An analysis was made to identify potential gravity-sensitive mechanisms that may be present in the single-cell growth system. Natural convection (density gradients, induced sedimentation, and buoyancy) is important in microbial systems. The absence of natural convection in the space-flight environment could provide an opportunity for new approaches for developments in industrial fermentation and agriculture. Some of the potential influences of gravity (i.e., convection, sedimentation, etc.) on the cell were discussed to provide insight into what experimental areas may be pursued in future space-flight research programs

Mayeux, J. V.

English

NASA Technical Reports Server

INFLUENCE OF ZERO-G ON SINGLE-CELL SYSTEMS AND ZERO-G W~~MENTER DESIGN 
CONCEPTS 
By Jerry V, Mayeux, Bio Innovar, Inc., Storm Lake, Iowa 
INTRODUCTION 
Exploratory biological studies on the effect of the space environment on biological 
systems, including microorganisms, have been carried out on the LJnited States' 
Discoverer, Biosatellite, Gemini, Apollo, Skylab, and ASTP programs and on several 
Russian satellites. In many cases, the results were incomplete or inconclusive; 
however, significant observations with respect to microbial interactions with the 
space-flight environment have been made. The data obtained have evoked considerable 
interest to continue a space-flight experimental program (ref. 1). 
The advent of the shuttle era presents the first multiple-flight opportunity for 
performance of comprehensive and well-controlled space-flight experiments. Early 
shuttle payloads such as the Long-Duration-Exposure Facility (LDEF) and Biomedical 
Experiment Scientific Satellite (BESS) may provide opportunities for bioscientists to 
initiate experimental programs that can be expanded and continued when the shuttle 
dedicated labs become operational. These first-generation experiments are a necessary 
step in the process of developing an orderly space microbiology application. Early 
investigations can be accomplished by passive growth experiments involving a selection 
of viable organisms over an extended period of time. The results of these experiments 
could lead to developments that would eventually provide beneficial exploitation of 
the space environment for unique application in a space microbiology program. 
An analysis has been made to identify potential gravity-sensitive mechanisms that 
may be present in the single-cell growth system (ref. 2). Natural convection (density 
gradients, induced sedimentation, and buoyancy) is important in microbial systems. 
The absence of natural convection in the space-flight environment could provide an 
opportunity for new approaches for developments in industrial fermentation and 
agriculture. 
Some of the potential influences of gravity (i. e., convection, sedimentation, 
etc.) on the cell will be discussed to provide insight into what experimental areas 
may be pursued in future space-flight research programs. 
INFLUENCE OF A CONVECTION-FREE ENVIRONMENT ON SINGLE CELLS 
Mass and energy transport depends primarily on diffusion and convection in 
single-cell systems (ref. 3). Migration or relocation of products and substrates from 
one point of production to a point of utilization can be critical to the cell, 
especially in synthetic processes (refs. 4, 5, and 6). Some of these requirements and 
their interdependence on diffusion and convection are shown in table I. 
Further evaluation of the cell functions identifies other potential convection- 
dependent functions at the subcellular level. Examples of these functions at the 
enzyme reaction site, for example, are as follows: heat dissipation, substrate 
transport, product removal, boundary layer formation by concentration gradients, enzyme 
induction or inhibition, and active or passive transport across membranes. 
https://ntrs.nasa.gov/search.jsp?R=19770010745 2020-03-22T10:36:13+00:00Z
INFLUENCE OF A CONVECTION-FREE MEDIUM ON THE CELL ENVIRONMENT 
Upon examination of the growth environment of the single cell, other convection- 
sensitive parameters can be identified. These parameters are in part determined by the 
nature of the growth system; i.e., air-water, air-water-particulate, air-water- 
hydrocarbon, or water-hydrocarbon. The major difficulty with these systems in unit 
gravity is maintaining homogeneity of the growth medium, because these nonmiscible 
materials are buoyant. The gravity-induced buoyancy affects nutrient transfer to the 
aqueous phase, sedimentation of cells, pH control, dissolved oxygen supply, and heat 
dissipation (refs. 4, 5, and 6). 
The theoretical advantages of low gravity in the space-flight environment may 
provide the opportunity to work with stable multiphasic fermentation systems that are 
not currently available to the fermentation scientists. These multiphasic fermentation 
systems could provide the cell with a continuous maximum interfacial contact of oxygen, 
aqueous medium, and insoluble substrates, with a minimum of agitation (ref. 7). The 
lack of buoyancy should allow the residence time of oxygen bubbles and oil droplets to 
be increased, with a concomitant decrease in shear forces (due to reaction in the need 
for agitation)(ref. 8). This result may allow fragile cells or shear-sensitive enzyme 
systems to be utilized in production processes requiring high levels of oxygen. 
The analysis of convection-dependent cellular functions and environmental growth 
factors has led to speculations about what would happen in the absence of gravity- 
induced convection (ref. 2). Intracellular biochemical reactions could exhibit an 
abnormal periodicity due to the time delay for translocation in regulatory substrates 
or metabolites. Multiphasic microbial fermentation systems that are stable, though 
not emulsified, could be prepared and maintained as a uniform mixture without the 
interference of natural convection (ref. 2). Single cells, suspended in a suitable 
liquid medium, could be made to grow and reproduce to form discrete colonies, 
originating from a single parent cell or clumps of cells (ref. 2). 
ZERO-G FERMENTER DESIGN CONCEPTS 
The unique opportunity that zero gravity provides for fermentation developments 
also provides some unique problems in equipment design for zero g. 
Design Considerations 
The oxygen bubbler (or aerator) generally depends upon bubble buoyancy and 
agitation for dispersion. Because there is no buoyancy, the bubbles will not 
automatically shear when a critical size is achieved. A shear device must be developed 
that will provide generation of fine bubbles without vigorous agitation of the growth 
liquids (refs. 2 and 9 ) .  
The mixer used in the fermenter must be capable of uniform dispersion of bubbles 
as well as other nonmiscible substrates. But most important, it must avoid promoting 
an increase in bubble coalescence, and a means must be provided to prevent bubble 
attachment to solid surfaces. 
The oxygen, or other gas bubbles provided to the growth system, will serve as a 
reservoir for waste gases produced by the cells. Although carbon dioxide ( ~ 0 ~ )  has a
high solubility in water, it will establish an equilibrium with the gas bubble and 
thus prevent the bubble from decreasing in volume as the oxygen is utilized. A 
mechanism for collecting and removing CO from either the aqueous phase or the gas 
bubbles must be developed. 2 
A necessary assumption is that, during the early stages of zero-g fermenter 
development, most of the preparation will be made on the ground and the events sequence 
will be programed and automated. Some of the factors that must be considered in 
overall fermenter design for zero-g use are discussed briefly below. 
Mixer control and agitator size will depend on the nature of the growth 
environment. If a true fermentation system is used, substrate mixing and C02 removal 
are the main problems to be considered (ref. 9 ) .  The addition of oxygen or a gaseous 
substrate provides other unique problems already mentioned. 
The logistics for handling the inoculant, the substrate, and other nutrients must 
be considered. If the cells should be in a freeze-dried state, they must be uniformly 
resuspended, added to the fermentation medium, and uniformly dispersed. The same 
holds for the substrate and other nutrients. Some of the questions that arise are as 
follows: How and when should the nutrients premixed in the reaction tank; should all 
the substrate be added initially, or should substrate be added gradually over the 
growth phase; and if the nutrients and substrates are added incrementally, how is it 
accomplished? 
At the other end of the fermentation program, the opposite sequence of events must 
occur, The fermentation products, metabolites or cells, must be accumulated or stored. 
Can these be selectively recovered while the fermentation is in progress? Or, must 
they be allowed to accumulate for later recovery? If so, how are they handled - as 
a batch or can a continuous system be developed? 
The question of sterilization and contamination controls is one that has received 
much attention from NASA as well as the fermentation industry. It is probable that the 
technology for these problem areas is already developed for use in zero g. The 
operational controls, startup and handling procedures, and monitoring of the oxygen 
supply, pH, pressure, and other environmental parameters have been developed to a 
highly sophisticated level by the fermentation industry and for other space-flight 
programs. 
It is conceivable that the technology developed for handling liquid fuels in 
zero g will be directly applicable to the handling and storage of nutrients, 
fermentation liquids, and metabolites. 
Zero-G Fermenter Design: Cylindrical Chamber Concepts 
A considerable amount of design work on zero-g fermentor concepts has been 
conducted by the Martin Marietta Aerospace Corporation in Denver. The information on 
these designs has been provided by courtesy of Martin Marietta's Denver Division (ref. 
9 ) .  
A cylindrical chamber concept of a zero-g fermentor is shown in figures 1 and 2 
(ref. 9). As conceived, the fermentor chamber will initially contain water and possibly 
the required initial concentration of dissolved nutrients. The cell inoculant would be 
presuspended and pressure fed into the tank, together with the nutrients and substrate 
to start the process. 
Slow-speed paddles would provide uniform mixing of the cells and substrate. It 
is probable that the paddles need only operate at intervals. The oxygen would be fed 
with a special bubbler head and dispersed by the paddles, Bubble coalescence could be 
a problem in this design. 
The nutrients could be in the fermentation tank initially, and the nutrient level 
could be maintained by nutrient diffusion through the dialysis tube, with a partial 
shroud formed around the paddles. The metabolites could be removed by a separate set 
of dialysis membrane tubes. 
Carbon dioxide and other gaseous wastes could be removed by diffusion through 
Teflon, silicone, or other selectively permeable membrane walls. This gas could be 
collected and vented as appropriate. 
A microbial cell harvester is provided to collect cells for further processing in 
flight or for return to the ground-based laboratory. Preservation of the cells or 
extracellular metabolites could be accomplished by use of space vacuum for freeze 
drying. 
Most of the handling and movement of fluids would be accomplished by the use of 
bladder tanks that can be subjected to a pressure gradient to force fluid flow in or 
out of the reservoir or collector. 
This concept, although designed for use with oxygen in aerobic systems, may be 
more applicable to anaerobic systems. It does not appear that the concept will provide 
sufficiently small bubbles without vigorous initial agitation. The closed-loop 
concept discussed next appears to be more suitable for use with oxygen. 
Zero-G Fermenter: Flow Circulation Loop 
The flow circulation loop concept of a zero-g fermenter design is shown in figure 
3 (ref. 9 ) .  This unit is designed with a continuous flow fermentation chamber that 
would be initially filled with water and that could contain a basal nutrient medium. 
The water is gently circulated by two pumps operating in synchrony. 
The cell inoculum, substrate, and nutrients are introduced into the chamber vessel 
through a pressure-feed system. The oxygen required for growth of aerobic organisms is 
provided through the two sparger screens. These sparger screens are grids containing 
multiple bubbling sites. As the liquid flows across the bubbling sites, the bubbles 
are sheared off. The size of the bubbles can be regulated by the flow rate through 
the screen. 
The nutrient supply in the fermenter is maintained by pumping the concentrates to 
the counterflow mass exchanger, which consists of a series of membrane tubes, to 
increase cross diffusion. 
The carbon dioxide produced will be collected by a separator screen into a single 
large pocket at the acute angle between the screen and the closed-loop fermenter wall. 
Accumulation of the carbon dioxide will automatically activate an optical sensing 
device that will vent the C02 to the outside. 
The metabolites will be removed by a second counterflow mass exchanger and 
accumulated for later use. The cells can be harvested continuously when growth reaches 
a steady state. These cells can then be stored in the collection reservoir or 
processed further in flight or preserved for return to a ground-based lab. 
CONCLUSION 
These concepts are preliminary and have not been proven to work, or fail to work, 
in flight. However, they do serve as a point of departure for discussion and planning 
of space-flight mission objectives in the field of applied biology; There are many 
steps that need to be taken before embarking on a space-flight fermentation program. 
The most critical question still remains to be answered. "To what degree does space 
flight influence single-cell systems?" Many projections have been made, but the 
experimentation to verify these predicted responses still remains to be conducted. 
When the microbial behavior is determined and the facts bear out the predictions, 
then it is time to determine the advantages that may be gained from the space 
environment. Yet, it is not expedient to wait until the experimental results are 
collected and tabulated before proceeding with the conceptual design of the necessary 
hardware and the exploration of these new, theoretical possibilities for solving some 
very difficult engineering problems in the biosynthesis industry. 
REFERENCES 
1. Taylor, G ,  R,, ed.: Proceedings of Symposium: Microbial Response to Space 
Environment. NASA TM X-58103, 1973. 
2. Mayeux, J. V.:  Development of a Zero-g Microbiological Experiment for the 
Long Duration FXposure Facility. NASA-CR-132429, 1974. 
3. Casida, L. E., Jr. : Industrial Microbiology. John Wiley & Sons, Inc. ( ~ e w  ~ork) ,
1968. 
4. Wang, D. I-C.; and Humphrey, A. E.: Article in Vol. 8 of Progress in Industrial 
Microbiology, D. J. D. Hockenhull, ed., CRC Press (Cleveland), 1968, p. 1. 
5. Richards, J. W.: Article in Vol. 8 of Progress In Industrial Microbiology, 
D. J. D. Hockenhull, ed., CRC Press  levelan and), 1968, p. 143. 
6. Aiba, S.; Humphrey, A. E.; and Mills, N. F.: Biochemical Engineering. Academic 
Press ( ~ e w  York) , 1965. 
7. Wang, D. I-C. ; and Ochoa, A. : Article in Biotechnology and Bioengineering, vol. 14, 
1972, p. 345. 
8. Moo-Young, M.; Whitworth, D. A.; and Shimigis, T.: Proc. IV International 
Fermentation Symposium. Fermentation Technology Today, Society of Fermentation 
Technology (~a~an), 1972, p. 91. 
9. Hise, R. E.; and Jordan, R. T.: Apparatus and Method for Microbial Fermentation in 
a Zero Gravity Environment. U.S . Patent No. 3,769,176, 1973. Assignee : 
Martin Marietta Corporation. 
TABLE I.- MECHANISM FOR MASS AND ENERGY TRANSPORT IN CELLULAR PROCESSES 
a Gravity-dependent natural convection. 
Nutrient procurement 
Passive diffusion 
Active transport 
Nutrient utilization 
Heat dissipation 
Energy conversion 
Biosynthesis 
Waste-product removal 
Passive diffusion 
Active transport 
Respiration 
Gas exchange 
Radiation 
X 
a Convection 
X 
X 
X 
X 
X 
X 
X 
X 
Conduction 
X 
Diffusion 
X 
X 
X 
X 
X 
X 
X 
X 
OXYGtl ,  OR lV lNG SLOWS PEED 
NUTRIENT 
'SUPPLY 
RESERVOIR 
Figure 1.- Zero-g fermenter design,  c y l i n d r i c a l  chamber concept. (cour tesy  
Martin Mar ie t ta  Corp. ) 
Figure 2.- Concept for carbon dioxide removal by dialysis in the cylindrical 
fernenter design. (courtesy Martin Marietta Corp. ) 



Influence of zero-G on single-cell systems and zero-G fermenter design concepts

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