Research PapersKinetics and manipulation of hyphal breakage and its effect on antibiotic production
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
References (23)
- et al.
A method for increasing the success rate of duplicating antibiotic activity in agar and liquid cultures of Streptomyces isolates in new antibiotic screens
J Ferment Bioeng
(1993) - et al.
High performance liquid chromatographic determination of erythromycin
J Chromatog
(1978) - et al.
Effect of hyphal morphology on bioreactor performance of antibiotic-producing Saccharopolyspora erythraea cultures
Microbiology
(1996) - et al.
Effect of small scale culture vessel type on hyphal fragment size and erythromycin production in Saccharopolyspora erythraea
Biotech Lett
(1997) - et al.
Non-fragmenting variants of Streptomyces hyphae have enhanced activity of an enzyme (phospho-N-acetyl muramyl pentapeptide translocase) in peptidoglycan biosynthesis
J Ferment Bioeng
(1995) - et al.
Influence of engineering variables upon the morphology of filamentous molds
Biotech Bioeng
(1981) - et al.
Method for quantitative representation of the morphology of molds
Biotech Bioeng
(1981) - et al.
The growth unit of the mould Geotricum candidum
Archiv Mikrobiol
(1973) - et al.
Dependence of release of nucleotides from fungi on fermenter turbine speed
Biotech Bioeng
(1980) - et al.
The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum
Bioproc Eng
(1998)
Turbulent breakage of filamentous microorganisms in submerged culture in mechanically stirred bioreactors
Chem Eng Sci
Cited by (11)
Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology
2017, Journal of Bioscience and BioengineeringCitation Excerpt :Among the tested pH range, the shorter, thicker and multi-branched hyphae at pH 5.0 corresponded to the highest yellow pigment production (Fig. 1c). In filamentous fungi, the biomass increases through tip extension and the extension of new tips by branching can facilitate the utilization of the nutrients (29,30), which can explain the observation that multi-branched hyphae result in a higher yellow pigment production. Although the exactly underlying mechanism is still not clear, multi-branched and swollen hyphae indicate favorable growing status, which is due to a rapid growth rate associated with reduced pellet size and an increased yield of yellow pigment production.
Correlation of pigment production with mycelium morphology in extractive fermentation of Monascus anka GIM 3.592
2017, Process BiochemistryCitation Excerpt :Therefore, a relationship between the physiological state of the mycelia and pigment yields existed in extractive fermentation. The multi-branched morphology of mycelia could facilitate nutrient utilization, as well as biomass growth, to guarantee highly efficient pigment biosynthesis [32]. Additionally, highly branched hyphae also promised a fermentation broth with low viscosity, which was beneficial for mass or oxygen exchange [33].
Fungicides affect the production of extracellular mucilaginous material (ECMM) and the peripheral growth unit (PGU) in two wood-rotting basidiomycetes
2006, Mycological ResearchCitation Excerpt :The HGU not only regulates fungal growth and the morphology of the colony, but may also have a direct or indirect effect on the production of metabolites by the organism. A reduction in antibiotic production has been observed in the presence of highly branched mycelia (Makagiansar et al. 1993; Wardell & Bushell 1999). High impeller speed in bioreactors is also generally associated with a decrease in the length of the HGU (and a consequent increase in branching ratio) and this is associated with a negative effect on antibiotic production.
Improving mycelial morphology and adherent growth as well as metabolism of Monascus yellow pigments using nitrate resources
2020, Applied Microbiology and BiotechnologyChanges in morphogenesis and carotenogenesis to influence polygalacturonase secretion in Aspergillus carbonarius mutant
2020, Archives of MicrobiologyInvestigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation
2020, Applied Microbiology and Biotechnology