Introduction (ngebahas fermentasi)

Many of today's biotechnology companies rely on fermentation to produce their

products.In the typical process, a particular microorganism is used to produce a valuable
compound or product.Fermentation permits us to scale-up that production.Instead of a single cell
producing our "molecule of interest", we use hundreds, thousands andthen millions and millions
of these tiny micro-factories to produce many different products on a commercial scale.In
fermentation, there are two ways in which the product of interest is produced by the cells :
1. Extracellular production, is when the cell "secretes" the product of interest.
Examples of this could be certain amino acids, enzymes or monoclonal antibodies. In
the case of extracellular production, the cells -- or biomass - are discarded at the
beginning of separation and the medium -- which contains the product - is kept for
further processing
2. Intracellular production is when the productof interest produced by the cells is
"kept" inside the cell...and during the early stages of separation, the biomass is
collected and then disruptedso that the product is released.In our treatment of
fermentation, we concentrate on intracellular fermentation.

The Basics of Recovery

The final product of fermentation is called broth. This broth contains the "molecule of
interest". However the molecule is still locked inside a host cell and millions of these host cells
are suspended in a pool of depleted media and metabolic waste products.
Two important functions In the Recovery
1. separating the cells -- the solids in our broth - from the liquid
2. then separating the molecules of interest from their host cells. Exactly where
within the production flow those technologies are employed varies significantly from product to
product.
Recovery tools (to isolate the product) :
1. centrifuges
2. cell disrupters
3. microfiltration
 In this program, we are going to look at a typical recovery process used in the manufacture
of GFP - (Green Fluorescent Protein). GFP is broadly used as a biological marker which - if
attached to a drug - could provide researchers with a visual story of where the drug goes. It is a
fluorescent dye that's verywell tolerated by most cells and doesn't interfere with normal cellular
function.

Recovery in brief
Fermentation broth harvested cells separated Cells washed separated again cells
resuspended cells disrupted (homogenized) debris removed sent to purification
After Fermentation is complete, the broth is harvested and sent to Recovery. Here the
E.coli host cells are separated from the liquid broth. Then suspended in a new solution to wash
the cells. Separated again. Resuspended in a new solution. Homogenized to break open the
cells. The cell debris is removed. Once Recovery is complete, the product issent to Purification
to be refined and concentrated.
But to really understand Recovery, we'll need to closely examine our process so we can
appreciate what each step accomplishes, and why it's important to the process.

Tools, Materials, and Preparation

Let's start with the tools we'll use in the Fluorescent Green Protein Recovery process. Those
would include :
1. Disc Stack Centrifuge to separate solids from liquids.
2. A Homogenizer to break open the E.coli cells
3. a.0.22 micron filter to separate any remaining solids in the product solution
Our materials include the Broth from our fermentation process are :
1. High Purity Water that has been reverse-osmosis filtered, de-ionized and UV
sterilized
2. buffering solution to help stabilize the pH of our product and keep it in suspension and
prevent the product from degrading.

The Recovery process is managed through the use of a Batch Process Record (BPR). The
Batch Record leads the operator through the process, step-by-step with each step requiring a
sign-off and separate verification by a second operator. This record also includes : spaces for
documenting times, activities, operation steps, and instrument readings.
Before the process can begin, the Recovery area must be cleaned and organized, so :
1. Any unnecessary equipment or materials should be removed and the area must be
cleaned and disinfected to decrease the level of microorganisms
2. All equipment must be cleaned, sanitized, and set up as required by Standard
Operating Procedures (SOPs)
3. All required materials and documentation must be gathered and prepared
4. any updates to the Process Control software should be made and verified.

The Recovery
1. GFP Recovery begins with the arrival of the broth tank : (broth harvested)
2. A sterile hose is run from the broth tank to the Disk-Stack Centrifuge and the tank is
pressurized to drive the broth from broth tank into the centrifuge.
3. After the centrifuge has reached a stable running speed, the inlet valve is opened and broth
enters the bowl.
4. The centrifugal force of the rotation forces the denser material (the solids) to the sides of the
bowl (much like the spin cycle on your clothes washer!), while the liquid flows through and
out of the centrifuge : (cells separated)
5. As more broth enters the bowl, it displaces the now clarified (solids removed) liquid to the
top of the bowl where it exits, while the cells continue to build up on the bowl surface.
Notes:
*The centrifuge has an integrated RPM monitor. If the unit is not rotating at a stable running
speed, the controller will alarm and shut it down.
*The liquid leaving the bowl is known as the "clarified stream", because almost all the solids
have been removed.
*A sensor monitors the clarified stream for "percent solids". When this value rises it
indicates that the bowl is at capacity, and the solids must be removed before processing
more broth.
*The solids are the E.coli cells, and they contain the product
6. When the bowl has reached capacity for solids, the bowl opens and the solids are discharged
into an appropriate container for collection.
7. Once the solids are discharged, the centrifugation step can resume while the clarified liquid
is waste. At this point, the cells are in a paste form,and although most of the liquid has been
removed, our cell paste is still about 40% liquid weight.
8. The remaining liquid contains high levels of metabolites and salts that could complicate
downstream processing, so we're going to lower those levels by "washing" the cells.
9. The cell paste is suspended in a buffered solution and then run through the centrifuge again.
As the clarified liquid leaves the centrifuge this time, it carries many of the contaminants
from the fermentation step with it : (Cells washed)
10. The cells, once again in paste form, are ready for the next step Cell Disruption, also called
Lysing. The cells are (resuspended) in a buffered solution and then pumped at high
pressure, 900 bar- which is about 13,000 pounds per square inch - through the Homogenizer.
Inside the Homogenizer they are forced through a tiny orifice. Just like a balloon being
tightly squeezed, the cells can't take the stress - and they rupture and break apart : (cell
disrupted).
11. (debris removed) : to ensure that all the E.coli cells are ruptured, the solution is cycled
through the homogenizer a second time. After the second homogenization, the lysed cell
solution is pumped back through the centrifuge. But this time, our goal is different! Before
lysing, our product was held within the E.coli cells. Now -- with the cells broken apart, the
cell contents - including cytoplasm and green fluorescent protein- are mixed into the
buffered solution. The centrifuge again spins out the solids- which are primarily cell debris -
and it's the clarified liquid which contain sour product: GFP! This time we discard the solids
and keep the liquid -- which is now known as Lysate!
12. (sent to purification) : Although the centrifuge has removed almost all of the cell debris,
some small particles still remain. We'll remove those with our final Recovery process step:
Filtration.The centrifuge lysate is pumped through a 0.22 micron filter.This filter is fine
enough that it removes virtually all of the remaining solid materials.At this point, the process
stream is referred to as Clarified Lysate.The Recovery process is finished.The Clarified
Lysate is pumped into a vented, temperature-controlled transfer vessel.This Lysate tank then
moves downstream to the next series of process steps--Purification--where dissolved
impurities are removed from the GFP solution, and GFP is subsequently concentrated and
stabilized.