Transport phenomena in bioprocess

 

Biomass maintenance – the focal point of the process.

The growth of biomass either as a primary product (bakers yeast) or as a cell factory producing primary or secondary metabolites is the main aim of the bioprocess

Ensuring that ideal conditions for biomass growth and maintenance are in place is the engineers responsibility.

·         Biomass often requires oxygen (aerobic growth). Oxygen is sparingly soluble in aqueous solutions. Therefore the challenge is there to provide adequate levels of oxygen at all stages of the process

·         Providing adequate oxygen transfer requires considerable kinetic energy input. This energy will eventually become heat energy. This heat has to be removed in order to maintain the optimal temperature for biomass growth (30^oC for yeast and some bacteria, 37^oC for animal cells and microbial pathogens).

·         Microbial metabolism also results in heat evolution. This heat has also to be dealt with.

The specific oxygen uptake rate increases with increase in the dissolved oxygen concentration up to a certain point (referred to as C_crit) above which no further increase in oxygen uptake rate occurs. Thus, maximum biomass production may be achieved by satisfying the organism's maximum specific oxygen demand by maintaining the dissolved oxygen concentration greater than the critical level. If the dissolved oxygen concentration falls below the critical level then the cells may be metabolically disturbed.  Oxygen is normally supplied to microbial cultures in the form of air, this being the cheapest available source.

The transfer of oxygen from air to the cell during fermentation occur in a number of steps:

(i) The transfer of oxygen from an air bubble into solution.

(ii) The transfer of the dissolved oxygen through the fermentation medium to the microbial cell.

(iii) The uptake of the dissolved oxygen by the cell.

Defining the mass transfer problem

There are eight resistances to mass transfer in this system.

1. The gas film
2. The gas-liquid interface
3. The liquid film at the GL interface
4. The bulk liquid
5. The liquid film surrounding the solid
6. At the liquid-solid interface
7. In the solid phase
8. At the reaction sites in the solid phase

Reactions 1-7 are purely physical and occur in series.

Assuming that oxygen transfer to the bulk liquid phase is limited by the liquid layer at the gas-liquid interface and that the bulk liquid phase is well mixed.

Oxygen Transfer rate   

                                

Where….

 is the concentration of oxygen in the liquid layer adjacent to the gas-liquid interface at infinite time (saturation concentration)

C_L is the bulk liquid oxygen concentration

Lets call the constant of proportionality k_La

Integrating results in the following…

DETERMINATION OF KLa VALUES

The sulphite oxidation technique

The measurement of dissolved oxygen concentrations relies on the rate of conversion of a 0.5 M solution of sodium sulphite to sodium sulphate in the presence of a copper or cobalt catalyst:

Na₂SO₃ + 0.5O_z = Na₂SO₄

The rate of reaction is such that as oxygen enters solution it is immediately consumed in the oxidation of sulphite, so that the sulphite oxidation rate is equivalent to the oxygen-transfer rate.

The procedure is carried out as follows: the fermenter is batched with a 0.5 M solution of sodium sulphite containing 10^-3 M Cu₂⁺ ions and aerated and agitated at fixed rates; samples are removed at set time intervals (depending on the aeration and agitation rates) and added to excess iodine solution which reacts with the unconsumed sulphite, the level of which may be determined by a back titration with standard sodium thiosulphate solution. The volumes of the thiosulphate titrations are plotted against sample time and the oxygen transfer rate may be calculated from the slope of the graph.

Advantage is simplicity and, also, the technique involves sampling the bulk liquid in the fermenter and, therefore, moves some of the problems of conditions through the volume of the vessel.

However, the procedure is time consuming (one determination taking up hours, depending on the aeration and agitation and is mostly inaccurate.

Gassing-out techniques

Depends upon monitoring the increase in dissolved oxygen concentration of a solution during aeration and agitation. The oxygen transfer rate will decrease during the period of aeration as the solution get saturated

To monitor the increase in dissolved oxygen over an adequate range it is necessary first to decrease the oxygen level to a low value. Two methods have been employed to achieve this lowering of the dissolved oxygen concentration - the static method and the dynamic method.

THE STATIC METHOD OF GASSING OUT

In this technique the oxygen concentration of the solution is lowered by gassing the liquid out with nitrogen gas, so that the solution is 'scrubbed' free of oxygen. The deoxygenated liquid is then aerated and agitated and the increase in dissolved oxygen monitored using some form of dissolved oxygen probe.

THE DYNAMIC METHOD OF GASSING OUT

The respiratory activity of a growing culture in the fermenter is used to lower the oxygen level prior to aeration. The procedure involves stopping the supply of air to the fermentation which results in a linear decline in the dissolved oxygen concentration due to the respiration of the culture.

The dynamic gassing-out method has the advantage over the previous methods of determining the KLa during an actual fermentation and may be used to determine K L a values at different stages in the process. The technique is rapid and only requires the use of a dissolved-oxygen probe, of the membrane type.

A major limitation in the operation of the technique is the range over which the increase in dissolved oxygen concentration may be measured. It is Important allow the oxygen concentration to drop during the deoxygenation step.

The oxygen-balance technique

The KLa of a fermenter may be measured during fermentation by the oxygen balance technique which determines, the amount of oxygen transferred into solution in a set time interval. The procedure involves measuring the following parameters:

(i) The volume of the broth contained in the vessel

(ij) The volumetric air flow rates measured at the air inlet and outlet

(iii) The total pressure measured at the fermenter air inlet and outlet

(iv)The temperature of the gases at the inlet and outlet

(v) The mole fraction of oxygen measured at the inlet and outlet

The oxygen-balance technique appears to be the simplest method for the assessment of KLa and has the advantage of measuring aeration efficiency during fermentation. The sulphite oxidation and static gassing out techniques have the disadvantage of being carried out using either a salt solution or an uninoculated, sterile fermentation medium.

The effect of medium and culture rheology on KLa

Fluids may be described as Newtonian or non-newtonian depending on whether their rheology characteristics obey Newton's law of viscous flow.  A Newtonian liquid has a constant viscosity regardless of shear, so that the viscosity of a Newtonian fermentation broth will not vary with agitation rate. However, a non-Newtonian liquid does not obey Newton's law of viscous flow and does not have a constant viscosity.

A fermentation broth consists of the liquid medium in which the organism grows, the microbial biomass and any product which is secreted by the organism. Thus, the rheology of the broth is affected by the composition of the original medium and its modification by the growing culture, the concentration and morphology of the biomass and the concentration and rheological properties of the microbial products. Therefore, it should be apparent that fermentation broths vary widely in their rheological properties and significant changes in broth rheology may occur during fermentation.

Highly viscous non-Newtonian broths of fungal and streptomycete fermentations present major difficulties in oxygen provision.

The effect of the degree of agitation

It has been demonstrated to have a profound effect on the oxygen-transfer efficiency of an agitated fermenter.

(i) Agitation increases the area available for oxygen transfer by dispersing the air in the culture fluid in the form of small bubbles.

(ij) Agitation delays the escape of air bubbles from the liquid.

(iij) Agitation prevents coalescence of air bubbles.

(iv) Agitation decreases the thickness of the liquid film at the gas-liquid interface by creating turbulence in the culture fluid.

Bioreactors: Essential Requirements

• Support high biomass concentration
• Maintain sterile conditions
• Provide adequate aeration (if aerobic)
• Agitation (homogeneous suspension, mass transfer)
• Removal of generated heat (temperature control)
• Shear conditions appropriate to biological system

Mass transfer in the bioreactor

All biochemical reactors of significance involve hetrogenous systems generally with more than one phase

To ensure effective biotransformation, interphase mass transfer must occur between these phases

Typically the transfer of oxygen from the gas phase to the liquid phase (where it is sparingly soluble) is the most difficult mass transfer problem in the reactor.

Another mass transfer problem is the removal of CO₂, a byproduct of metabolism from the fermentation broth.

Definition of mass transfer coefficients

Example: oxygen