Svq

Calibration curve

so, measure either

• a biomass component that is not present in the substrate (protein, glucosamine, ergosterol)

• a biomass activity (consumption of O2 or production of CO2)

determine dry weight directly? No! It is almost impossible to separate the fungus and the residual solid substrate but typically the relationship with biomass does not remain constant throughout growth

The problem: Given a profile of glucosamine contents of the fermenting substrate, how much of the increase is due to the production of new biomass and how much is due to an increase in the glucosamine content of the biomass?

but typically the relationship with biomass does not remain constant throughout growth

time

Fig. 14.3. A comparison of the ease of establishing biomass dry weight profiles. (a) In submerged liquid fermentation. (b) In solid-state fermentation time

Fig. 14.3. A comparison of the ease of establishing biomass dry weight profiles. (a) In submerged liquid fermentation. (b) In solid-state fermentation

14.2.2 Indirect Approaches to Monitoring Growth

This section briefly mentions some of the direct approaches and various indirect approaches that can be used for monitoring growth in SSF systems. It is not intended to be an exhaustive review and it does not give protocols for the various methods. These should be searched for in original references. Some useful sources are given in the further reading section at the end of this chapter.

In some cases direct separation of the biomass is possible. With unicellular organisms it may be possible to dislodge the cells from the solid particles during a homogenization step and then to separate the solids from the suspended cells by sedimentation. However, some cells will remain adhered to the sedimented solids while some fine solid particles ("fines") liberated from the solids will not sediment. These fines will cause problems for determination of dry weight by filtration of the supernatant, since they will be erroneously counted as dry biomass. If viable count measurements are done on the supernatant, it is quite probable that the fines will have various cells adsorbed onto them, and these will give rise to only one colony per particle instead of one colony per cell.

In fungal fermentations, it is sometimes possible to digest the solid substrate within an aqueous enzyme solution, thereby allowing the mycelial biomass to be recovered by filtration. For example, this may be possible if the solid substrate is based on starch and contains little fiber, in which case the substrate can be hydro-lyzed with amylases. However, some of the dry weight of biomass may be lost in this procedure and some solid residues may remain in the filtered biomass fraction. The efficiency of the recovery could be checked by submitting known masses of fungal mycelium, for example, from membrane filter culture (Chap. 15.3.1), to the hydrolysis and recovery procedure.

Various indirect methods rely on measurement of biomass components such as:

• Ergosterol. This is the predominant sterol in the cell membrane of many fungi, and is typically not found in plant material. It can be quantitatively measured by gas chromatography, HPLC, or UV spectrometry.

• Glucosamine. This is produced by the hydrolysis of chitin, which many fungi contain in their cell wall. It is typically not found in materials of plant origin. The hydrolysis of the biomass and subsequent determination of glucosamine by the chemical method can be quite tedious. It may be preferable to determine the glucosamine in the hydrolysate by HPLC.

• Protein. Protein is a major cell component. However, it is present in many plant materials and, if present, it will be impossible to know the proportion of protein in the sample that comes from the substrate, and the proportion that comes from the biomass, since the microorganism will typically hydrolyze the protein during growth. Therefore use of protein determination as an indicator of growth is restricted to cases in which the substrate contains negligible protein.

Unfortunately, the content of all of these components within the biomass can vary with culture conditions and with the age of the fungal mycelium. This greatly complicates the conversion of indirect measurements into estimates of the dry weight of biomass.

Other indirect methods rely on detecting activities of the biomass. Of these, the consumption of O2 and production of CO2 are most important. Gas metabolism is potentially a very important growth activity, especially since the rate of heat evolution will typically be directly proportional to the O2 consumption rate, at least for an aerobic process. Further, the overall O2 consumption within a bioreactor can be used for on-line monitoring of the growth process, even though it is not necessarily a simple matter to convert the O2 consumption profile into a trustworthy biomass growth profile. Due to the importance of O2 uptake measurements, the experimental use of this method in growth kinetic studies is discussed in Chap. 15.

The above discussion shows that several questions must be answered when selecting an appropriate indirect method for estimating growth:

• Is the component that is to be measured also present in the substrate?

• What time and resources are required for processing of the samples?

• To what degree does the relationship between the activity or component and the amount of biomass present change during the fermentation?

It may or may not be desired to convert an indirect measurement into an estimation of the biomass itself. If it is desired to do so, then the measurement method must be calibrated. In other words, the organism must be grown in a system that allows the dry weight of biomass to be measured in addition to the component or activity. These issues are discussed in Chap. 15.3.

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