The operation and evaluation of a bioreactor designed for high intensity oxygen transfer in a microgravity environment is described. The reactor itself consists of a zero headspace liquid phase separated from the air supply by a long length of silicone rubber tubing through which the oxygen diffuses in and the carbon dioxide diffuses out. Mass transfer studies show that the oxygen is film diffusion controlled both externally and internally to the tubing and not by diffusion across the tube walls. Methods of upgrading the design to eliminate these resistances are proposed. Cell growth was obtained in the fermenter using Saccharomyces cerevisiae showing that this concept is capable of sustaining cell growth in the terrestial simulation

Dunlop, Eric H.

Petersen, G. R.

Seshan, P. K.

English

NASA Technical Reports Server

N90-13954
MODEL SYSTEM STUDIES WITH A PHASE SEPARATED MEMBRANE BIOREACTOR
G.R. Petersen and P.K. Seshan
Jet Propulsion Laboratory
4800 Oak Grove Drive
Pasadena, CA 91109
E.H. Dunlop
Department of Chemical Engineering
Colorado State University
Fort Collins, CO 80523
ABSTRACT
The operation and evaluation of a bioreactor designed for high intensity
oxygen transfer in a microgravity environment is described. The reactor itself
consists of a zero headspace liquid phase separated from the air supply by a
long length of silicone rubber tubing through which the oxygen diffuses in
and the carbon dioxide diffuses out. Mass transfer studies show that the
oxygen is film diffusion controlled both externally and internally to the
tubing and not by diffusion across the tube walls. Methods of upgrading the
design to eliminate these resistances are proposed. Cell growth was obtained in
the fermenter using Saccharomyces cerevisiae showing that this concept is
capable of sustaining cell growth in the terrestiai simulation.
INTRODUCTION
The use of a bioreactor as a fermenter in Controlled Ecological Life Support
Systems (CELSS) will likely occur in the food production or waste processing
subsystems. It is anticipated that a design for a fermenter for an operational
CELSS will be developed from models flown and tested on STS missions.
Probable _reas of use
There are three possible places a CELSS-type bioreactor could be used:
i. As redundancy or backup for the conventional food production systems
that would be available in space. It is clear that several systems could be
developed, probably using plants and/or animals. However there is always the
problem of catastrophic crop failure and if there is not enough stored food and
it would be necessary to activate emergency rations of food. One possible
source of this is microbial food which can be made available in two or three
days. We have done preliminary studies that show that in reasonable sized
fermenters it is possible to produce adequate quantities of edible types of
biomass, for example yeast, that can be processed into the necessary food
components.
ii. As supplements to conventional food production. The limiting amino
acids for human nutrition are tryptophan and lysine. One of the deficiencies
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in human foods such as wheat and similar materials is very easily satisfied by
microbial sources. Many bacteria, and some yeasts, could provide the
necessaryamounts of lysine, methionine and tryptophan. This is just one
exampleof a supplementand others may be possible.Also an analysisof
human food balances reveals that even when using wheat and high quality
foods humansare still short of carbohydrate.It is possible that it will always
be necessaryto have some calories from microbial carbohydrates.
iii. The area that will probably have first application for the bioreactor is
the production of valuable commodities(in this case, food) from inedible plant
waste. It is a consistentobservationfor all plants that about 50% of biomassis
inedible. Of the inedible biomass,about 40% is comprisedof cellulosics and
about 20-30% is found in hemicellulosics(pentose sugars).Thesetwo
components are readily separatedwith mild hydrolysis and fractionation
methods. The further hydrolysis of these components into monosaccharides
suitable for direct use or fermentation by microorganismsprovides additional
food sourcesfor CELSS food production subsystems.
APPARATUSANDMETHODS
Thc main problem with carrying out fermentations in microgravity is of
course that the bubbles will not rise in the fermenter thus preventing gas
liquid disengagement(1). One reasonablesolution is to avoid the need to solve
the separationproblem by not having a gas phase to disengage.The apparatus
is designed to explore this concept for high-rate oxygen-transfer intensive
microbial growth in a CELSS environment.
The gas and liquid phasesare kept separatewhen the reactor contains
about 10% by volume of silicone tubing in a zero-headspacefermentation
configuration and passing the gas (air or oxygen) through the inside of the
tubes. Oxygen and carbon dioxide are highly permeableto silicone rubber and
diffuse rapidly through it. It is also possible to have liquid silicones saturated
with oxygen passing through the tubes to act as oxygen carriers. Carbon
dioxide can be readily removedfrom the off-gassesby adsorption in a sink
such as monoethanolamine.A potentially attractive alternative to a fixed CO2
sink isreversible adsorption by redox-switched absorbers such as substituted
metallocenes and quinones (2).
Such a system is essentially gravity-independent and can be readily
examined under terrestial conditions.
The terrestial model tested was constructed from plexiglass in the form of a
cylinder containing a total of 8.7 liters volume. The working volume was about
7.7 liters, the other liter being occupied by tubing and support frames. Thus
88% was available for culture.
150 feet of silicone tubing was wound round a support frame. The tubing
had an internal diameter of 0.104 inches and an external diameter of 0.192
inches.
Stirring was provided to the center of the liquid by a marine impeller
revolving at 200-400 revolutions per minute. Air flow to the inside of the tube
could be varied by a mass flow controller from 2.5 to 20 liter per minute gas
flow at an applied pressure of between 3 and 10 psig.
A 1.5% innoculum of Saccharomyces cerevisiae PEP4 was added to a
synthetic medium (Yeast carbon base- YCB) supplemented with YM (1%) and
0.1% tryptone.
178
100 ml of an overnight culture of the yeast are added. The head spaceis
removedby adding enough YM broth to fill up the reactor, and then all the
probes are inserted.
Oxygen transfer measurementswere made by degassing the fermenter
with nitrogen and following the rise of dissolved oxygen on a chart recorder
as air was reintroducedthrough the tubes. Measurementswere made with a
New Brunswick galvanic oxygen probe.
RESULTS& DISCUSSION
Yeast growth
In order to evaluate the reactor under actual growth conditions, cultures of
yeast were grown under a variety of reactor conditions. Figure 1 was an initial
run at low gas pressure but high flow rate. There was no attempt to control pH
or temperature. The data showed us that the apparatus and sterilization
techniques could be employed to culture yeast cells. The effect of lowering the
flow rate by one half and increasing the pressure is shown in figure 2. Again,
the system worked well and the rate of cell growth increased as is shown by
the quicker depletion of oxygen (20 hours vs. 30 hours).
Since oxygen was apparently supplied at adequate levels, an attempt was
made to evaluate the lower working point of the apparatus. The time at which
oxygen depletion occurred as a function of reactor conditions was used as the
basis for evaluating the lower working limit of the apparatus. The initial
experiment in this series is shown in figure 2. With only 50 feet of tubing, 7.5
psig. and 1 liter/min flow rate, the reactor reached oxygen depletion after
about 14-15 hours. However, the possibility that glucose depletion was the
cause for lowered oxygen consumption could not be ruled out. In the
experiment shown in Figure 3, the flow rate was lowered even more and the
glucose measurements were taken more frequently. The results showed that
glucose depletion had not occurred simultaneously with oxygen depletion. This
indicates that the cells are growing at a rate that was a direct function of
oxygen supply. The other observation was that by lowering the flow rate to 0.5
liter/min, the point at which oxygen was depleted was shifted to 16-17 hours.
This is slightly higher than the value shown in Figure 2 and is consistent with
the fact that airflow was half that of the value used in the Figure 2 experiment.
The cells are still healthy and normal. The maximum cell count is 1.3 grams
per liter. This is not a high density, but it is encouraging for our first design.
These simple experiments showed the following:
1. The reactor could be sterilized, operated and maintained using the simple
equipment employed (i.e. no temperature or pH control) to provide
meaningful results.
2. Oxygen limitation can be reached in a relatively short time permitting
quick analysis of the system.
3. Measurements of oxygen transfer rates will need to be conducted in
order to estimate actual maximum operating limits.
179
Qxyggn transfer studies
The oxygen transfer data from the step response studies were analyzed by
the method of Ruchti et al. (3), and expressed as the product of the overall mass
transfer coefficient and the surface area per unit volume of reactor, Kla. The
measurements were taken for a range of air flow rates and stirrer speeds and
are given in Table 1.
The biological experiments demonstrated that modest cell dry weights could
be obtained with this design of fermenter before oxygen limitation was
reached. While these results are encouraging they clearly are not adequate
for a practical system. To overcome the inherent limitation of the preliminary
equipment design, the oxygen transfer studies were initiated. The values of Kla
obtained were some 50-100 times lower than in conventional stirred
fermenters operating under terrestiai conditions. They correspond to oxygen
transfer intensities of around 0.04 kg O2/m3/hr.
Three main effects can be expected to contribute to the low oxygen transfer
intensities observed in this study:-
a. film diffusion resistance in the tube containing the gas
b. external film diffusion into the bulk liquid
c. oxygen diffusion across the silicone tubing wall
For laminar flow of the gas and liquid, resistances a and b above will be
reduced as the flow rate past the tubes is increased while resistance c will be
unchanged. From fluid mechanics it is known that the mass transfer
coefficient will vary inversely with the square root of the flow rate. A
common way of therefore assessing the relative importance of the
contributions is to plot the reciprocal of Kla vs. the reciprocal of the square
root of the flow rate, extrapolate to zero on the axis, i.e., infinite velocity
which removes the film resistance and compare the magnitude of the residual
mass transfer coefficient (4).
1 1
K1 (velocity, internal )1/2
+ (velocity, external )1/2
+ (membrane diffusion resistance ) ....... (1)
Figure 4 shows this procedure for the internal flow rate variation
experiment. The graph shows a marked slope implying that indeed the
internal diffusion resistance in the tube is substantial and that major
improvements in oxygen transfer can be expected simply by increasing the
flow rate, perhaps with recycle, through the tubes.
180
The residual mass transfer resistancescan now be subtractedout and the
effect of external film resistancesexamined.Figure 5 shows the samekind of
graph,this time produced by changing the stirrer speed. Again a substantial
slope is observedwith the regression line passing through the origin of the
graph,, i.e., at infinite stirrer speed the mass transfer coefficient becomes
infinite. The interpretation of this is that the external fluid resistancesare
extremely high compared to which any resistance from the oxygen diffusion
across the membrane is negligible.
These results are very reassuringas they imply that redesign of the
equipment can be done in ways that will result in very substantial increases
in oxygen transfer efficiency that will permit large increasesin cell mass to
be obtained long before the diffusion resistancesin the tubes themselvesstart
to become important.
The reactor will be reconfigured to reflect these findings.
CONCLUSIONS
1. Yeast can be successfullygrown in a phase separatedfermenter that
should be capable of operation independentof gravity.
2. The current design limitations can be overcomeand will result in
substantial increases in oxygen transfer intensities which in turn will
support greater cell massesto provide a practical test facility for a CELSStest
bed.
TABLE 1 Mass transfer coefficients (Kla) as a function of system variables.
Airflow Stirrer K la
Rate Speed
(lit/min) (rpm) (hr- 1)
9.5 325 4.20
7.5 325 2.75
5.0 325 2.64
2.5 325 2.08
7.5 275 3.47
7.5 275 2.75
7.5 225 2.77
7.5 110 1.97
7.5 155 1.29
181
REFERENCES
Bell, W.L., A. Miedaner,J.C.Smart,D.L. Dubois and C.E. Drostow. 1988. Synthesis
and Evaluation of electroactive CO2 carriers. NASA SAE Technical Paper
Series. Paper 881078.
Dunlop, E.H., and R. Williams. 1978. Physicochemical aspects of Protein bound
substances. Part II- kinetics of removal. Medical & Biological Engineering
16:350-362.
Ruchti, G., I.J. Dunn and J.R. Bourne. 1981. Comparison of Dynamic Oxygen
Electrode Methods for the Measurement of Kla, Biotech. Bioeng. 23 277-290
Seshan, P.K., G.R. Petersen, B.J. Beard and E.H. Dunlop. 1986. Design Concepts
for Bioreactors in Space in CELSS " NASA Technical Manual 88215.
182
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HOURS OF OPERATION
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-I
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Fig. 1. Initial evaluation of the fermenter. The lowest working point of the
fermenter was established. With only 50 feet of tubing, 7.5 psig. and 1
liter/min flow rate the reactor reached oxygen depletion after about 14-15
hours.
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Fig. 2. At even lower flow rate glucose depletion does not occur
simultaneously with oxygen depletion, indicating that the cells are growing at
a rate that was a direct function of oxygen supply.
183
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80 l
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Fig. 3. Further reduction in the flow rate shows that the cells were growing
at a rate that was a direct function of the oxygen supply. Thus oxygen will
remain the limiting nutrient in these experiments.
184
20OO
1500
_iooo
500
"_UBE-SIDE MASS TRANSFER RESISTANCES
RESIDUAL EXTERNAL RESISTANCES @ 325 rpm
I I I I
.2 .4 .6 .8
1/(FLOWRATE) 0"5
Fig. 4. The procedure for assessing the internal flow rate resistances. The
graph shows a marked slope implying that indeed the internal diffusion
resistance in the tube is substantial and that major improvements in oxygen
transfer can be expected simply by increasing the flow rate.
2000
_'1500
o.71000
500 LIQUID SIDE MASS TRANSFER
J RESISTANCES
_ _ESISTANCES (RESIDUAL)
I I I I I
.2 .4 .6 .8 .10
1/(rpm) 0'5
Fig. 5. The residual mass transfer resistances from Figure 4 are subtracted
out and the effect of external film resistances examined by changing the
stirrer speed. Again a substantial slope is observed with the regression line
passing near the origin of the graph, i.e. at infinite stirrer speed the mass
transfer coefficient becomes infinite, implying that the external fluid
resistances are extremely high compared to which any resistance from the
oxygen diffusion across the membrane is negligible.
185



Model system studies with a phase separated membrane bioreactor

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