The problems of scaling up a growth, from 200 ml to 10,000 l

Parameters that must be precisely regulated

• temperature
• pH
• rate and nature of mixing
• oxygenation
• sterility and containment

On having the right medium - shall I get "super" or "turbo"?

What if I don't know how?

Service, with discretion

Step-wise scale-up of culture

• 5-10 ml stock
• 200-1000 ml shake flask
• 10-100 l seed fermenter
• 1,000 - 100,000 l production fermenter

  Example - a Picchia fermenter

The black line represents the log of the number of cells as a function of time. What does a straight line on this plot represent (e.g. the segment marked "log")? It means that the segment could be described as a linear function of the form y=mt+b, where "t" is the "time" axis. Remember the "y" in this case is the log base 10 of the cell number rather than the cell number itself.

Where the black line can be expressed in the form y=mx+b, the derivative dy/dt is a constant m. That means that the rate of growth of (the logarithm of cell number) is constant whenever the black line is straight.

Types of cultures

• Batch
• Fed batch
• Continuous (e.g. Fig 12.7, p. 286 for indigo production)

How are these defined?

and an analogy to freeway traffic...

Fed-batch fermentation A comparison of glucose with Gatorade© drink powder written as a term project during the Fall 1995 semester for Intro to Biochemical Engineering. Theory of fed-batch fermentation Kurt M. Fritzsche "Fed-batch fermentation is described in BASIC Biochemical Engineering (Bungay, 1993) as the type of system where "nutrient is added when its concentration falls below some set point." Usually the addition is of the nutrient is controlled by a computer for precision. The best way to control the addition of the feed is to monitor the concentration of the nutrient itself in the fermenter or reactor vessel, so you know exactly how much more of it needs to be added. The nutrient is added in several doses, to ensure that there is not too much of the nutrient present in the fermenter at any time. If too much of a nutrient is present, it may inhibit the growth of the cells. By adding the nutrient a little bit at the time, the reaction can proceed at a high rate of production without getting overloaded." http://www.eng.rpi.edu/dept/chem-eng/Biotech-Environ/LAB/theory.htm "Here is the graph of how much carbon dioxide was leaving the reactor flask each minute. The graph has been corrected to incorporate the fact that the glucose feed solution ran out at 900 minutes, and the Gatorade feed solution ran out at 600 minutes. The glucose is the solid line with squares, and the Gatorade is the dotted line with circles. The graph itself is not very indicative of how much carbon dioxide was totally produced. The total amount of carbon dioxide produced is important, since it directly correlates to how much ethanol was produced. The great the amount of carbon dioxide generated, the greater the amount of ethanol produced. To determine the total amount of ethanol produced, one must add up the area under each curve. Doing so, one will find that the area under the glucose curve sums to greater than 20,000 milliliters of carbon dioxide generated, while the area under the Gatorade curve sums to just less than 17,000 milliliters of carbon dioxide produced. Therefore, one can deduce that glucose produces more ethanol than an equivalent amount of Gatorade brand drink powder for this particular fermentation." http://www.eng.rpi.edu/dept/chem-eng/Biotech-Environ/LAB/correct.htm

Continuous fermentation http://www.eng.rpi.edu/dept/chem-eng/Biotech-Environ/Contin/ethanol.htm OPERATING COST FOR FUEL ETHANOL PRODUCTION BY CONTINUOUS FERMENTATION AND STRIPPING AND COMPARISON WITH EXISTING TECHNOLOGY Author(s): TAYLOR FRANK MCALOON ANDREW J CRAIG JR JAMES C "Approximately 10% of the gasoline now sold in the U. S. is a mixture of 10% ethanol and 90% regular gasoline. Most new cars can run on this fuel mixture. Ethanol is produced from corn in fermentors, brewing reactors where yeast microorganisms consume the glucose sugar in starch extracted from corn, and synthesize the alcohol. The problem is that this process is more costly than that used for making gasoline. This paper presents a detailed cost analysis of a new fermentation process that requires much smaller and less expensive fermentors. The results suggest that, compared with the current fermentation process, a savings of 3.5 cents per gallon of ethanol can be realized with the new process. Opportunities therefore exist for making ethanol production more competitive with gasoline. " http://www.nal.usda.gov/ttic/tektran/data/000007/33/0000073382.html

Advantages of continuous fermentation (p. 405):

• smaller bioreactors
• smaller scale of equipment for cell harvesting
• avoids "down time" between batches
• uniform physiological state of cells

Disadvantages:

• 500-1000 hours duration
• overgrowth of variants lacking plasmid
• maintenance of sterility over long term
• batch to batch variation of medium is problematic

Dissolved oxygen

Agitation

- particularly a problem of scaled-up procedures

Optimal agitation

• Adequate mixing of nutrients
• Prevention of accumulation of toxic metabolites
• Uniformity of pH and temperature
• Rate of transfer of oxygen from bubbles to liquid medium

Excessive agitation

• Hydromechanical stress (shear)
• Temperature increase in culture

Optimization of growth medium to generate high-density cultures (pp. 407-8)

Inhibition of growth of E. coli can be caused by:

> 50 g/l glucose

>3 g/l ammonia

>1.15 g/l iron

>8.7 g/l magnesium

>10 g/l phosphorus

>0.038 g/l zinc

Oxygen delivery rate

sparging rate

agitation rate

use of pure oxygen instead of air

Temperature - removal of excess heat of fermentation by cooling coils (notwithstanding the problem of "fouling", p. 410)

Example: Exponential feeding rates in fed-batch culture

Bioreactors

Stirred-tank reactors (STR)

Bubble column reactors

Internal- and External-loop airlift reactors

Advantages of STR

flexible operation

readily available

efficient gas exchange

extensive base of experience

Advantages of pneumatic reactors (e.g. bubble and airlift)

agitation is caused by rising air - more energy efficient

less shear force

Typical large-scale fermentation systems

The problem: IPTG, temperature shifts, etc. are used to induce the expression of genes from specific promoters. How do you induce a 10-100 liter culture uniformly and quickly?

Solution: Two-stage airlift reactors (e.g. Figure 16.5). Example: Production of T4 DNA ligase (a commercial product) under control of PL promoter and CI⁸⁵⁷ inducer.

Cell stress

The codon bias problem

An example of a Candida protein in production

Harvesting of cells

High-speed centrifugation (including continuous flow centrifugation)

disadvantages:

stopping and re-starting

cost of equipment

potential release of aerosols or cells in spent liquid

Micro-filtration

disadvantages:

decrease in flow rate with time

Cell disruption

• chemical methods (high pH, organic solvents, detergents)
• enzymatic lysis
• osmotic shock,
• freeze-thaw,
• sonication,
• wet milling (bead beaters),
• high-pressure homogenization (french press)

Examples:

• Design and Production of recombinant subunit vaccines
  
  
• Pichia

Moving up - production in macroorganisms

• Plants -Meristem Tech.

• Baculovirus

• Optimizing the CHO cell system

• Cows give milk