Research Papers
Kinetics and manipulation of hyphal breakage and its effect on antibiotic production

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Abstract

Erythromycin production in 1.5 l continuous cultures of Saccharopolyspora erythraea, was correlated with the mean mycelial fragment size, which was influenced by culture agitation (750–1 500 rev./min marine/disk turbine impellors). Kinetic parameters for hyphal breakage were determined, and revealed that more highly branched mycelial fragments (hyphal growth unit ∼14 μm, compared to ∼21 μm) were more resistant to breakage. Branching rate depended on growth rate, the more highly branched mycelium being observed at the higher dilution rate. The breakage rate was inversely related to the mycelial fragment diameter at all dilution rates. Manipulating the size distribution of mycelial fragment by using an ultrasonic filter improved specific antibiotic productivity by 33%.

Introduction

We have reported that antibiotic production in liquid culture is correlated with mycelial fragment diameter in actinomycete cultures [1], [2]. Our findings implied that mycelial fragments with a diameter of less than 80–90 μ are not productive. Smaller fragments appear to grow at the same rate as larger particles but are incapable of significant antibiotic production [2]. This phenomenon appears to account for loss of biosynthesis in liquid culture in species able to produce antibiotic on agar [3], differences in productivity in different, small-scale culture vessels [2], and morphology related effects on productivity in fermenter culture [1]. We obtained evidence that resistance to hyphal breakage depends on the activity of a peptidoglycan-synthesizing enzyme, phospho-N-acetylmuramyl pentapeptide translocase, the activity of which appears to determine hyphal tensile strength [4].

These findings led us to postulate that the site of antibiotic production is at a fixed distance from the growing hyphal tip. Mycelial fragments too small to have developed this site would be incapable of producing antibiotic.

Relationships between bioreactor performance and mycelial morphology have been studied mainly with fungal cultures. The mean hyphal fragment length in Penicillium chrysogenum increases with growth rate and decreases with specific power input [5]. Metz et al. [6] concluded that hyphal fragment length and hyphal growth unit [7] were the most useful parameters for assessing the impact of bioreactor conditions on mycelial morphology, and Ujcova et al. [8] established the use of nucleotide release as an indicator of hyphal breakage in a range of species of filamentous fungi. Values for these parameters were shown to depend on both growth rate and stirrer speed in P. chrysogenum [9], but this study also indicated that the rate of mycelial circulation through the zones of high-energy dissipation in the bioreactor was probably more influential on hyphal breakage. This was borne out by studies [10] which showed that hyphal breakage was a first-order kinetic process whose rate depended on the mean energy dissipation rate and impellor circulation time. The most successful quantitative model to date for agitation-induced fragmentation of fungi is that of Justen et al. [11]. By using a population balance model, mycelial breakage rate in P. chrysogenum could be correlated with either impellor tip speed or the energy dissipation/circulation function, but the commonly used power input per unit volume did not correlate adequately.

Equivalent work on Streptomyces species has not examined these phenomena in the same depth. Fragmentation in Streptomyces clavuligerus was affected by stirrer speed although growth and productivity was not significantly influenced [12].

In this paper, we present a study of the susceptibility of hyphae to fragmentation at different stirrer speeds and at different branching rates, and we describe the use of ultrasonic filtration for enriching cultures for larger (more productive) mycelial fragments.

Section snippets

Strains and culture media

Saccharopolyspora erythraea NRRL 2338 was used throughout. Antibiotic concentrations were measured routinely by bioassay by using a strain of Arthrobacter citreus (GL1) obtained from the Shell Laboratories Culture Collection (Shell Laboratories, Sittingbourne, Kent, UK). The chemically defined (nitrogen-limited) antibiotic production medium for S. erythraea contained the following major nutrients: (g/l in reverse osmosis purified water) glucose 15, NaNO3, 1.19; KH2PO4 3; K2HPO4, 7; and the

Effect of stirrer speed on hyphal branching rate

Values for hyphal growth unit (hgu) observed at a growth (dilution) rate of 0.04/h were, in general, higher than those obtained at 0.1/h (Fig. 1) indicating a more highly branched morphology at the higher growth rate. It is thought [18] that changes in hgu in response to changes in nutrient status are a consequence of evolution to allow filamentous micro-organisms to exploit rapidly any nutrient-rich microhabitats encountered by the growing tip. When a nutrient-rich microhabitat is

Conclusion

Chemostat culture provided a means of systematically examining the effect of stirrer speed on mycelial fragment size, and the relationship between size and antibiotic production under comparable conditions.

The antibiotic productivity of the cultures increased as the mean diameter of the minimum bounding circle of the hyphal fragments in the culture increased. This is in line with our previous findings in bioreactor and flask culture that led us to speculate that a minimum bounding circle

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