- Biomass is a critical parameter in monitoring and control of SSF

- Is difficult to estimate on-line.

- Especially with filamentous fungi, where the hyphae penetrate and bind tightly
to the solid substrate.

- Indirect methods are available

- estimation of cell-specific components such as ergosterol
- Glucosamine
- Proteins and nucleic acid
- Spores

- Biomass is also correlated with biological activity like

- estimating ATP,
- respiratory activity
- enzyme activity, metabolite production, nutrient consumption
- Disadvantages, samples to be withdrawn continuously from the reactor bed
- this compromise sterility , analysis is time consuming and costly
- difficult to obtain, well mixed or homogenous samples
- If sample are selected from matrix grid, volume and number increases

- For modular mixed bioreactor system, wherein hundreds of modules may be

stacked together, measurement and analysis is a challenge

- Measuring respiratory activity is economic

- Exhaust gases are rich in information about organism metabolic activity and in
general bioreactor performance.

- O2 uptake rate (OUR) and CO2 evolution rate (CER) provide convenient way of
on-line monitoring of microbial activity in an SSF bioreactor
- How Will Growth Be Measured Experimentally?
- The Problem of Measuring Biomass in SSF

- In order to measure the dry weight of biomass directly, it is necessary to separate

the biomass from solids.
- Due to the penetration of the mycelium into the solid substrate, it is often
impossible to remove the biomass quantitatively from the substrate

- Even in fermentations that involve unicellular organisms,

- Many of the cells are adhered to the particle surface
Various indirect methods rely on measurement of biomass components such as:

Ergosterol.
- Predominant sterol in cell membrane of fungi, not found in plant material.
- Can be quantitatively measured by GC, HPLC, UV spectrometry.

Glucosamine.
- Produced by hydrolysis of chitin, not found in plants origin.
- Hydrolysis of biomass and subsequent determination is tedious.
- So, determine glucosamine in the hydrolysate by HPLC.

Unfortunately, the content vary with culture conditions and age of mycelium.
This complicates measurements
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.
Measurable parameters

Bed temperature - heterogeneity of the bed - several temperature sensors placed

inside the solid bed to get a reliable estimation of its average temperature.
- Growth and production rates affected if hot spots inside the bed
are not addressed

Bed water content - Water content should be measured periodically to avoid bed
overdrying, especially during the exponential growth phase.

Bed porosity - growth of filamentous fungi reduces bed porosity,

lowering CO2 and O2 transfer
limiting metabolic heat removal.
Inlet air conditions – air flow rate, and humidity of the inlet air stream need to be
measured. The only way to control temperature

CO2 and O2 concentration (respirometric variables). - exhaust gases -physiological

state of the culture and allow us to detect if the microorganism is under stress.

CO2 Evolution Rate (CER), O2 Uptake Rate (OUR), and the respiratory
quotient (RQ)

Volatile metabolites. Monitoring volatile metabolites in the gas outlet stream can
provide an indication about the production rate and metabolic activity or help in
defining operating policies to improve process reproducibility
pH - measuring pH inside solid beds is difficult - so prepare the substrate with a
buffered solution, so that pH does not change too much during the fermentation.

Biomass concentration – indirect calculations

Substrate concentration - Establishing the initial concentration of the limiting

substrate is usually enough to ensure normal operation in batch bioreactors.

However, in the production of secondary metabolites it may be necessary to

measure the limiting substrate concentration periodically so that optimal fed-batch
policies can be established.

In SSF this might be difficult, especially if the substrate contains several carbon or
nitrogen sources that the microorganism is capable of using.
Product concentration.