he has obtained a linear relationship between the cube root of mycelium weight and time. The present investigation was undertaken to test the validity of Emerson's findings and to use the data obtained to determine the growth characteristics of a number of filamentous microorganisms. To make the estimation more sensi- 1 Contribution from the Department of Agronomy, New York State College of Agriculture, Cornell University, Ithaca, New York, as Agronomy Paper no The investigation was supported in part by a grant from the United Fruit Company. tive and to permit observing changes in short time intervals, a manometric technique rather than mat weight was utilized. MATERIALS AND METHODS Six organisms were used in this study, Fusarium oxysporum forma cubense, Aspergillus niger, Aspergillus flavus, Cunninghamella elegans, Streptomyces sp., and Nocardia sp. Spore suspensions of the fungi were prepared in 5 ml of sterile tap water from 2-day-old slant cultures grown at 30 C on a modified Czapek agar (Difco) containing 0.1 per cent yeast extract and from 3-day-old cultures of Streptomyces sp. and Nocardia sp. grown on nutrient agar (Difco). Except where indicated, the spore suspensions were diluted approximately 0-fold in sterile water before use. Growth rates were determined by measurement of oxygen uptake by the microorganisms growing in a complete medium in Warburg microrespirometer flasks. This technique has been successfully employed in characterizing the growth of Azotobacter spp. (Burk, Lineweaver, and Horning, 1934; Wyss and Wilson, 1941). The procedure serves as an indicator of multiplication using metabolic activity as the criterion rather than total cellular mass, but the two estimates give the same results, at least with the bacteria investigated. Before use, the 15-ml Warburg vessels were plugged and steamed for 30 min to reduce the chances of overgrowth of the slower growing microorganisms by contaminants. After steaming, each vessel received 1.0 ml inoculum, 2.0 ml sterile medium, and 0.2 ml of 20 per cent KOH in the center well. Czapek-Dox broth (Difco) plus yeast extract (1.0 g per L) was used for the fungi and nutrient broth for the actinomycetes. The gas phase in all instances was air. Oxygen uptake was measured in the conventional manner (Umbreit, Burris, and Stauffer, 1957) at a temperature of 30 C. All flasks were shaken at a speed of 112 cycles/min at an amplitude of 4 cm. 412

2 19601 GROWTH OF FUNGI AND ACTINOMYCETES 413 RESULTS For each organism tested, several means of expressing the kinetics of oxygen utilization were considered. Arithmetic expressions of 02 uptake were never linear during the active stages of development, but linearity was approached during the late phases, at periods when some limitation to maximal growth was apparent. This linearity probably was a consequence of an inadequate supply of 02. From the plot of the logarithm of oxygen uptake, it was evident that growth of these filamentous microorganisms was not exponential as well. Figure 1 represents the results obtained with Nocardia sp., but analogous growth patterns were observed with the fungi and the streptomycete. With none of the organisms tested did logarithmic, square root, or arithmetic expressions of the data appear linear during active stages of proliferation. A straight line relationship was observed in the cube root plots, however, an indication that growth was three dimensional (figure 2). At the later stages of the cube root expressions, a period of declining oxygen utilization was observed. This phase appears to be comparable to that found following the logarithmic portion of the bacterial growth curve, that is, a time during which nutrient deficiencies, inadequate oxygen supply, or toxic metabolites affect the organism. It is during the period in which some limitation to maximal growth is apparent in the cube root transformations that the arithmetic expression of 02 disappearance approaches linearity. It is of interest that a linear relationship is found when the data presented by Gottlieb and Legator (1953) for Streptomyces venezuelae is subjected to the cube root transformation. 2.5r LOG /AL 0O 2.0- t HOURS Figure 1. Logarithmic plot of during growth of Nocardia sp. (r O r a VWL08 to HOURS Figure 2. Cube root plot of oxygen uptake during growth of Nocardia sp. (r = ) _ X vs Y 20 _ X vs log,0y tor --lb nR I00 Volues of r Values of r 30 t30 20 X Vs X Vs ~Y i Loo.975 S f I00O Volues of r Values of r Figure S. Frequency histograms of linear correlation coefficients between four expressions of growth rate and time. A total of 74 individual estimations of the oxygen uptake patterns of the microorganisms was performed. To summarize the data obtained with the four fungi and two actinomycetes, linear correlation coefficients (r) between growth expression and time were determined for four methods of data presentation. The distribution of correlation coefficients exceeding is summarized in figure 3. Oxygen consumption is designated Y and time, X. The values of r are consistently high, but the frequency histogram shows clearly that the cube root presentation most clearly represents the growth kinetics; i.e., fungal and actinomycete development under unrestricted conditions is three dimensional. Thus, 59 per cent of 30 ' 40 the correlation coefficients for the cubic method exceed whereas 18, 1, and 0.0 per cent oxygen uptake exceed this value for the square root, arithmetic, = ). and logarithmic means of presentation.

3 414 MARSHALL AND ALEXANDER [VOL. 80 TABLE 1 Correlation coefficients between oxygen uptake and time for several fungi and actinomycetes No. of Determinations Average Value of r for X vs. Y X vs. logio Y X vs. /V Xvs.'YF Fusarium oxysporum Aspergillus niger Aspergillus flavus Cunninghamella elegans Nocardia sp Streptomyces sp TABLE 2 Influence of inoculum size on the correlation coefficients between oxygen uptake and time Dilution Average Correlation Coefficient for X vs. Y X vs. logio Y X vs. XY X vs.iy Fusarium oxysporum / / / / Aspergillus niger / / / / The same relationship applies when the six organisms are considered individually (table 1). In each instance, the growth rate is seen to be essentially cubic. The greatest deviation of the correlation coefficients from unity is seen with F. oxysporum, but even here r is The fit, with F. oxysporum at least, is a function of the size of inoculum, the closest fit being observed at the highest inoculum levels (table 2). This effect of dilution on the fungus may prove important in a study of factors associated with growth. No such relationship was clearly apparent for A. niger. The filamentous microorganisms tended to form spherical pellets when shaken in the liquid medium, particularly the aspergilli, Cunninghamella elegans, and Streptomyces sp. Where bacterial contamination was encountered, the culture fluid rapidly developed turbidity rather than the typical hyphal pellets, and the rate of oxygen utilization was logarithmic. The method of estimating growth manometrically has also been used TABLE 3 Growth rate constants of several filamnentous microorganisms m Value Fusarium oxysporum Aspergillus niger Aspergillus flavus Cunninghamella elegans Nocardia sp Streptomyces sṗ with several common soil bacteria, and the rate of increase was exponential for each of the isolates. From the algebraic expression for a straight line, y = mx + b the slope of the line, m,in the cube root plots may be written as

4 19601 GROWTH OF FUNGI AND ACTINOMYCETES 415 _A/Y2 - _/yl m = - where Y1 is the oxygen uptake at time X1 during the period of cubic growth and Y2the corresponding value at some later time X2 in the same phase. The displacement of the line from the origin, b, is not required in this rate expression. The value of m may be considered as a growth rate constant. Calculated m values for the microorganisms used in this investigation are presented in table 3. In general, the growth rates of the fungi studied exceeded that of the streptomycete whereas the nocardia was the least active. The m value of F. oxysporum was consistently lower than those of the other fungi investigated. DISCUSSION Emerson's (1950) contention that the growth of neurospora in unrestricted systems is three dimensional has been confirmed by the results reported. This phenomenon is not restricted to the fungi, moreover, since representatives of two genera of actinomycetes behave in the same fashion. Mycelium weight was used by Emerson as a criterion of growth whereas the rate of oxygen consumption was the criterion in the present study. The latter procedure is more sensitive and has the further advantage of permitting continuous observations on individual culture vessels. The evidence obtained in the present investigation is in contrast to the reports that growth of neurospora and actinomycetes approximates a logarithmic function (Zalokar, 1959a; Haines, 1932). A fundamental question arising from these findings is how to account for the three dimensional growth patterns of filamentous microorganisms. Smith (1923), Plomley (1959), and Zalokar (1959b) have noted that elongation takes place only at the tips of the branching hyphae of fungi, and Emerson (1950) has envisaged growth in unrestricted systems as an increase in the outer surface of a sphere as a result of the elongation of hyphal tips in all directions from a central point. This three dimensional growth could account for the cubic increase in both mycelial weight and oxygen uptake. Gottlieb and Anderson (1947) point out that the mycelium of streptomycetes grown in shake culture forms spherical clumps having a peripheral zone of young, actively growing hyphae. This observation is apparently analogous to the type of hyphal extension of filamentous fungi. In the experiments reported herein, oxygen utilization appeared to be cubic from the time at which the gas exchange could be measured. It may be that the inability to demonstrate any transition from a lag period to the rapid growth phase was the result of a lack of sensitivity at this initial stage. The eventual decline in rate late in the growth cycle was probably associated with limitations in oxygen diffusion through the system since the nutrient supply was far in excess of the demand. The results obtained by the use of this manometric technique appear to be quite reproducible, thereby permitting the calculation of growth rate constants for individual microorganisms. The method provides a ready means of comparing the effects of various treatments on the proliferation of filamentous microorganisms in systems allowing relatively unimpeded vegetative development. The large experimental error in measuring gas exchange in the first few hours tends to introduce an error into calculation of the slope so that these initial values should be ignored. For determination of m values, it is deemed better to use only that portion of the curve in which 02 uptake is rapid. SUMMARY A linear relationship between the cube root of oxygen consumption and time has been observed in growing cultures of a number of fungi and actinomycetes, thereby confirming the observation that fungus growth in liquid media is three dimensional. The basis of this phenomenon is discussed. An expression for the growth rate constant has been derived, and the growth rates of a number of filamentous fungi (Fusarium oxysporum, AspergilUus niger, Aspergilus flavus, Cunninghamella elegans), a Streptomyces sp., and a Nocardia sp. have been determined. REFERENCES BURK, D., H. LINEWEAVER, AND C. K. HORNER 1934 The specific influence of acidity on the mechanism of nitrogen fixation by Azotobacter. J. Bacteriol., 27, EMERSON, S The growth phase in Neurospora corresponding to the logarithmic phase in unicellular organisms. J. Bacteriol., 60, GOTTLIEB, D., AND H. W. ANDERSON 1947 Mor-

5 416 MARSHALL AND ALEXANDER [vol. 80 phological and physiological factors in streptomycin production. Bull. Torrey Botan. Club, 74, GOTTLIEB, D., AND M. LEGATOR 1953 The growth and metabolic behavior of Streptomyces venezuelae in liquid culture. Mycologia, 45, HAINES, R. B The influence of temperature on the rate of growth of saprophytic actinomycetes. J. Exptl. Biol., 9, PLOMLEY, N. J. B Formation of the colony in the fungus Chaetomium. Australian J. Biol. Sci., 12, RYAN, F. J., G. W. BEADLE, AND E. L. TATUM 1943 The tube method of measuring the growth rate of Neurospora. Am. J. Botany, 30, SMITH, J. H On apical growth of fungal hyphae. Ann. Botany (London), 37, SMITH, J. H On the early growth of the individual fungus hypha. New Phytologist, 23, STADLER, D. R Chemotropism in Rhizopus nigricans. The staling reaction. J. Cellular Comp. Physiol. 39, UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER 1957 Manometric techniques. Burgess Publishing Co., Minneapolis. Wyss, O., AND P. W. WILSON 1941 Mechanism of biological nitrogen fixation. VI. Inhibition of Azotobacter by hydrogen. Proc. Natl. Acad. Sci. U. S., 27, ZALOKAR, M. 1959a Enzyme activity and cell differentiation in Neurospora. Am. J. Botany, 46, ZALOKAR, M. 1959b Growth and differentiation of Neurospora hyphae. Am. J. Botany, 46, Downloaded from on October 31, 2018 by guest