mg of component dry weight of biomass plot mg of component per g of biomass plot mg of component per g of biomass

Fig. 15.6. The use of membrane filter culture for calibrating methods of biomass determination that involve the measurement of a component of the biomass

medium containing the key nutrients of the solid substrate, or it may even be possible simply to press the moist solid substrate into a compact slab. Various slabs within appropriately-sized petri dishes are then overlaid with sterilized or pasteurized membrane filters, inoculated evenly across their surfaces with a spore suspension, and placed in an incubator. Typically it will be necessary to provide a high humidity within the incubator to prevent the cultures from drying out. In more sophisticated systems, each individual culture has its own chamber. At various sampling times, one or more of these replicate plates can be removed and processed.

The membrane filter is chosen with a pore size (e.g., 0.2 |im) that is sufficiently small to prevent the fungus from penetrating through the membrane into the substrate slab. It is then a simple matter to peel the mat of biomass off the filter. It can then be processed as desired, for example, it can be dried for dry weight determination. Alternatively, it might be processed for the determination of a biomass component such as glucosamine, ergosterol, or protein. As long as the membrane allows extracellular enzymes to diffuse into the substrate, this method can be used with polymeric carbon sources.

Beads of an ion exchange resin, impregnated with nutrient medium, have also been used as a system that allows direct biomass measurement, since the biomass can be easily dislodged from the beads (Auria et al. 1990). However, it has only been used with soluble nutrients, presumably because it is difficult to impregnate the resin with macromolecules such as starch.

15.3.2 Conversion of Measurements of Components of the Biomass

If the biomass composition remains constant for samples removed at different times during the growth curve, then it is a simple manner to convert an indirect measurement into an estimate of the biomass.

cf where CCA is the concentration of the component in a sample removed during the fermentation (mg-component g-IDS-1), CF is the relationship between the component and the biomass, as determined in the calibration experiments (mg-component g-biomass-1), and CXA is the calculated biomass content of the sample (g-biomass g-IDS-1).

If the level of the component in the biomass varies during the fermentation, it will be necessary to use different conversion factors for samples removed at different times. One example where this has been done is in the work of Nagel et al. (2001b). They measured the glucosamine content as a function of time in a system where their fungus was grown on a membrane overlaid on a slab of pressed, ground wheat. They used non-linear regression of a curve plotted in the manner described in Fig. 15.6 to obtain the following equation for the glucosamine content of the biomass (Gx, mg-glucosamine mg-dry-biomass-1) as a function of time:

where A. is the lag time (h). Obviously this kind of approach can be adapted to other systems.

15.3.3 Limitations of these Calibration Methods

A problem with these calibration methods is that it is not possible to be sure that the conversion factor (or temporal relationship) that holds in the calibration system will be followed in the real SSF system. The more the substrate used in the calibration system can be made to mimic the substrate used in the SSF system then the more likely it is that the conversion factor will be reliable, but some doubt will always remain. Also, the calibration must be redone, even with the same microorganism, if the substrate is changed, or if environmental or nutritional conditions are varied significantly from those under which the relationship was determined.

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