Scott Stevens, Ph.D.
Use of a large-scale fermentor for gene expression study advances research in directions previously thought unimaginable.Scott Stevens, Ph.D., assistant professor of molecular genetics and microbiology,
Its definitely the exception to the rule for university-based research facilities to conduct fermentation studies utilizing the capabilities of a 500 liter fermentor. Recently BioPharm International capitalized on an opportunity to chat with noted cell culture researcher Scott Stevens, who currently is growing a diverse group of cultures with the use of just such a fermentor at his facility at the University of Texas at Austin, with noteworthy results.
BioPharm: Describe the work youre doing in the laboratory at University of Texas at Austin and what you hope to accomplish.
Stevens: In my laboratory we study ribonucleoprotein
structure and function, specifically
the structure and function of those
ribonucleoproteins involved in gene expression.
The small nuclear ribonucleoproteins
(snRNPs), which are our primary interest, are
present in relatively low abundance (200 to
500 molecules per cell in yeast) making their
isolation and structural characterization a
particular challenge. We hope to determine
the low-resolution structural arrangement of
these snRNPs by cryoelectron microscopy
and eventually, the high-resolution structure
by X-ray crystallography. To produce milligram-
scale quantities of these complex molecules
that contain dozens of proteins and
several small RNA molecules, we require multiple
kilograms of yeast. The strains we use for
these experiments contain specific modifications
in several genes to facilitate the snRNP
purifications, so we are unable to use commercially
available wild-type yeast cakes.
BioPharm: What cultures do you grow in the laboratory?
Stevens: We primarily grow Saccharomyces
cerevisiae in our laboratory, but we also grow
Schizosaccharomyces pombe and Escherichia coli.
BioPharm: For what purpose(s) do you grow the cultures?
Stevens: For our large-scale microorganism
growth, we generally use the product for
protein or macromolecular complex purification.
For S. cerevisiae and E. coli we
perform preparative purifications of single
polypeptides ( E. coli ), or multi-protein
complexes ( S. cerevisiae ). For S. pombe we
perform exploratory analyses of macromolecular
BioPharm: Describe the processes and technology you originally employed to grow cultures and the progression to those you use today?
Stevens: Like most academic investigators
performing similar experiments, we began
experimentation using small-scale cultures
of one to six liters in shake-flasks. We continue
to perform small-scale experiments
to troubleshoot and optimize our purification
procedures. We now are able to routinely
produce 400 liters of culture in a
fermentor (500L modular BioFlo Pro, New
Brunswick Scientific [NBS]) in an overnight
experiment. We find that although using
the fermentor typically allows for
faster and more dense culture
growth, the procedures scale directly
and the resulting product is identical
to what is produced from
smaller-scale cultures. We are currently
attempting to optimize culture
conditions with oxygen
supplementation to increase the biomass
yield at the end of the culture
growth. With the equipment we use,
there have been no challenges to
overcome in scaling to this level,
other than learning how to operate
BioPharm: How customary is it for a college or university laboratory to use a fermentor to grow culture?
Stevens: In researching the purchase of the equipment for our facility, we found that many academic laboratories, departments, and colleges that used large-scale fermentation at one time have discontinued their use in the last several years. In the past decade or more, laboratories interested in milligram quantities of protein have dispensed with large-scale growth of microorganisms as bacterial over-expression systems have progressed to the point where hundreds of milligrams of product can often be purified from one to five liters of culture. In the current age of structural genomics where the low-hanging fruit of easily expressed single polypeptides will have their structures determined rapidly, these smallscale cultures are ideal.
For investigators like myself who
study polypeptides of up to 280 kD
contained in macromolecular complexes
composed of dozens of
polypeptides and several RNAs,
those structures will only be determined
through the brute-force techniques
we use to purify them from
their natural hosts. Several factors
prevent the conventional overexpression
of snRNP polypeptides in
bacteria. First, many individual
snRNP proteins are insoluble in the
absence of their binding partners.
Second, many snRNP proteins are
100 to 280 kD in mass, making
their overexpression in E. coli particularly
difficult, if not impossible.
Lastly, their structural context is lost
when looking at single polypeptides.
Only when looking at the
whole assembly purified from their
native organism can we be sure that
we are studying the functional proteins
and their arrangement in the
intact snRNP. Their low abundance
in yeast requires that we grow massive
culture volumes to purify sufficient
quantities of these complex
molecules. I feel that after the lowhanging
fruit has been picked, the
future of structural biology will be
found in large, often hard-to-isolate
complex protein assemblies that
will necessitate the use of large-scale
BioPharm: Why did you invest in a fermentor?
Stevens: When I began my first independent
position three years ago, I
knew that my work would require
the use of this type of fermentor. I
was very fortunate to receive the
funds from the University of Texas
at Austin to build our fermentation
facility. The benefits of this type of
facility are the speed with which we
can grow the yeast (approximately
400 liters per day) and the quantity
of material we can obtain (tens of
kilograms per week when running at
full capacity). The setup we have
installed in our facility makes it easy
for one operator to perform the culture
preparation, culture growth,
harvesting of the culture, and processing
of the resulting biomass.
BioPharm: How much time did it take from ordering the fermentor to its delivery?
Stevens: Our BioFlo Pro instrument
was the first one of this size and
design, so the design and build-out
of the machine was performed in
conjunction with NBS over the
course of about six months. Im not
aware of how long it would currently
take to produce this
machine. (Editors note: NBS states
it now takes about 12 weeks from
order to delivery.)
BioPharm: Did you receive training to operate the fermentor? Is help readily available if you experience difficulties?
Stevens: We received two days of
training on this machine, which was
more than adequate. We were able
to proceed through an entire fermentation
run, from media preparation
through harvesting of the
BioPharm: Who takes care of the maintenance of the fermentor?
Stevens:We maintain the entire fermentation
facility with the staff in
my laboratory unless we encounter
difficulties or have questions that
require the manufacturer, who is
BioPharm: How large is the staff that it can maintain the entire facility?
Stevens: The equipment has been
designed so that one individual can
operate it. That said, the individual
must be able to lift rather heavy supplies
and equipment, making some
of the steps (e.g., removing the full
centrifuge rotor) much easier with
BioPharm: How did you convince the university to fund a fermentation facility?
Stevens: I included the facility costs in
my negotiations for my startup funds
before coming to the university.
BioPharm: What was the cost of the fermentation facility?
Stevens: Although the cost of all the
major equipment was approximately
$250,000, a facility build-out will
depend on the extent to which the
facility is made user-friendly and the
needs of the individual investigator.
BioPharm: How long did it take to build the facility?
Stevens: The build-out of
the facility took about six
months from the time the
plans were finalized. A
total of about one year was
required for all of the planning,
building, and equipment
BioPharm: Can you modify the fermentor if your work requires it? Do you envision ever scaling up the volume youre producing? What would scale-up entail with the fermentor?
Stevens: One of the first
considerations I addressed
with NBS was the modularity
of the machine. The
design is such that we were able to
add options during manufacture and
after installation. Based on the scalability
of our processes from shakeflask
to 400 liters, I am confident
that further scalability to larger volumes
could occur should our experiments
call for it.
BioPharm: How have your processes and results changed since using a fermentor?
Stevens: Using the fermentor has
allowed my research to advance in
directions previously thought
unimaginable prior to obtaining
the fermentor. Although it is possible
to grow hundreds of liters of
cells without a fermentor (albeit an
extremely tedious and time consuming
proposition), we find that
the reproducibility and dynamic
monitoring of variables such as dissolved
oxygen, pH, temperature,
and agitation speed allow us to predictably
and reproducibly generate
useful biomass for our experiments.
BioPharm: Is the fermentor integrated with any other technology in the laboratory/ facility?
Stevens: Our facility has been designed for maximum flexibility and ease of use. In addition to the fermentor, there is a process steam generator (Electro Steam Generator). We use a CEPA Z-60 continuous-flow centrifuge for biomass harvesting. This centrifuge allows us to harvest 400 liters of culture volume in under an hour. We also have installed a 12-liter, smallscale fermentor (NBS) that can be connected through an in situ sterilizable connector (custom part, NBS) to feed the large fermentor.
To process the material obtained
from the fermentor, we have
the option of high-pressure pneumatic
homogenization using a
Microfluidics 110-EH unit, which
homogenizes about one liter of resuspended
cells (about 300 grams) in
under three minutes. We are also
installing a bead mill (Glen Mills) for
continuous cell disruption under
chilled, low-pressure conditions. The
facility also houses a whole-room
chilled water system which allows us
to tap into an in-room heat exchanger
(System III, Thermo Electron) at any
point along the walls to cool any of
the devices we have installed to ~5C.
BioPharm: If a colleague is considering using your strategy (a fermentor), what are your recommendations?
Stevens: I would strongly recommend
that investigators interested
in this type of facility installation
meet with representatives from the
manufacturers and consult architects
to determine their needs and
the best way to address them. For
example, an important consideration
for NIH protocol compliance
was the containment of a bioproduct
breach, either from a fermentor
or centrifuge spill, or from a processing
catastrophe. This necessitated
the design of a containment
system incorporated into the room,
essentially making the whole room
a waterproof containment pool.
Containment breaches and routine
facility cleaning and maintenance
are performed by chemically sterilizing
the entire facility before passing
inactivated material down an
otherwise sealed floor drain.
BioPharm: Does your laboratory have any business partnerships within the fields of biopharmaceuticals, traditional pharmaceuticals, or government?
Stevens:We are not currently in any
business partnerships, nor are any
planned in the near future. We do
offer fermentation services and product
homogenization to our local colleagues
for a very reasonable rate. As
our laboratory expands and our personnel
increase, we hope to make
these services available to laboratories
outside our university.
BioPharm: As you believe the future of structural biology lies in the hard-toisolate complex protein assemblies, do you feel that youre at the beginning of a trend in returning to the use of largescale fermentors?
Stevens: It may very well be some years down the road, and certainly each large macromolecular complex will present its own technical challenges, but I think that there will be a number of structural biologists and collaborators who will choose to go this route. Roger Kornberg at Stanford University reported that crystallization of the RNA polymerase II complex from yeast required 10,000 liters of fermentation to produce the required amount of protein complex. Clearly the payoff was huge. I can see that this will be required in many other cases and that large-scale cultures will be the only way to address interesting biological problems. Investigators who employ brute-force approaches will be the ones who solve large, interesting structures.