Outline

Scale up issues

Overview of reactor types Operating issues that affect reactor design

Heat transfer Foaming Sterility Oxygen transfer

Overview of Reactor Types

Reactors with internal mechanical agitation (impellers, turbines) Bubble columns, which rely on gas sparging for agitation Loop reactors, in which mixing and liquid circulation are achieved by the motion of an injected gas, by a mechanical pump, or by a combination of the two

Bioreactor Types: (A) Stirred-Tank Reactor, (B) Bubble-Column Reactor

Reactors with Internal Mechanical Agitation

Most common type of bioreactor, highly flexible Mechanical agitation:

Disperses gas bubbles throughout the reactor Increases the residence time of bubbles Shears large bubbles to form smaller bubbles Provides high kLa values

Air is supplied by a sparger Mixing is accomplished using disk- or turbinetype impellers

Baffles also augment mixing

Liquid Flow in Bottled Tanks

A Rushton radial flow impeller (A) is a disc with 6-8 blades, designed to pump fluid in a radial direction

This was the predominant impeller design til the 1980s, and is still the most common type found in industrial and laboratory fermenters

An axial flow hydrofoil impeller (B) is designed to pump liquid either up or down (i.e. in an axial direction)

These impellers have been shown to provide superior performance compared to Rushton impellers, in terms of lower energy outputs required for equivalent oxygen transfer Also produce less shear stress

Bubble Columns
Disperse gas bubbles through the fermenter, using perforated plates to enhance gas dispersion and mixing Provide a low-shear environment, although cells often accumulate at the surfaces of bubbles, and bubble bursting can damage or destroy cells Energy efficient (low power input required to transfer a given amount of oxygen, relative to stirred tank reactors) Absence of mechanical agitation reduces costs and eliminates one potential entry point for contaminants

Loop Bioreactors: (C) Airlift Loop Reactor, (D) Propeller Loop Reactor, (E) Jet Loop Reactor

Airlift Loop Bioreactors

Airlift systems are most common type of loop bioreactor

Bubbles rising in a draft tube cause mixing The motion of the gas carries fluid and cells up a draft tube At the top, gas leaves the liquid, and the degassed liquid (which is heavier than the gassed liquid) descends in the ring outside the draft tube At the bottom of the reactor, the descending fluid encounters the gas stream is carried back up the draft tube The largest fermentations (>200,000 L) are usually carried out in airlift bioreactors, as higher oxygen transfer rates and better cooling can be achieved in these systems versus mechanically agitated reactors at very large scale (the largest airlift bioreactor in the world is 1,500,000 L, and is used to produce single cell protein)

Reactor Geometry, Layout and Construction

Reactors can be constructed of glass or stainless steel

Typically, glass vessels are only used for

volumes <50L; structural properties of glass make it unsafe to use glass vessels at volumes >500L 316 stainless steel is used for the full range of fermenter volumes 304 ss (slightly less corrosion resistant) is used for vessel covers and jackets

Typical mechanically agitated 100,000 L fermenter

Height to diameter ratio of 2-3 Sterile air inlet and sparger Baffle plates and impellers Cooling coils Foam breaker Working (liquid) volume ~ 75% of the total vessel volume

100,000L fermenter installed in a plant. The steam lines permit in-place sterilization of valves, pipes and seals. The input air can be sterilized by heat or filtration. S = steam C = condensate W = water A = air

Heat Transfer
In large reactors, the two main limitations on size are the abilities of the design to provide an adequate supply of oxygen and to remove metabolic heat efficiently Large reactors use either internal coils or a jacketed vessel for heat removal

Internal coils provide advantages over cooling jackets, as they provide a larger surface area for heat transfer In some systems the coils can rapidly become fouled by microbial growth, decreasing heat transfer and often adversely affecting mixing

Foaming
Many microorganisms produce foam as a byproduct of growth

If foam escapes from the fermenter, it can wet (and block) air filters, decreasing influent air flow, and/or causing pressure to build up in the reactor if the effluent filters are blocked Wet filters also become a potential entry point for contaminating organisms Foam can be controlled by either mechanical breakers or chemical agents (antifoam) Reactors are typically operated at a working volume 0.75 total vessel volume to prevent foam-over and allow for volume increase due to aeration and agitation

Sterility
Preserving the sterility of the vessel contents throughout a fermentation run is a prime consideration in reactor design

Pressurized steam is used for in-place sterilization (SIP) of the reactor and all seals, probes and valves All surfaces that will be in contact with the vessel contents must be smooth and readily cleanable

Crevices in the tank surface, pipes or valves can trap

large quantities of particulate organics and contaminating organisms; such clumps of cells increase the chances for a contaminant to survive the sterilization procedures

Cleaning
Cleaning is generally performed in-place (CIP) Highly alkaline detergents are typically used (often at high temperatures), and this factor dictates the selection of materials (e.g. 316 ss) Materials that will be in contact with fermentation broth are typically electropolished to ensure smooth and uniform surfaces In a typical fermentation run, CIP is performed at the end of a run, and then again before a new run is initiated, after which SIP procedures are performed, and then the new run can begin

Oxygen Transfer
Recall that the equation for oxygen uptake rate (OUR) is:

OUR = X qO2 = k L a (C C L )
*

(10.1)

Where qO2 is the specific uptake rate of oxygen (mol O2/g-h)

The value of qO or OUR is the demand side of 2 the equation; typical values of OUR in largescale culture are 40 to 200 mmol O2/l-h, with most systems in the 40-60 mmol O2/l-h range

Typical Respiration Rates of Various Organisms in Culture

Oxygen Transfer
On the supply side, the critical parameter is kLa (h-1), the volumetric transfer coefficient

kLa is a measurable parameter

A variety of correlations exist to predict values for kLa, but none consistently produce accurate estimates Existing correlations neglect the effects of medium components The presence of salts and surfactants can significantly alter bubble size and liquid film resistance around the gas bubble; these factors can also affect the maximum oxygen solubility (C*) Temperature and pressure also affect both kLa and C* Four approaches are commonly used: unsteady state, steady state, dynamic and the sulfite test

The Unsteady State Method

A reactor is filled with water or medium and C* is measured (saturated concentration of oxygen) The medium (or water) is then sparged with nitrogen to remove the dissolved oxygen Air is then introduced, and the change in DO is monitored until the solution is essentially saturated: dC L (10.4a) = k L a C * CL dt d C * CL or = kLa (10.4b) * dt C C L

or

ln C * C L = k L a t

( (

) )

(10.5)

The Unsteady State Method

(a)Typical data for unsteady-state accumulation of oxygen in a large stirred tank (b)Calculating kLa from a semi-log plot of (C*- CL) vs time

The Sulfite Method

SO32, is an oxygen scavenger, which, in Sulfite,

the presence of Cu2+ removes O2 from the medium to form sulphate, SO42 The rate of sulphate formation is monitored, and is proportional to the rate of oxygen consumption, as shown in the following equations: 1 (10.6) dCSO4 dt = k L aC * 2 1 dCSO4 dt kLa = or (10.7) * 2 C

The Steady State Method

Considered the best method for determining kLa Uses actual fermenter culture (containing cells) Requires the accurate measurement of oxygen concentration in all gas exit streams and a reliable measurement of CL A mass balance on O2 in the gas streams allows the rate of O2 uptake, OUR, to be calculated, which then provides an expression for kLa: OUR (10.8) kLa = * C CL

The Dynamic Method

Like the steady state method, the dynamic method also uses a fermenter containing active cells

It is simpler than the steady state method, as it requires only a DO probe and a chart recorder, rather than off-gas analyzers + a DO probe (as required in the steady state method) The dynamic method requires that the O2 supply be turned off for <5 min and then turned back on various parameters can then be calculated from a plot of DO vs time (see graph) The primary advantage of the dynamic method is that kLa can be measured under actual fermentation conditions

The Dynamic Method