The metabolic performance of a microbial culture in the bioreactor depends strongly on complex interactions of the various operating conditions.

For example, the agitation intensity, the microbial species being cultured, the nutrients type and supply determine the bulk rheology and cellular morphology. Rheology in turn affects supply of nutrients, specially oxygen, and the ease of mixing of the broth. Rheologymorphology relationships are particular relevant in fermentations involving filamentous fungi and bacteria. The specific growth morphology produced under given conditions depends on several factors including the fungal strain, the method of initiation of culture (e.g. spores, pellets, dispersed mycelium , the nature of the growth medium, and the hydrodynamic regime in the bioreactor !"#$. %xcessive hydrodynamic shear stresses are known to damage mycelial hyphae and pellets !&$, but much lower shear stresses are sufficient to influence growth morphology. The available literature on the effects of mechanical forces on fungal morphology is mostly focused on mycelial cultures consisting of clumps and freely dispersed hyphae !'($. )n general, little is known about the influence of shear stresses on pelleted fungal cultures !"*$. +elleted growth produces broths that are relatively less viscous and therefore easy to mix and aerate in comparison with filamentous growth

The pellet diameter and compactness were affected by the agitation intensity of the broth. pelleted growth may result in reduced cell mass due to substrate and oxygen limitation in the dense core of the pellet when the pellets exceed a ,critical radius-..,"' /xygen depletion in the center of the pellets will cause autolysis of the cells and eventually the formation of a hollow center. Example0 the relationship between the pellet morphology, as influenced by agitation intensity, and the bulk rheology of broths of the filamentous microfungus

Aspergillus terreus. These effects were examined in a conventional stirred tank fermenter and a fluidi1ed bed bioreactor. )n both systems, the dissolved oxygen concentration was held constant at &**2 of air saturation, to separate the influence of agitation from the oxygen transfer effects. The stirred tank reactor was operated at #**, 3** and .** rpm. Fluidi1ed bed reactorwas aerated at " vvm. The pellet morphology and bulk broth rheology were measured for various agitation regimens in the bioreactors. The following specific conclusions can be drawn for pelleted submerged fermentations of A. terreus0 (" )n fluidi1ed beds and stirred tanks agitated at up to #** rpm (impeller tip speed up to ".*4ms5" , the hydrodynamic shear regimens are comparable and permit stable existence of pellets of up to4#**6min diameter. (4 )ntense shear forces in stirred tanks (73** rpm8 impeller tip speed of 74.*#ms5" fragment pellets of"4**6m initial si1e to a stable si1e of 9(**6m. (# The compactness or fluffiness of pellets are affected by the agitation intensity. :mall, dense pellets form under intense agitation (73** rpm . (& The broth rheology parameters K and n are influenced by the concentration of the biomass, the average pellet si1e and, to a lesser extent, the fluffiness of the pellet. ;ll these effects are completely independent of any oxygen transfer related effects, as within a given reactor the biomass concentration is not affected by the agitation intensity over the range investigated. For both types of reactors, irrespective of the agitation speed and the mode of aeration (air or oxygen enriched air used, the pellet diameter correlated with the specific power input according to the following e<uation (Fig. 4 0

where Dp is the average pellet diameter, Pg is the total power input (including that from expansion of the sparged gas and V is the volume of the broth in the bioreactor. Filamentous0 ; fungal fermentation system is widely recogni1ed as a complicated multi=phase, multi=component process in which numerous problems can occur. >rowth of the cultured microorganisms and product formation are determined by a wide range of parameters, including culture medium, fermentation p?, temperature, dissolved oxygen tension, shear stress, and fungal morphology. Thus, more knowledge of the effects of morphology and operation parameters on fungi is needed to understand and perform the fermentation process properly and effectively. Filamentous growth is common in industrial fermentation. ?owever, reduced extracellular protease secretion was found in pelleted growth and it is beneficial for heterologous protein production. )t has been shown that high biomass concentration in a fungal culture system might greatly decrease the oxygen mass=transfer coefficient, k@a. Example0 recombinant Aspergillus niger strain, ;A&."!pgpdA>@;>F+$B"", which carries a glucoamylase (>@; =green fluorescent protein (>F+ fusion gene. study the effects of bioprocess parametersCagitation intensity, initial glucose concentration, initial yeast extract concentration, and dissolved oxygen tension (D/ The broth became more viscous, and poor mixing was observed at the lower agitation rate (4'* rpm . ;gitation intensity and dissolved oxygen tension also affected the pellet

porosity significantly. ?igher agitation and D/ level resulted in lower pellet porosity. The highest >F+ production and lowest protease activity were obtained in trial ab, ie the run with higher levels of agitation and D/ and lower levels of initial glucose and yeast extract concentrations. )ncreasing agitation intensity enhanced the mechanical forces on the fungal cells as well as the gasli<uid mass transfer. +ellet si1e and hair length in a submerged fermentation were controlled by mechanical forces. ;gitation also determines the mixing conditions in the reactor which greatly influence the substrate mass transfer. ?owever, it was observed that with the agitation rate higher than &** rpm, fungal hyphae were severely damaged by shear stress which resulted in poor biomass development. Thus, an agitation rate of &** rpm is considered to be the optimal rate for >F+ production. ;lthough agitation was not the strongest parameter affecting protease activity, the protease activity was lowered when the agitation rate was increased (Table ' . The increase in >F+ production could also be a benefit resulting from the decrease in protease activity. Dissolved oxygen tension affects productivity, cell autolysis, the rigidness of the cell wall, and many other features of fungal fermentation.4" The effect of D/ on protein production is profound, as reported in the literature. /xygen can potentially impact fungal cultures in a number of ways. )t may enhance growth and protein secretion directly. )t may also indirectly influence these processes by altering the morphology of the culture, either positively or negatively. )t has been shown that oxygen enrichment in the gas supply resulted in a higher percent of ,active lengths(cytoplasm filled compartment, production andEor

secretion region in the hyphal elements.4# :ince protein synthesis occurs in the cytoplasmic regions 4& and protein secretion takes place mainly at the hyphal tips, an increase in the activity of native glucoamylase was obtained.4',43 ?owever, most of these studies were based on filamentous growth. )n the pelleted growth, mass transfer in the pellets might exert critical effects on the protein production as well. )n this study, protease activity was significantly affected by the D/ level in the broth (P=value F *.*#4 . ?igher D/ tension resulted in higher protease activity. The dissolved oxygen level did not significantly affect the pellet si1e. ?owever, pellet structure was considerably influenced by D/Ca higher D/ level resulted in denser pellets. )n summary, a set of recommended cultivation conditions for this reactor can be as follows0 agitation intensity &** rpm, initial glucose concentration 4' g dm5#, no yeast extract, and D/ tension of "'2. Gnder these conditions, the specific >F+ production was the highest.