Ergosterol
Ergosterol is the predominant sterol in fungi. Glucosamine estimation was therefore compared with the estimation of ergosterol for the determination of growth of Agaricus bisporus (Matcham et al, 1985). In solid cultures, directly proportional relationships for glucosamine and ergosterol against linear extension of the mycelium were obtained. Determination of ergosterol was claimed to be more convenient than glucosamine. It could be recovered and separated by HPLC and quantified simply by spectrophotometry, providing a sensitive index of biomass at low levels of growth. HPLC was necessary to separate the ergosterol from sterols endogenous to the solid substrate. However, Nout et al., (1987) showed that the ergosterol content of Rhizopus oligosporus varied from 2 to 24 micrograms per mg dry biomass, depending on the culture conditions, aeration and substrate composition, concluding that it was an unreliable method for following growth.
Physical measurement of biomass
Peñaloza (1991) evaluated mycelial growth, based on the difference in the electric conductivity between biomass and the substrate. Good correlation with biomass was obtained and a model was proposed.
Auria et al.,(1990) monitored the pressure drop in a packed bed during SSF of Aspergillus niger on a model solid substrate consisting of ion exchange resin beads. Pressure drop was closely correlated with protein production. Pressure drop is a parameter that is simple to measure and can be measured on-line. Further studies are required to determine whether the use of pressure drop is generally applicable for monitoring growth in SSF bioreactors under forced aeration. An interesting point of this physical technique resides in the fact that it is sensible to conidiation: early conidiophore stage makes the pressure to drop drastically and a breaking point can be easily observed.
In conclusion, the measurement of biomass in SSF is important to follow the kinetics of growth in relation to the metabolic activity. Measurement of metabolic activity by carbon dioxide evolution or oxygen consumption can be generally applied, whereas extracellular enzyme production will only be useful when enzyme production is reasonably growth-associated.
Vital staining with fluorescein diacetate has potential in providing basic information as to the mode of growth of fungi on complex solid surfaces, as this method can show the distribution of metabolic activity within the mycelium. But it cannot be measured on line.
On the other hand, in the production of protein enriched feeds, the protein content itself is of greater importance than the actual biomass concentration, and the variation in biomass protein content during growth becomes less relevant.
Overall, oxygen uptake and carbon dioxide evolution methods are probably the most promising techniques for biomass estimation in aerobic SSF as they provide on-line information. The monitoring and computing equipment is relatively expensive and will not be suitable for low technology or rural applications. No method is ideally suited to all situations, so the method most appropriate to a particular SSF application must be chosen in each case on the basis of simplicity, cost and accuracy. A good strategy can be a combination of several techniques based on the determination of different parameters that can correlate actual biomass with the material balance.