O2

position

Fig. 2.9. Changes that take place during the fermentation with respect to concentration profiles along a radius that extends through the particle into the inter-particle gas phase. The example is given for the growth of a biofilm of a unicellular microorganism on a polymeric carbon source, in a situation where this polymer also provides the physical structure of the particle, such that the particle shrinks during the fermentation. For clarity, enzyme concentrations are not shown. In reality, the interface between the biomass and the substrate particle may be less distinct than is indicated here. This figure is based on modeling studies undertaken by Rajagopalan and Modak (1995) and Rajagopalan et al. (1997). Key: (-)

Polymer concentration; (---) soluble hydrolysis product; ( ) O2. For clarity, enzyme profiles are not shown, but are similar to those shown in Fig. 2.8

The original extension of the germ tube is fueled by reserves in the spores, but the continued growth depends on nutrients from the substrate. In the case that the carbon source is a polymer, this requires the secretion of the appropriate enzyme or enzymes. Enzymes diffuse away from the site of secretion into the particle. The speed of diffusion depends on the size of the enzyme and on the internal structure of the substrate particle. The enzymes begin hydrolyzing the polymer, and the soluble hydrolysis products then diffuse through the substrate. Oxygen consumption causes diffusion of O2 through the static gas layer to the biomass and any initial O2 within the substrate also diffuses to the biomass.

Soon hyphae from neighboring microcolonies meet one another, which causes negative interactions between the extending hyphae at the tips. For example, hy-phae may change their growth direction or even cease to extend. During this time some hyphae will also have extended above the surface of the liquid film and others will have penetrated into the substrate (Fig. 2.7). During these very early stages, there is a sufficiently high O2 concentration within the substrate to support this penetration: given the low biomass, the rate of O2 uptake is low and diffusion can replenish O2 reasonably effectively. Also due to the low biomass, the overall rate of heat production is very low, so it may still be necessary to warm the bed to provide the optimum temperature for growth.

So, early in an SSF process, growth is essentially biologically limited. Growth occurs at the maximum specific growth rate at which the organism is capable of growing on a solid surface at the prevailing temperature, pH, and water activity, although the extent to which this is true depends on how quickly enzymes are produced to liberate hydrolysis products from polymers. This period of biologically-limited growth can potentially be quite short once active growth has begun, possibly of the order of 2 to 10 hours, depending on the process.

The mid-stages of the process. The situation quickly changes as the biomass density increases, since the overall growth rate increases as the biomass increases, causing increases in the rates of growth-associated activities, such as nutrient and O2 consumption and heat production. The consumption of O2 and nutrients by the fungus decreases their concentrations in the immediate environment of the biomass (Fig. 2.8), and these changes typically occur more rapidly than O2 and nutrients diffuse towards the biomass. The nutrient and O2 concentrations within the biofilm continue to fall until they reach concentrations that are sufficiently low to decrease the growth rate. In this case, the process is limited by mass transfer.

During this phase the biomass density per unit surface area of substrate increases. The biomass may continue to penetrate into the substrate, although this might be relatively slow due to O2 limitations. The production of aerial hyphae may contribute significantly to the overall increase in biomass density, but the density of the biomass in the biofilm may also increase. In an unmixed bed, the fungal hyphae form a network within the inter-particle spaces. Depending on the strength and density of this network, the substrate bed may be bound into a compact "cake". In a bed that is agitated, even if only intermittently, these aerial hy-phae may be squashed onto the surface of the particle by the mixing, and may be damaged sufficiently to reduce growth. Mixing may also prevent sporulation, by damaging the developing aerial conidiophores before sporulation begins. Typically the mycelium squashed onto the particle surface is surrounded by a liquid film and is therefore considered as biofilm biomass. In this case the situation is closer to that presented in Fig. 2.9.

During this phase the rate of heat production soon exceeds the rate at which heat can be removed, such that the temperature of the substrate bed rises (Fig. 2.10). It continues to rise as long as the overall rate of heat production is greater than the overall rate of heat removal. Under operating conditions that have typically been used in SSF bioreactors, even if the inlet air and water jacket temperatures are maintained at the optimum temperature for growth, the temperature may increase over a period of several hours to values 10 to 20°C above the optimum, at least in some regions of the bioreactor. This is typically sufficient to affect growth deleteriously.

Therefore, during the mid-phases of an SSF process, growth can be limited by unfavorably high temperatures, or low concentrations of nutrients, soluble hydrolysis products or O2. The major limiting factor depends on the growth rate of the microorganism, the properties of the substrate and the type of bioreactor used and how it is operated. Even for a single organism-substrate-bioreactor system, it is possible for different factors to be limiting at different times or even at the same time but at different locations within the bed.

If the polymer being hydrolyzed by the fungus is a structural polymer of the particle, then the particle properties will change. If other structural polymers are present that are not attacked, then the particle may simply lose strength. In many cases the size of the particle diminishes as the polymer is hydrolyzed. This might be accompanied by shrinkage of the bed, that is, a decrease in either or both of the bed height and width. With a fungal fermentation the "cake" may pull away from the walls. Note that these changes can affect the bulk scale transport processes. For example, the filling of the inter-particle spaces with biomass can cause increased pressure drops in a static bed with forced aeration (Fig. 2.10).

Fig. 2.10. Important variations throughout the whole fermentation at the macroscale

The latter stages of the process. Continued stress on the fungus due to high temperatures, low O2 or lack of nutrients may trigger processes such as sporulation, termination of cell growth or death. As a result, the growth decelerates, and the rate of heat production falls. As the heat production rate falls, the temperature of the substrate bed falls.

This period may be quite important if the desired product is spores or a secondary metabolite, but for other products the process might typically be harvested at the onset of this phase. Depending on the process, this phase can be relatively short, consisting of a few hours, or quite long, consisting of days to weeks, such as can occur with the production of secondary metabolites or spores.

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