Hyphal vacuolation and fragmentation in batch and fed-batch culture of Aspergillus niger and its relation to citric acid production

Abstract

The relationship between hyphal vacuolation, fragmentation and citric acid production by Aspergillus niger, was investigated in batch and fed-batch culture. Quantitative information on morphology and vacuolation was obtained by image analysis. Time profiles of the morphology parameters — mean perimeters of clumps, P, lengths of filaments, L — and vacuoles, together with, specific growth and production rates, were used to establish a link between vacuolation, fragmentation and product formation under various agitation conditions and glucose levels. Under intensive agitation conditions and during the early fermentation stages, the characteristics observed were the increased specific growth rates and hyphal branching, along with low vacuolation levels. These were followed by fragmentation of the highly vacuolated parts of filaments and regrowth at later stages, a process that maintained that the mycelium grew old and highly vacuolated, with limited renewal, conditions that did not favour increased biosynthetic activity. Increased vacuolation and low specific production rates were observed at low glucose levels in fed-batch culture. The results indicate that vacuolation weakened the hyphae and low glucose levels created the conditions that favoured fragmentation and made the mycelium more susceptible to it when exposed to increased agitation.

Introduction

Many industrially important metabolites, such as organic acids, antibiotics and enzymes, are produced by filamentous fungi. The morphology of these micro-organisms in submerged culture varies between the pelleted and the free filamentous form, depending on the strain and the culture conditions [1]. The fungal morphology for these fermentations is of great importance since it affects the rheological properties of the fermentation broth and consequently its mass heat and transfer characteristics. The free filamentous form can lead to very viscous fermentation broths with pseudoplastic behaviour, while pellets ensure low viscosities and Newtonian behaviour. The main disadvantage of the pelleted form is that the centre of the pellets is subjected to autolysis, due to oxygen limitation, and does not participate in product synthesis.

Culture conditions affect both gross morphology and fine structure of the mycelium of filamentous micro-organisms. Among other factors, agitation has been studied extensively and shown to account for significant changes in morphology and productivity. The mycelium seems to undergo a complete transformation as agitation levels increase. Studies have shown changes in cell wall composition and certain structures on cell walls have been suggested to be involved in mechanisms of resistance to shear [2]. Works with a number of different filamentous fungi have reported reduced pellet size and filament lengths, number of tips and hyphal growth units under intensive agitation [3], [4], [5], [6]. Agitation induced morphological forms are often linked to increased productivities and the pelleted form is generally considered as the most desirable for citric acid production [7]. However, the opposite effect has also been reported, with low metabolite production and specific growth rates resulting from increased stirrer speeds [8], [9], [10]

Intensive agitation may also control the morphology of filamentous micro-organisms by actual breakage of filaments, as was observed in the work of Belmar-Beiny and Thomas [11] with S. clavuligerous, where a cycle of fragmentation and regrowth of mycelium predominated at high stirred speeds. In filamentous fermentations the direct estimation of the effect of agitation is complicated by the natural process of vacuolation, which seems to weaken the filaments and expose them to damage [3]. It is known that in the growing hyphae the size of vacuoles increases with distance from the apex, i.e. the age of compartments [12]. Increased vacuolation corresponds to decreased biosynthetic activity, since the total number of mitochondria and nuclei decreases [13] in highly vacuolated hyphae. Certain conditions also seem to contribute to the natural process of vacuolation and result in increased fragmentation, as observed by Righelato et al. [14] with P. chrysogenum in continuous culture when glucose was supplied at the maintenance rate.

Works on the effect of agitation on morphology are mostly empirical, giving little understanding to the physiological processes that underline the observed morphological changes. This lack of knowledge represents a difficulty in scale-up studies of filamentous fermentations, where correlations of the physiological state of cells with physical conditions would be valuable. Attempts have been made to use both physiological and morphological criteria in scaling-up a submerged fermentation for citric acid [8]. However reports of quantitative studies on morphology of citric acid producing Aspergillus niger are limited, despite the commercial importance of the citric acid fermentation. In this work, employing image analysis to quantify the observations on morphology, we have investigated the development of morphology and vacuolation of A. niger in batch and fed-batch cultures at different agitation and glucose levels. Time profiles of the morphological parameters and vacuoles were used to establish a link between vacuolation, fragmentation and citric acid production in submerged culture.