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NASA TECHMCAL N6EMORmE)UR.I

NASA T M X- 5819 1 January 1977

BIOPROCESSING IN SPACE

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Proceedings of the 1976 NASA Colloquium on Bioprocessing i n Space Houston, Texas, March 10-12, 1976 Compiled by Dennis R. Morrison

(NASA)

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NATIONAL AERONAUTICS AND SPACE ADMINISTRATION Lyndon B. Johnson Space Center

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Houston, Texas 77058

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BIODROCESSING IN SPACE

s R. Morrison

pace Administration

ctor waived the use of the International System of Units (SI) for this technical because, in his judgment, the use of SI units would impair the usefulness of result in excessive cost.

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to introduce this subject to academic and industrial scientists who have gravity-induced biological research problems o r an interest i n the possibilities of processing o r manufacturing biologicals i n space. The meeting was intended to establish a cooperative effort among Government and non-Government researchers to examine the potential of bioprocessing in space. The program included general sessions and formal presentations on the following topics : NASA's Space Shuttle, Spacelab, and space-processing programs; the known unusual behavior of materials in space; space-processing experiment results; cell biology, gravity sensors i n cells, space electrophoresis of living cells, new approaches to biosynthesis of biologicals from cell culture in space, and zero-g fermentation concepts; and upcoming flight opportunities and industrial application planning studies already underway. The proceedings include the summaries of workshops held during the colloquium to allow participants to discuss new ideas, future biological research areas, and gravity-related problems i n the a r e a s of (1) industrial biosynthesis, (2) pharmaceutical research, (3) biotechnology , and (4) cell biology.

Processing (biological)

'For sale by the National Technical Information Service, Springfield, Virginia 22151

NASA TM X- 58191

BIOPROCESSING IN SPACE Dennis R. Morrison, Ph. D. Lyndon B. Johnson Space Center Houston, Texas 77058

FOREWORD The NASA Lyndon B. Johnson Space Center was particularly pleased to conduct this colloquium because it was the first opportunity for the life sciences elements of NASA to focus the attention of a broad spectrum of scientists on this new research area - bioprocessing. More than 170 academic scientists and industrial researchers attended the colloquium. This document is a compilation of the papers presented at the symposium and brief summaries of the discussion workshops held during the meeting. Acknowledgment is made of the following individuals, who were directly responsible for the organization and conduct of this symposium. The colloquium committee included Dr. Wayland Hull, Bernard Mieszkuc, Dr. Dennis Morrison, and Dr. Peter Whittingham from the Johnson Space Center; and Bruce Goss of the Boeing Company.

I particularly wish to thank Dr. Whittingham for his invaluable assistance in the detailed preparation and conduct of this colloquium. Additionally, I wish to thank Dr. Merlyn Bissell and Dr. Gerald Taylor for their suggestions and assistance. Finally, I wish to thank the authors and the numerous other individuals of the participating organizations who provided active support in the preparation of this colloquium.

Lawrence F. Dietlein, M.D. Acting Director of Life Sciences NASA Lyndon B. Johnson Space Center

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SESSION LEADERS General Sessions Space Shuttle and Space Processing Chairman: Dr. Dennis R. Morrison, Lyndon B. Johnson Space Center, Houston, Texas Materials Behavior and Space Processing Chairman: Dr. Peter Wittingham, Lyndon B. Johnson Space Center, Houston, Texas Cellular Research and Biological Processing Chairman: Mr. Bernard J. Mieszkuc, Lyndon B. Johnson Space Center, Houston, Texas Biosynthesis Chairman: Dr. Wayland E. Hull, Lyndon B. Johnson Space Center, Houston, Texas Flight Opportunities and Potential Programs Chairman: Dr. Dennis R. Morrison, Lyndon B. Johnson Space Center, Houston, Texas Workshops Biotechnology Cochairmen: Mr. John M. Walsh, Beckman Instruments, Inc., Anaheim, California; and Dr. Robert E. Allen, George C. Marshall Space Flight Center, Huntsville, Alabama Cell Biology Cochairmen: Dr. Jerry V. Mayeux, Bio Innovar, Inc., Storm Lake, Iowa; and Dr. Gerald R. Taylor, Lyndon B. Johnson Space Center, Houston, Texas Industrial Biosynthesis Cochairmen: Dr. W. E. Brown, Squibb Institute, Princeton, New Jersey; and Dr. R. E. Sparks, Washington University, St. Louis, Missouri Pharmaceuticals Cochairmen: Dr. E. John Staba, University of Minnesota, Minneapolis, Minnesota; and Dr. Takeru Higuchi, University of Kansas, Lawrence, Kansas

CONTENTS Page

Section

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INTRODUCTION Dennis R. Morrison

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General Sessions Space Shuttle and Space Processing SPACE SHUTTLE AND LIFE SCIENCES John A. Mason NASA's SPACE PROCESSING PROGRAM James H. Bredt

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Materials Behavior and Space Processing e

................ SPACE PROCESSING ON SKYLAB AND ASTP . . . . . . . . . . . . . . . . . . . . . R. S. Snyder THREE MODEL SPACE EXPERIMENTS ON CKESIICAL REACTIONS . . . . . . . . . . . . .

BEHAVIOR OF FLUIDS IN A WEIGHTLESS ENVIRONMENT Dale A. Fester, Ralph N. Eberhardt, and James R. Tegart

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Philomena Grodzka and Barbara Facemire

Cellular Research and Biological Processing SURVEY OF CELL BIOLOGY EXPERIMENTS IN REDUCED GRAVITY Gerald R. Taylor

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GRAVITY AND THE CELL: INTRACELLULAR STRUCTURES AND STOKES SEDIMENTATION Paul Todd

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BIOPROCESSING: PROSPECTS FOR SPACE ELECTROPHORESIS Milan Bier

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ELECTROPHORETIC SEPARATION OF HUMAN KIDNEY CELLS AT ZERO GRAVITY Grant H. Barlow, S. LaVera Lazer, Annemarie Rueter, and Robert E. Allen

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ELECTROPHORESIS FOR BIOLOGICAL PRODUCTION Louis R. McCreight

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Biosynthesis SOME QUESTIONS OF SPACE BIOENGINEERING Laszlo K. Nyiri

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INFLUENCE OF ZERO-G ON SINGLE-CELL SYSTEMS AND ZERO-G FERMENTER DESIGN CONCEPTS Jerry V. Mayeux

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Flight Opportunities and Potential Programs SPACE SOLAR POWER SYSTEMS Curt Toliver

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Section

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B E N E F I C I A L USES OF SPACE Harold L. Bloom

INTRODUCTION D e n n i s R. M o r r i s o n

BIOTECHNOLOGY WORKSHOP SUMMARY John M. W d s h and R o b e r t E . Allen

CELL BIOLOGY WORKSHOP S W Y Jerry V . M a y e u x and G e r a l d R . T a y l o r

I N D U S T R I A L B I O S Y N T H E S I S WORKSHOP SUMMARY W. E . B r o w n and R o b e r t E . Sparks PHARMACEUTICAL WORKSHOP SUMMARY E . John Staba and T a k e r u H i g u c h i

Appendixes

............................ A P P E N D I X B - B I O P R O C E S S I N G BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . A P P E N D I X C - RELATED CONFERENCES AND S T U D I E S . . . . . . . . . . . . . . . . . APPENDIX A

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ATTENDEES

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BIOPROCESSING IN SPACE By Dennis R. Morrison Lyndon B. Johnson Space Center INTRODUCTION Experiments conducted in space onboard Apollo, Skylab, and the recent Apollo-Soyuz Test Project flight have established that many materials exhibit unique properties in virtual weightlessness, a condition impossible to duplicate on Earth. These results have led to new, Earth-based research and development efforts in areas of fluid mechanics, metallurgy, crystal growth technology, acoustic levitation and manipulation of materials without containers, silicon refining for semiconductors, and several innovations in electrophoretic separation of different types of biological cells. Materials processing onboard orbiting manned spacecraft can take advantage of weightlessness, readily available solar energy, and a vacuum source of unlimited capacity to purify, isolate, or manufacture materials that are too difficult, costly, or impossible to produce on Earth. The Space Shuttle and Spacelab flight in the 1980's will be used to explore manufacturing processes that benefit from convectionless behavior of weightless fluids, new techniques for production and manipulation of immiscible mixtures, and containerless processing of biological materials in liquid media, and mass transfer in liquids that is wholly controlled by diffusion. The possible utilization of the unique space environment for the manufacturing and processing of biological materials could be very significant to pharmaceutical research and manufacturing. In March 1976, the National Aeronautics and Space Administration (NASA) sponsored a 3-day colloquium on "Bioprocessing in Space'' at the Lyndon B. Johnson Space Center, Houston, Texas. The main purpose of the colloquium was to introduce this subject to academic and industrial scientists who may have gravity-induced research problems or an interest in the possibilities of processing or manufacturing biologicals in space, but who were uninformed about the behavior of materials in weightlessness and the forthcoming opportunities to fly experiments by means of NASA's sounding rocket and Space Shuttle payloads program. The general sessions included presentations on (1)the unusual behavior of materials in a weightless environment, (2) research facilities and opportunities for flight onboard the Space Shuttle and Spacelab, (3) results of pertinent experiments conducted in space, and (4) potential research and industrial applications of the space environment. Workshops in related areas of biotechnology, cell biology, industrial biosynthesis, and pharmaceutical research also were held to generate new ideas and principles for solving biological research or manufacturing problems that are gravity dependent. More than 170 participauts, mostly from the biological- or pharmaceutical-related nonaerospace industry and academia, attended the colloquium. The interest level was quite high concerning new opportunities to perform experiments in orbit. Many participants made requests for literature indices to specific space experiment results, more payload user information, and future meetings on related space biology topics. The following proceedings include the formal papers presented at the colloquium and brief summaries of the discussions and noteworthy ideas from the different scientific workshops.

General Sessions

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SPACE SHUTTLE AND LIFE SCIENCES By John A. Mason Chief, Bioseienee Pay1 oads O f f ice Life Sciences Directorate Lyndon B. Johnson Space Center Houston, Texas ABSTRACT The Space Shuttle, America's f i r s t reusable spacecraft system, will have many diverse functions : I t will launch satel l i t e s geared for such tasks as communications, weather observations, pol 1ution monitoring, Earth-resource studies, and world wide navigation. I t will retrieve sate1 1 i t e s for Shuttle-based repairs or return to Earth. And the Shuttle will carry the f i r s t laboratory dedicated t o the study of l i f e processes in space. The Spacelab, being designed and constructed by the European Space Agency, will serve as a general research facil i ty for the exploration and eventual industrial ization of space. During the 19801s, some 200 Spacelab missions will be flown in Earth-orbit. Within these 200 missions, i t i s planned that a t least 20 will be dedicated t o 1i f e sciences research, projects which are yet to be outlined by the l i f e sciences community. Discussions within the paper cover objectives of the Life Sciences Shuttle/Spacelab Payloads Program; also discussed are major space 1i f e sciences programs including space medicine and physiology, clinical medicine, l i f e support technology, and a variety of space biology topics. The Shuttle, Spacelab, and other l i f e sciences payload carriers are described. Concepts for carry-on experiment packages, mini -1 abs , shared and dedicated space1 abs , as we1 1 as common operational research equipment (CORE) are reviewed. Current NASA planning and development includes Space1ab Mission Simulations, an Announcement of Planning Opportunity for Life Sciences, and a forthcoming Announcement of Opportunity for Flight Experiments which wi 11 together a s s i s t in forging a Life Sciences Program in space. INTRODUCTION I t i s my pleasure t o present t h i s paper: "The Space Shuttle and Life Sciences." The paper i s divided into 6 parts: 1 ) Space Shuttle Program Description, 2) Spacelab Description, 3 ) Life Sciences Program, 4 ) Life Sciences Payload Carrier Characteristics, 5 ) Development of Program Planning and Operational Approaches, and 6 ) Summary. 1 ) SPACE SHUTTLE PROGRAM DESCRIPTION The Space Shuttle i s the f i r s t true blending of the manned and unmanned space programs. The Shuttle will fly approximately 20 years a f t e r the United States f i r s t entered space in January 1958. The Shuttle i s designed t o support Spacelabs (which will be discussed l a t e r ) and t o 1aunch sate1 1i t e s , as well as space probes with propulsion stages. A1 1 of these, wi 11 support applications and technology as well as scientific experiments. I t i s anticipated that not only NASA personnel b u t a1 so individual s from other government agencies, uni vers i t i e s , and industry, as well as the international community will use the Shuttle (Figure 1 ) (1 1.

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Program Objecti ves The Space Shuttle Program Objectives are twofold: 9 ) to reduce the cost of space operations, and 2 ) t o provide a capability designed to support a wide range of scientf f i e , applications, defense, commercial and international uses as mentioned e a r l i e r . This i s to help establish a national space transportation capabi 1 i ty (Figure 2). The Space Shuttle System as i t i s called i s composed of 4 major parts. The Shuttle Orbiter (an aircraft-like structure), the lar e external hydrogenloxygen tank, and the 2 rocket engines. The Orbiter i s 122 feet (37.2 my in length, and i t has a wing span of 78 feet (23.8 m ) . In comparison t o other known a i r or spacecraft, the Shuttle i s slightly larger than the DC9. I t i s approximately 22 feet longer than the Boeing 737, half the height of the Saturn V (the rockets that took the Apollo crews t o the moon). '

Space Shuttle Mission Profile I n i t i a l l y , the Shuttle will be launched from the Kennedy Space Center. Approximately 2 years 1a t e r i t i s planned that the Western Test Range, located a t Vandenberg Air Force Base, will be added as a launch s i t e . The Shuttle Orbiter will be taken into near Earth orbit by the solid rocket engines which will be jettisoned and returned to Earth by a parachute system for reuse. The large external hydrogen/oxygen tank's propellants will continue to carry the Shuttle Orbiter into orbit and then the tank will be jettisoned. I t will not be returned by parachute system and will not be reused. Mainly because of i t s large size and configuration, i t will not be able t o reenter the Earth's atmosphere without damage, so the crash point for the tank will be programmed for a remote place in one of the oceans. The orbiter will then remain in Earth orbit, nominally 100 t o 200 miles from the Earth's surface. I t will remain there from one week i n i t i a l l y up t o a maximum of 30 days during which time the various pay1 oads will be activated, tested, exposed or launched. The cargo bay doors may be opened t o allow for launch or to place spacecraft into orbit or, i f there i s a Spacelab attached within the cargo bay, i t will remain and work will be performed in the Spacelab. After the mission i s completed, the Orbiter will enter the Earth's atmosphere with a high angle of attack and will coast with the minimal assistance of non-airbreathing engines. I t will coast in a preprogramed f l i g h t plan and will land a t Kennedy Space Center or Vandenberg Air Force Base. I t i s planned that there will be no more than a 2-week turnaround time between the time of landing and the next launch of a scheduled Shuttle mission (Figure 3) (1, 2). Space Shuttle Program Activities The Space Shuttle Program i s made up of a number of planned a c t i v i t i e s . The development phase i s now underway. Some of the key events are scheduled as follows: The f i r s t Space Shuttle Orbiter will be "brought out of the hangar" in the f a l l of 1976. The f i r s t captive f l i g h t t e s t a t Edwards Air Force Base i s scheduled for the spring of 1977. Approach and landing t e s t s also will be conducted a t Edwards Air Force Base starting in the l a t e summer of 1977. The f i r s t manned orbital t e s t flights are scheduled for 1979. The operational flights then will begin early in the 1980s (Figure 4). Space Shuttle Operations The Space Shuttle i s planned for a number of different operations. I t will a s s i s t in orbital missions where we will have', for example, a telescope t o permit astronomers to view heavenly bodies from above most of the Earth's atmosphere. The Shuttle Orbiter may also

serve as the propulsion stage f o r delivery and retrieval of orbiting spacecraft. I t may . replace the f i r s t two launch stages f o r a spacecraft t h a t will be launched into deep spacei I t will a l s o allow r e p a i r and servicing of s a t e l i i t e s t h a t are already in Earth o r b i t , thus providing an extension of the l i f e of spacecraft in o r b i t . I t w i l l also permit the carrying of passengers and crewmembers t o near Earth o r b i t f o r a space s t a t i o n when i t i s developed In addition, rescue operations may be conducted i f a crewmember and becomes operationai were t o become i11 o r when there i s a problem in a spacecraft (Figure 5 ) .

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2) SPACELAB DESCRIPTION

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a pressurized volume and/or a p a l l e t , The Spacelab actually may have 2 major parts carried separately o r together (Figure 6 ) . These are contained and carried into near Earth o r b i t in the cargo bay of the Shuttle Orbiter. The Spacelab remains attached in the cargo bay f o r the e n t i r e mission since i t r e l i e s on the Shuttle Orbiter f o r many of the l i f e support sys terns. The Spacelab has been developed through international cooperation by the European Space Agency (ESA). ESA i s made up of representatives from a number of European countries, with the major contributor, Germany, contributing over 53% of the financial backing. The United Kingdom, I t a l y and France are other large contributors, although nowhere near the percentage t h a t Germany has contributed. ESA has i t s headquarters i n Paris, and ESTEC (the space center which might be compared t o the Johnson Space Center) i s located i n the Netherlands. This i s where the Spacelab Program i s managed. The major contractor working and coordinating the work of other contractors f o r Spacelab i s ERNO, located i n Bremen, Germany. I t turns out t h a t the nations contributing money i n t o the program, t o a large extent, have contractors receiving funds approximately equal t o the amount t h a t the country has contributed t o ESA (Figure 7). Experiment Accommodations of Spacelab (Pressurized Module) The Spacelab will be i n o r b i t i n i t i a l l y 7 days, as I mentioned before, and i t i s planned t h a t i t may be extended t o 30 days. The experiment weight t h a t i t will arry in o o r b i t will be from 4600-16700 1bs. (2100-7600 kg). The experiment volume i s 790 f t S (22.3111 ). The volume planned i s f o r biological specimens. I t cannot carry an elephant but could carry primates, man o r his surrogates. The atmosphere i s t o be an Earth-like environment, i . e . , 20% oxygen i n nitrogen a t 14.7 psi o r 760 mmHg pressure. The temperature will be selectable and will be a shirtsleeve environment, 18-27OC. The uti 1i t i e s avail able a r e v i r t u a l l y those t h a t can be expected i n an Earth laboratory. There will be e l e c t r i c a l power, thermal cont r o l , data processing as needed f o r t e s t s currently considered. There will be support equipment, and storage, i . e . , places t o s t o r e film so t h a t i t can be used e f f e c t i v e l y , and there will be viewports t o view the Earth o r heavenly bodies from the pressurized volume of the Space1 ab.

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This has been a brief review of what the Spacelab i s and plans t o be t o support s c i e n t i f i c endeavors.

3) LIFE SCIENCES PROGRAM

Five main o b j e c t i v e s are c u r r e n t l y i d e n t i f i e d f o r t h e L i f e Sciences Program. The f i r s t i s t o explore and r e s o l v e t h e preblems associated w i t h f u t u r e ventures o f man i n t h e e x p l o r a t i o n , e x p l o i t a t i o n , and eventual c o l o n i z a t i o n o f space. Next i s t o expand our knowledge o f l i f e sciences, i.e., l i f e sciences as we perceive i t t o be r e l a t e d t o Earth organisms. Another i s t o develop technology. This i s something t h a t we w i l l be l o o k i n g i n t o . Coupled w i t h t h i s i s an improvement i n man's l i v i n g c o n d i t i o n s , both from an ecologi c a l and a b i o l o g i c a l standpoint. L a s t l y , prepare f o r space manufacturing and processing of biochemical s and b i 01o g i c a l m a t e r i a1 s. For instance, c e r t a i n pharmaceuti c a l s may be produced i n space more e f f e c t i v e l y , purer, and l e s s expensively than on Earth. L i f e Sciences Program Overview From the standpoint o f p r e s e n t i n g an overview o f t h e L i f e Sciences Program, i t can be d i v i d e d i n t o 3 segments. The f i r s t segment might be c a l l e d t h e "Preparation Decade," i.e., The " I n v e s t i g a t i o n Decade" would extend from 1981 t o 1991 , and beyond from 1971 t o 1981 t h a t , from 1991 t o 2001, would be t h e " E x p l o i t a t i o n Decade." During t h e p r e p a r a t i o n decade, i.e., from 1971 t o 1981 , you can f u r t h e r d i v i d e t h a t i n t o 2 p a r t s . That p a r t b e f o r e 1974 had a number o f programs t h a t have l e d t o t h e planning and developing o f t h e S h u t t l e and t h e Spacelab. The Mercury, Gemini, Apollo and Skylab Programs a l l have c e r t a i n l y given us extensive i n f o r m a t i on. There have been mission models and planning s t u d i e s (which are a c t u a l l y payload d e f i n i t i o n s t u d i e s ) t h a t have aided. The second p a r t o f t h i s p r e p a r a t i o n decade can be i d e n t i f i e d as t h e "Development Period" and t h a t i s where we are r i g h t now. We a r e l o o k i n g a t t h e kinds o f experiments which can b e n e f i t from t h e space environment and what support i t wi 11 take t o execute these experiments. Common Operational Research Equipment (CORE) i s being i n v e s t i g a t e d which w i l l support t h e experiments and o p e r a t i o n a l procedures. This w i l l be described l a t e r . Some o f t h e o t h e r a c t i v i t i e s t h a t are t a k i n g place are t h e examination o f carry-on l a b o r a t o r i e s . What can be done i n t h e crew q u a r t e r s of t h e S h u t t l e O r b i t e r i n t h e way o f experimentation i n the very l i m i t e d space t h a t i s a v a i l a b l e ? What can be done i n m i n i - l a b s when another d i s c i p l i n e than l i f e sciences i s using t h e pressurized volume o f t h e Spacelab? What can be done i n m i n i - l a b s ? What are those t h i n g s t h a t can be done l i f e sciences-wise when t h e pressurized volume o f t h e Spacelab i s completely dedicated t o l i f e sciences? What are those experiments t h a t can best f l y on t h e proposed Biomedical Experiments S c i e n t i f i c S a t e l l i t e (BESS)? These are t h e kinds o f questions being considered now and w i l l continue t o be u n t i l t h e Shuttle/Spacelab becomes o p e r a t i o n a l i n t h e 1980s. Then the i n v e s t i g a t i o n decade w i 11 begin when o p e r a t i o n a l f 1 ights w i 11 be avai 1able f o r 1i f e s c i e n t i s t s t o f l y experiments. Probing type experiments and then in-depth e x p e r i ments w i l l n a t u r a l l y b u i l d on these f o r e x p l o i t a t i o n and b u i l d i n g i n breadth and depth of l i f e sciences knowledge (Figure 8).

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Space Transportation System Pay1oad Program Elements Not a l l i n d i v i d u a l s are aware o f t h e various areas t h a t can b e n e f i t from t h e space environment through t h e use o f t h e S h u t t l e Spaqelab. They range from Space Physics, M a t e r i a l s Sciences, Earth and Ocean Physics, Solar Physics, Astronomy, Earth Observations, Comnuni cations/Navigation, Techno1 ogy, and High Energy Astrophysics, t o L i f e Sciences. So many people do n o t appreciate t h a t L i f e Sciences i s n o t merely biomedical s t u d i e s b u t ranges beyond biomedical s t u d i e s t o i n c l u d e areas such as t h e study o f vertebrates, i n v e r t e b r a t e s ,

plants, c e l l s , t i s s u e s , bacteria and viruses, as well as environmental control and man/ systems integration studies. These are areas of Life Sciences t h a t we currently visualize that can benefit from studies in the space environment. There may be others. Major Space Life Sciences Research Areas The major Life Sciences research areas that are predicted t o benefit from the space environment experimentation are Space Medicine, Clinical Medicine, Life Support Techno1 ogy, and Space Biology. These a r e the 4 major categories t h a t include areas i n Life Sciences mentioned e a r l i e r (1, 2, 3 , 4 , 5). S ace Medicine.- In Space Medicine t o date the general conclusion i s t h a t man can adapt t o weight essness with good health f o r extended periods of time with appropriate exercise, sleep, d i e t , working, and recreation. So f a r , no major physiological problems have been encountered, but we do need t o understand and we do need experiments which will permit investigation of the mechanisms of changes t h a t are occurring. Some remedial o r preventive measures may be required f o r missions longer than the experience of 84 days t h a t we had man in a weightless condition u p t o now. For voyages of man t o Mars or other regions in space where he will be in the weightless condition as long as 9-12 months or perhaps several years, we certainly must look a t longer term remedial o r preventive measures.

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Shuttle research emphasis i n space medicine will probably focus on the vestibular, cardiovascular, mineral / f l u i d bal ance/electrolytes, and hematology areas, i . e . , from the perspective of our experience on Skylab and the other manned programs. Clinical Medicine.- In Clinical Medicine, the e f f e c t s on physical condition will be studied. We must be prepared t o t r e a t fractures t h a t might occur in a space environment and 1 earn how heal i ng wi 11 progress. We w i 11 examine wound heal i ng , burn therapy, cardi ovascul ar disease and therapy, and emergency and dental therapy. These are a l l conditions t h a t are t o be expected in individuals who will be i n space. They are t o be expected in individuals on Earth, and i f man i s going t o be in space f o r extended periods, we must know how t o handle these kinds of problems. In addition, there will be opportunities f o r new research in areas where we may have ailments of vision, muscle or cardiovascular function. Life Support Technology.- In Life Support Technology, biological research involving both the crew and passengers as well as biological specimens will be examined. Regenerative l i f e support systems involving atmospheric r e v i t a l i z a t i o n , reusing water t h a t will be there in the spacecraft as a by-product and looking a t sources of food t h a t should be grown will demand attention. Spade Biology. - In Space Biology, vertebrates, invertebrates, c e l l s and t i s s u e s and plants will be studied in the weightless context and radiobiologically. Again, t h i s i s a projection from past research e f f o r t s t h a t have not been directed i n t o an organized space research program. Sciences Laboratory Traffic Model

NASA-Shuttle/Spacelab/Life

The NASA-Shuttle Spacelab Traffic Model, or actually the projected plan of opportunities t o f l y in space during the decade of the 1980s, from the perspective of today, i s q u i t e encouraging. I t i s planned t h a t approximately 10%of a l l the Shuttle f l i g h t s will be f l i g h t s

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which will include Life Sciences research. To be s p e c i f i c , in the current plan, the Shuttle i s t o go into space 501 times during the years from 1979 through 1991. Of those 276 f l i g h t s will contain Spacelabs, and Life Sciences can expect t o have 22 dedicated laboratories and 25 missions where we will have a mini-lab aboard a f l i g h t t h a t would be primarily dedicated t o another discipl ine (Figure 9 ) . 4 ) LIFE SCIENCES PAYLOAD CARRIER CHARACTERISTICS

Earlier in the paper, ~ a y l o a d sc a r r i e r s were b r i e f l y mentioned. There are 3 main c a r r i e r s t h a t will be contained within the Shuttle and remain there the e n t i r e mission (Figure 10). The other i s the BESS o r the Biomedical Experiments S c i e n t i f i c S a t e l l i t e which will be launched and remain in Earth o r b i t f o r 6 months t o perhaps 1 year.

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Carry-on experiments. Carry-on laboratory (COL) experiments wi 11 be contained in the crew compartment of the Shuttle Orbiter (Figure 11 ). They will be small experiment packages weighing l e s s than 23 kg (50 Ibs.), and will require minimum involvement of the crewmembers and an insignificant amount of power. They will be carried when weight and space are available i n the crew compartment. Mini-labs.- Mini-labs will be contained in racks i n s t a l l e d in the pressurized volume of the Spacelab, perhaps in 1 rack, 19 inches wide, o r 2 equalling approximately 1 meter (Figure 12). As indicated, t h i s mini-lab would be flown on a shared d i s c i p l i n e mission. I t would generally weigh l e s s than 500 kg and there could very well be a s i g n i f i c a n t i n t e r f a c e with the Spacelab o r o r b i t e r as f a r as power, heat balance, command, control, o r electronic countermeasures are concerned. A payloads o r mission speci a1 i s t , who would oversee the mini-lab, would not necessarily be trained i n a Life Sciences discipline. Dedicated 1aboratories. - The next and perhaps most important i s the dedicated 1aboratory where up t o /,600 kg of laboratory equipment would f l y (Figure 13). There would be extens i v e interfaces with o r b i t e r as f a r as power, thermal control, e t c . are concerned, and up t o 3 discipline s p e c i a l i s t s operating 12 hourslday on a 7-day mission might f l y . I t could be extended up t o a 30-day mission as our experience and capabi 1i t y i s extended (2). Biomedical Experiments S c i e n t i f i c Sate1 1i t e (BESS). - The Biomedical Experiments S c i e n t i f i c S a t e l l i t e i s a free-flying s a t e l l i t e which i s under development now and which will become operational some time l a t e r than the Spacelab, perhaps around 1983. I t will provide a 6-month t o 12-month time period i n o r b i t f o r specimens. I t will be deployed from the cargo bay of the Shuttle Orbiter and will allow f o r longer duration, zero gravity exposures f o r both animals and plants with a built-in p a r t i a l or one-g experiment control capability provided by a specimen centrifuge. I t i s planned t h a t the specimens will be inserted into the BESS from the spacecraft a f t e r o r b i t i s achieved. Checkout will occur t o insure t h a t a l l specimens are i n good order. If some problem should e x i s t , a correction could be made even a f t e r release of the BESS into o r b i t . Upon r e t r i e v a l , specimen examination could be made in a Spacelab t h a t might be carried a l o f t a t the time of r e t r i e v a l . I t i s a very exciting capability t h a t i s now being planned. Earlier the Common Operational Research Equipment (CORE) was mentioned. CORE can be f u r t h e r subdivided i n t o 3 areas (Figure 14). Regular equipment items such as a microscope, o r a mass measuring device, which would be used in weightlessness t o obtain measurements, wi 11 be used over and over again and may be required by many d i f f e r e n t biological experiments. CORE Intermi t t e n t would be i tems t h a t would support several d i f f e r e n t experiments b u t would not be as universally useful as those mentioned under the regular equipment i tems. Examples would be lower body negative pressure device o r certain racks f o r t e s t tubes. Another piece of CORE equipment might be a rotating l i t t e r chair t o exercis.e orientation and balance organs which very few experiments would require.

5 ) DEVELOPMENT OF PROGRAM PLANNING AND OPERATIONAL APPROACHES

An examination of the phases of a Life Sciences Payloads Mission would show i t can be divided into 4 major segments or phases: Experiment Selection Phase, Preparation Phase, Flight Operations Phase, and Postflight Phase. Each of these can be further subdivided and then examined. In the Experiment Selection Phase, formulation of an overall plan takes place; solicitation, receiving of proposals, and selection takes place. In the Preparation Phase, mission scheduling, experiment development, experiment testing and training, and integrated simulations are some of the major areas. In the Flight Operations Phase, a t prelaunch there i s checkout, mating of the experiment carrier with the Orbiter, launch, flight operations, f l i g h t support, as well as landing operations. The Postflight Phase i s composed of debriefing, examination of the data and distribution of the data and samples to the investigators for analysis and reporting by the experimenter. This i s an example in a generalized way of what takes place in all flights. I t i s presented to give a concept of those things that must be considered for space flight. Spacel ab Mission Simulation Some of the aspects of the mission have been investigated and reviewed through a Spacelab Mission Simulation. The current simulation plan a t JSC projects a total of 5 Spacelab Mission Simulations or SMS. Two simulations have been conducted, 3 more are to occur, planned before the operational period of the Shuttle beginning in 1980 or 1981. Each one of the scheduled simulations will look a t and emphasize specific aspects of operational planning. Total ly , we wi 11 be considering science community invol vement, management systems, experiment and pay1 oad processing, mission operation systems and approaches, equipment and faci 1i t i e s , supporting documentation, and Spacelab configurations as well as roles of organizations and key personnel

.

Involvement of Non-NASA Scientists The simulation that we are preparing for now will involve visiting scientists, those from organizations other than a t the Johnson Space Center and other elements of NASA. The Spacelab Mission Simulation Program i s considered key in preparing for the operational period that will soon be upon us. Through an Invitation t o Participate in Life Sciences Space Program Planning, an announcement of planning opportunity was issued in the spring of 1975. The announcement was sent to over 27,000 scientists in this country. As of early this year, over 1,400 responses with over 2,500 ideas for experiments were received. The responses are being used by NASA to determine the range and types of biological specimens that will be required. The data wi 11 be used for determining the scientific disciplines (special t i e s ) that we must accommodate in the Shuttle, and t o determine and identify the laboratory equipment that will be required. An Announcement of Flight Opportunity will be issued l a t e r in 1976. The f i r s t call will be for the f i r s t US/European Space Agency Spacelab flight scheduled for 1981. A1 though experiments wi 11 be somewhat 1imi ted for this Spacel ab flight , 1ater a general announcement of flight opportunity will be made for Life Sciences (6,7).

6) SUMMARY

In summary, i t can be said t h a t the Space Transporta"con System Program i s we11 under way and will provide a broad new capability by the 1980s; a capability which will permit flying many i i Pe Sciences experiments. The development of the Spacelab/Common Operational Research Equipment and the Biological Specimen Holding Faci 1i t y will a1 low f o r the involvement of many s c i e n t i s t s with minimum hardware costs. There will be a broad L i f e Sciences Program which i s currently in the conceptual phase (some few ideas are now represented by preliminary designs). Perhaps the most important point i s t h a t f o r the diverse program t o be a success the participation of l i f e s c i e n t i s t s such as you i s required.

REFERENCES 1.

Mason, J. A. ; and Heberl i g , J . C. : The Space S h u t t l e and I t s Support f o r Space Bioresearch. BioScience, v o l e 2 3 ( 5 ) , 1973, pp. 307-310.

2.

Mason, J . A.:

Opportunities f o r Biological Research i n Space During t h e 1980's. 26 (5), 1996, pp. 325-329.

BioScienee, vol

.

3.

National Academy of Sciences, Space Science Board: Shuttle. 1974. Washington, D. C.

4.

Winter, D. L. : Man i n Space-A Time f o r Perspective: Crew Performance on Space S h u t t l e Space1 ab Program. Astronautics and Aeromutics, v01. 13(1 0 ) , 1975, pp. 28-36.

5.

National Aeronautics and Space Administration, Life Sciences: Final Report o f t h e Space S h u t t l e Payload Planning Working Groups. U .S. Government P r i n t i n g Office Document 1972-735-969-740, vol 4, 1973, Washington, D. C.

S c i e n t i f i c Uses o f the Space

.

6.

National Aeronautics and Space Administration: An I n v i t a t i o n t o P a r t i c i p a t e i n Planning t h e NASA Life Sciences Program i n Space. U . S. Government P r i n t i n g Office Document 1975-628-637-268, 1975a, Washington, D. C .

7.

Leeper, E. M.:

NASA Seeks Help f o r B i o l o g i s t s .

BioScience, vol. 25(4), 1975, p. 284.

TRENDS OF THE 1980's

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INTEGRATED SPACE OPERATIONS

MANNED a 'UNMANNED SPACE SYSTEMS @ NASA CENTERS @ OTHER GOi/T AGENCIES @ UNIVERSITIES @ INDUSTRY

.. - -

FAMILY OF LAUNCH VEHICLES FOR UNIQUE MISSIONS

GEN'L-PURPOSE LAUNCH S Y S T E M REUSABLE HARDWARE

Figure 1.- Space Shuttle era (~AS~-S-74-5358~).

TO ESTABLISH A NATIONAL SPACE TRANSPORTATION CAPABILITY THAT WILL O SUBSTANTIALLY REDUCE THE COST OF SPACE OPERATIONS AND O PROVIDE A CAPABILITY DESIGNED TO SUPPORT A WIDE RANGE OF SCIENTIFIC, APPLICATIONS, DEFENSE, COMMERCIAL AND INTERNATIONAL USES Figure 2.- Space Shuttle Program objective (NASA-S-74-5359~).

ATMOSPHERIC EN

RNAROUND MAINTENANCE

MANEUVER CAPA

LAUNCH PAD

UNPOWERED LAMDING

Figure 3.- Space Shuttle mission profile (NASA-S-75-868).

Figure

4.-

Space Shuttle Program activities ( NASA-S-75-15027).

SPACE SHUTTLE

SPACELAB

mj . .

EUROPEAN SPACE AGENCY ( ESA 1

53.1% W. GERMANY

* ESA MEhlBER BUT NOT INVOLVED I N SPACE LAB "* NON NMEMBEROF ESA

=%2b

Figure 7.- Extent of international cooperation in manned space flight (NASA-S-74-3431~).

I

PREPARATION DECADE

DEVELOPMENT PAYLOAD DEFINITION STUDIES

0 EXPERIMENTS 0 CORE (I)

(I)

CARRY-ON LABS

PAYLOAD PROGRAM DEDICATED LABS

0 MISSION MODELS

I INVEST1 GATION DECADE I PROBETYPE EXPERIMENTS

@ BESS

EXPLOITATION DECADE

@ REFINEMENT AND

EQCllPMENT

QUANTIFICATION OF SPACE INFORMATION

SOFTWARE

0 DEVELOPMENT OF TECHNOLOGY

* PROGRAM PLANNING

0 EXPANSION OF LIFE EXPERIMENTS

SCIENCES KNOWLEDGE

t FABRICATION

* TRAINING t TESTS AND SIMULATIONS

0 IMPROVEMENT OF MAN'S LIVING CONDITIONS

- ECOLOGICAL - BIOLOGICAL

@ PREPARATION FOR CONTINUED SPACE UTILIZATION AND EXPLORATION

Figure 8.- L i f e science program overview ( N A S A - S - ~ ~ - ~ O ~ ~ ~ A ) .

I

i'

(1) FLIGHTS CONTINUE AT 2,"fEAR THRU 1987. THEN 3 B E A R T H R U 1 9 9 1 F O R A T O T A L O f f 26 L S L DEDICATED SPACELAB MISSIONS (4;7 DAY AND 22;30 DAY) Figure 9.-

Space Shuttle/Spacelab/Life Sciences Laboratory (LSL) traffic model (NASA-S-76-10161).

@

@ @ @

CARRY -ON ORBITER CREW COMPARTMENT LESS THAN 23 K g (50 LB) MINIMAL INTERFACES POWER FLIGHTS OF OPPORTUNITY 1 TO 7-DAY

M I N I -LAB 9 SHARED M I S S I O N GENERALLY LESS THAN 500 K g 9 ONE TO SEVERAL RACKS OF EQU IPMENT SIGNIFICANT INTERFACES WITH SPACELAB CDMS, POWER, THERMAL, ECS 9 SHARED PIL SPECIAL1 ST 7 TO 30-DAY M I S S I O N S

DEDICATED LABORATORIES

9 UP TO 7,600 Kg FULLY DEDICATED SPACELAB MISSION 9 EXTEN S l VE l NTERFACE S W ITH SPACELAB - CDMS, POWER, THERMAL, ECS UP TO 3 DISCIPLINE SPECIAL1 STS, 12-MRIDAV ON 7-DAY M I S S I O N S 7 TO 30-DAY M I S S I O N S

Figure 10.- Life science payload characteristics ( ~ ~ ~ ~ - ~ - 7 6 - 1 0 1 5 7 ) .

*-

URINE ACQUISITION KIT URINE STORAGE BAGS 200 cc SYRINGE ( 3 )

/

NEEDLES

b

/

BLOOD ACQUISITION KIT SYRINGES

6 1 ,,: $.

COTTON SWABS A LC0 HO L TOURNIQUET BAND-AIDS LANCETS PRESTAINED SLIDES

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HEMOGLOBINOMETER T'YPICAL ELASTIC SEPARABLE KIT UNITS (4) PHYSICAL EXAMINATIOK "" r r r t a n r BLOOD P R t > > u n t \I CUFF STETHESCOPE DOPPLER FLOW METER OTO-OPHTHALMASCOPE TUNING FORK THERMOMETER

-\

L \NA CTF

1

~ ATTACHMENT ~ POINTS FOR EQUIPMERIT RESTRAINT

STORAGE RESTRAINT

Figure 11.- Conceptual design sketch of biomedical COL (NASA-S-76-10158).

~

Kl TS LINEAR MEASUREMENT DISSECTION MICROBIOLOGY OTHERS

CHEMICALS STAINING SYSTEM AND MISCELLANEOUS

MICROSCOPE STORAGE

MMB PLANT CAGE CRE:W WORK STATION COLONY COUNTER CRYOGENIC FREEZER

47i?LdQJi! ! \

LOW-TEMPERATURE FREEZER

MASS MEASUREMENT DEVICE (MICRO) AND MISCELLANEOUS

F i g u r e 12.- T y p i c a l m i n i l a b f o r b i o l o g i c a l specimen examination and experimentation.

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-.=-

NASA's SPACE PROCESSING PROG

...

-

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By James H. ~ r e d t ,NASA Office of Applications, Washington, D.C. ABSTRACT

The primary goal of NASA's Space Processing Program is to initiate utilization of space flight capabilities for economically beneficial activities in all branches of materials science and technology. It is expected that the condition of virtual weightlessness obtained in space flight will permit unprecedentedly precise control over many known processes and development of many novel processes to manipulate and prepare biological materials intended for use on the ground. This expectation has already been verified in the case of electrophoretic separation of living cells through a sequence of experiments performed on the Apollo, Skylab, and Apollo-Soyuz Test Project (ASTP) missions. It is believed that further applications will be found as well, and that future reductions in the costs of space operations will make a large increase in the scope of space experimentation possible. Plans are now being made for payload equipment to implement materials processing experiments on the missions of the Space Transportation System (STS). This equipment is intended to support a diversified program of NASA-sponsored materials processing experiments by all classes of scientists, as well as pilot activities by non-NASA sponsors. It appears feasible to organize payload systems that can implement a wide variety of activities without undue constraints from spacecraft resources, and on this basis we expect that STS payloads can begin to provide supporting services for research and applications more or less routinely fairly early in the 1980's.

PREEDING PAGE BLANK NOT F E

'Phe NWSA Space processing Program i s conducted by t h e Office of Applications t o develop uses of space f l i g h t t h a t w i l l support research e f f o r t s and manufacturing operations on t h e ground by I t i s expected t h a t the unique processing m a t e r i a l s i n space. conditions t h a t a r e a v a i l a b l e i n space w i l l provide a b a s i s f o r a wide v a r i e t y of economically b e n e f i c i a l s e r v i c e s t o science and i n d u s t r y i n f i e l d s such as metallurgy, e l e c t r o n i c m a t e r i a l s , g l a s s technology, f l u i d physics and chemistry, and i n b i o l o g i c a l m a t e r i a l p r e p a r a t i o n a s well. The primary advantage t h a t such a p p l i c a t i o n s w i l l seek t o e x p l o i t i s t h e condition of v i r t u a l weightlessness obtained i n a f r e e l y moving s p a c e c r a f t during unpowered f l i g h t . In low Earth o r b i t , which has been t h e usual s e t t i n g f o r m a t e r i a l s processing experiments i n space, t h e g r a v i t a t i o n a l f i e l d i n t e n s i t y i s only a l i t t l e l e s s than it i s on t h e E a r t h ' s s u r f a c e . However, i n unpowered f l i g h t a s p a c e c r a f t and everything i n i t a r e f r e e l y a c c e l e r a t e d by t h e f o r c e of g r a v i t y , and t h e s e a c c e l e r a t i o n s a r e a l l equal with an accuracy of t h e order of one p a r t i n t e n m i l l i o n . Therefore, g r a v i t y e f f e c t s cannot produce appreciable r e l a t i v e v e l o c i t i e s between o b j e c t s i n t h e s p a c e c r a f t . For example, a lump of lead t h a t was released very c a r e f u l l y s o a s t o avoid imparting any impulse t o i t would t y p i c a l l y d r i f t a meter o r s o from i t s o r i g i n a l s t a t i o n and then r e t u r n during t h e o r b i t a l period of approximately 90 minutes, because i t s o r b i t would d i f f e r s l i g h t l y from, t h a t of t h e s p a c e c r a f t unless t h e two b o d i e s ' c e n t e r s of mass happened t o coincide. The f o r c e s needed t o hold t h e lump of lead i n a c o n s t a n t p o s i t i o n r e l a t i v e t o t h e i n t e r i o r of t h e s p a c e c r a f t would be very small, and i n f a c t they could be provided e a s i l y by a i r c u r r e n t s , a c o u s t i c r a d i a t i o n p r e s s u r e , o r electromagnetic i n t e r a c t i o n s . In l i q u i d media t h e r e l a t i v e v e l o c i t i e s t h a t can be produced by g r a v i t y e f f e c t s a r e much s m a l l e r , because t h e very small f o r c e s t h a t a r e involved have t o a c t a g a i n s t viscous drag f o r c e s t h a t a r e t y p i c a l l y a hundred times a s g r e a t a s those found i n gases. For a l l p r a c t i c a l purposes, t h e r e f o r e , t h e d r i v i n g forces f o r thermal convection, sedimentation and o t h e r buoyancy e f f e c t s a r e completely removed from l i q u i d systems i n space. This i s a f a c t o r of considerable s i g n i f i c a n c e f o r chemical and b i o l o g i c a l procedures, because it becomes p o s s i b l e t o work with heterogeneous chemical systems under experimental conditions where t h e only h e a t and mass t r a n s p o r t e f f e c t s t o be considered a r e h e a t conduction and chemical d i f f u s i o n . Since both of t h e s e e f f e c t s a r e p r e c i s e l y c a l c u l a b l e according t o a r e l a t i v e l y simple mathematical theory, it should be p o s s i b l e t o apply extremely p r e c i s e techniques of c o n t r o l and measurement t o experiments with such systems i n space.

The e f f e c t s of weightlessness have been exploited t o study a v a r i e t y of processes i n s o l i d i f i c a t i o n , c r y s t a l growth, f l u i d physics, and physical separation methods i n space f l i g h t experiments conducted over P l i g h t s t h a t have c a r r i e d space processing experit h e p a s t f i v e years. ments a r e i n d i c a t e d i n t h e t o p s e c t i o n of the schedule c h a r t shown i n Figure 1. They include the Apollo 14, 16 and 1 7 lunar missions, t h e four f l i g h t s of t h e Skylab program, and l a s t y e a r ' s Apollo-Soyuz Test These f l i g h t s have c a r r i e d a combined t o t a l P r o j e c t (ASTP) mission. of 41 experiments and demonstrations r e l a t e d t o m a t e r i a l s processing. In o r d e r t o continue experimentation through t h e r e s t of t h e 1970's, t h e Space Processing Program has undertaken a s e r i e s of rocket missions t h a t w i l l c a r r y payloads on b a l l i s t i c f l i g h t s t h a t each a f f o r d between f i v e and ten minutes of experiment time. This p r o j e c t i s c a l l e d t h e Space Processing Applications Rocket (SPAR) p r o j e c t , and it i s planned t o conduct t h r e e f l i g h t s p e r year u n t i l t h e p r o j e c t i s superseded by experiment operations using the Space S h u t t l e and Spacelab. The f i r s t I t c a r r i e d nine SPAR f l i g h t was c a r r i e d out on December 11, 1975. experiments, thus bringing t h e t o t a l f o r t h e whole program t o 50; t h e d i s t r i b u t i o n of experiments and demonstrations among t h e missions flown t o d a t e i s given i n Table I. Five of t h e program's experiments have been d i r e c t e d toward developing new methods f o r b i o l o g i c a l preparations. In t h e s e experiments, e l e c t r o p h o r e t i c s e p a r a t i o n s have been performed i n aqueous media t h a t were s t a b i l i z e d a g a i n s t convection by weightlessness r a t h e r than by porous supports o r laminar flow, and p r o t r a c t e d s e p a r a t i o n runs have been accomplished on p a r t i c l e s t h a t would have sedimented very r a p i d l y on Earth. These e a r l y experiments have mainly served t o e s t a b l i s h t h e technology needed f o r more advanced work i n t h e f u t u r e , b u t t h e i r r e s u l t s i n d i c a t e t h a t f u r t h e r development can be expected t o r e s u l t i n r e f i n e d and powerful s e p a r a t i o n methods t h a t should be capable of q u a n t i t a t i v e l y predictable results. A s t h e second s e c t i o n of Figure 1 i n d i c a t e s , t h e development

(Phase C/D) of t h e Space S h u t t l e was i n i t i a t e d a t about t h e time of t h e Apollo 16 f l i g h t , and t h e Pxeliminary Design Review (PDR) was held s h o r t l y a f t e r t h e ASTP mission. The f i r s t S h u t t l e Orbiter is scheduled t o be r o l l e d o u t toward t h e end of t h i s year and w i l l be f l i g h t t e s t e d within t h e atmosphere during 1977. Thus t h e design phase of t h e S h u t t l e p r o j e c t has been going forward concurrently with t h e experiments t h a t NASA has been performing t o v e r i f y t h e promise of m a t e r i a l s processing i n space.

During t h e d e s i g n work on t h e S h u t t l e , t h e Space P r o c e s s i n g Program h a s performed s u b s t a n t i a l l y continuous s t u d i e s o f p o t e n t i a l modes However, it h a s been n e c e s s a r y o f S h u t t l e and Spacelab u t i l i z a t i o n . t o w a i t u n t i l t h e experiment r e s u l t s from t h e manned m i s s i o n s o f t h e 1 9 7 0 ' s were a v a i l a b l e and t h e S h u t t l e d e s i g n was s u b s t a n t i a l l y complete b e f o r e embarking on f i n a l d e f i n i t i o n o f Space P r o c e s s i n g A p p l i c a t i o n s (SPA) payloads f o r t h e S h u t t l e m i s s i o n s . Phase 1/11 payload d e f i n i t i o n s t u d i e s a r e now i n p r o g r e s s and a comp e t i t i v e procurement w i l l b e h e l d i n t h e l a t t e r p a r t o f 1976 f o r payload d e s i g n and development work which w i l l b e g i n i n t h e f i r s t q u a r t e r o f 1977. The development of s p e c i f i c equipment i t e m s w i l l be phased t o p r o v i d e f o r d e l i v e r i e s a t t h e approximate p o i n t s shown i n t h e t h i r d s e c t i o n o f F i g u r e 1, comprising two i n i t i a l p a y l o a d s compatible w i t h t h e t e s t f l i g h t s of t h e S h u t t l e and two payloads f o r o p e r a t i o n a l f l i g h t s w i t h t h e Spacelab i n 1981. I n d e f i n i n g payload equipment f o r t h e Space S h u t t l e and Spacelab we have followed approaches i n t e n d e d t o g i v e e x p e r i m e n t e r s e a s y a c c e s s t o s p a c e and maximize t h e s c i e n t i f i c o u t p u t o f t h e i r experiment w h i l e minimizing t h e c o s t s of o p e r a t i n g them i n s p a c e . ~ y p i c a lpayload c o n f i g u r a t i o n s t h a t have been d e r i v e d t o implement t h i s d e s i g n p h i l o s o I n g e n e r a l s p a c e p r o c e s s i n g payload phy a r e i l l u s t r a t e d i n F i g u r e 2 . systems w i l l b e b u i l t up o f modular, r e u s a b l e equipment t a k e n from a s t a n d a r d i n v e n t o r y of a p p a r a t u s and s u p p o r t i n g equipment, s o t h a t each i t e m o f equipment can s e r v e many i n v e s t i g a t o r s and l i t t l e a p p a r a t u s w i l l need t o b e developed s p e c i f i c a l l y f o r i n d i v i d u a l experiments. The u s u a l mode o f o p e r a t i o n w i l l b e t o f l y s p a c e p r o c e s s i n g payloads on m i s s i o n s s h a r e d w i t h o t h e r d i s c i p l i n e s , and t h e modular n a t u r e o f t h e equipment w i l l make i t p o s s i b l e t o t a k e advantage o f a wide v a r i e t y o f s h a r e d m i s s i o n o p p o r t u n i t i e s because each payload w i l l o n l y need t o i n c l u d e t h e equipment n e c e s s a r y t o i t s m i s s i o n . Payloads w i l l a l s o b e o r g a n i z e d f o r maximum p r o d u c t i v i t y , s o t h a t t h e The u n i t c o s t s o f p r o c e s s i n g m a t e r i a l samples w i l l b e minimized. system is b e i n g d e s i g n e d t o t h a t a l l of t h e equipment i n a given payload Since t h i s can b e o p e r a t e d d u r i n g a l l o f t h e t i m e i t spends i n space. c o n c e p t r e q u i r e s many experiments t o o p e r a t e a t once, t h e s p a c e proc e s s i n g equipment i n v e n t o r y w i l l i n c l u d e an A u x i l i a r y Payload Power System (APPS) t h a t p r o v i d e s power from S h u t t l e t y p e f u e l c e l l s and h a s a r a d i a t o r t o dispose of t h e r e s u l t i n g heat. The system w i l l b e c a p a b l e o f s u p p l y i n g 15 kw o f e l e c t r i c power, and i t i s e x p e c t e d t o f r e e s p a c e

processing experiment operations s u b s t a n t i a l l y from c o n s t r a i n t s due t o l i m i t a t i o n s on t h e resources of t h e S h u t t l e , Investment and o p e r a t i n g c o s t questions are c r i t i c a l f o r m a t e r i a l s processing i n space because one of NASA's goals i s t o introduce a c t i v i t y sponsored by o t h e r government and p r i v a t e sponsors i n t o space. This w i l l be p o s s i b l e only i f t h e c o s t s of performing space experiments a r e within t h e range t h a t such sponsors a r e accustomed t o pay f o r research. The r e s u l t s of t h e Space Processing Program's c u r r e n t payload d e f i n i t i o n s t u d i e s i n d i c a t e t h a t t h i s may be achieved i f t h e number of i n v e s t i g a t o r s served on each mission i s l a r g e enough, and t h e r e f o r e t h e program i s planning f o r payloads t h a t w i l l be capable of processing l i t e r a l l y hundreds of samples f o r a d i v e r s i f i e d community of u s e r s on every f l i g h t . This type of operation i s unprecedented i n space experimentation, and it w i l l obviously provide f l i g h t o p p o r t u n i t i e s f o r l a r g e r than usual ~ n i t i a l l yt h e s e i n v e s t i g a t o r s w i l l perform numbers of i n v e s t i g a t o r s . sponsorship t o develop experimental t h e i r experiments un e r NASA's methods and demonstrate t h a t space f l i g h t provides c a p a b i l i t i e s f o r f r e s h approaches and new d i s c o v e r i e s i n t h e i r f i e l d s . The Space processing Program i s employing a v a r i e t y of means t o engage t h e i n t e r e s t s of q u a l i f i e d experimenters and d e f i n e worthwhile a p p l i c a t i o n s ; among t h e s e means a r e meetings such a s t h e p r e s e n t one, working group a c t i v i t i e s i n support of t h e payload planning e f f o r t , and p a r t i c i p a t i o n i n t h e SPAR p r o j e c t . In t h e e a r l y years of space processing experiments on t h e S h u t t l e and Spacelab, we expect of the o r d e r of 100 i n v e s t i g a t o r s t o conduct research p r o j e c t s t h a t use space a c t i v i t i e s t o achieve t h e i r objectives. Among t h e s e w i l l be p r o j e c t s d e a l i n g with t h e f e a s i b i l i t y of b i o l o g i c a l and biomedical a p p l i c a t i o n s of space f l i g h t , some of which may be based on ideas brought o u t by t h i s Colloquium. Assuming t h a t these p r o j e c t s a r e as f r u i t f u l and inexpensive a s planned, we b e l i e v e t h a t t h e i n v e s t i g a t o r s w i l l soon f i n d o t h e r sponsors wishing t o support t h e i r work a s well. I f t h i s i s t h e case, then by t h e l a t e 1980's we can expect t o f i n d t h a t space experiments w i l l have become a more o r l e s s r o u t i n e resource i n t h e kinds of b i o l o g i c a l and o t h e r work f o r which they o f f e r s u b s t a n t i a l advantages.

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APOLLO FLTS SKYLAB FLTS

C/D SIGNED

1 S T MOF 2 N O M O F

SRR DEVELOPMENT

4 C I D A W A R D PRR

-a,

4

SRR

PDR

CDR

DEL E/M

1 ST FLT ITEM

DEVELOPMENT

F i g u r e 1.- Overview s c h e d u l e s f o r Space T r a n s p o r t a t i o n System, Space P r o c e s s i n g A p p l i c a t i o n s , and Spacelab.

FURNACE AND BIOLOGICAL

AUXILIARY PAYLOAD POWER SYSTEM (APPS) SUPPORT STRUCTURES

ELECTROMAGNETIC

Figure 2.-

Space processing of Spacelab payloads.

BEHAVIOR OF FLUIDS I N A WEIGHTLESS ENVIROWNT By Dale A. F e s t e r , Ralph N . E b e r h a r d t and James R. T e g a r t Martin M a r i e t t a C o r p o r a t i o n , Denver, Colorado ABSTRACT F l u i d b e h a v i o r i n s p a c e d i f f e r s markedly from t h a t i n a gravity-dominated environment. T h i s must be c o n s i d e r e d f o r a l l f l u i d usage, whether f o r v e h i c l e o p e r a t i o n s o r payload exNumerous i n v e s t i g a t i o n s have shown t h a t t h e b e h a v i o r of f l u i d s i n a low-g e n v i r periments. onment i s c o n t r o l l e d p r i m a r i l y by s u r f a c e t e n s i o n f o r c e s . C e r t a i n f l u i d and system c h a r a c t e r i s t i c s determine t h e magnitude of t h e s e f o r c e s f o r b o t h a f r e e l i q u i d s u r f a c e and l i q u i d i n c o n t a c t w i t h a s o l i d . These c h a r a c t e r i s t i c s , i n c l u d i n g s u r f a c e t e n s i o n , w e t t a b i l i t y o r c o n t a c t a n g l e , system geometry, and t h e r e l a t i o n s h i p s governing t h e i r i n t e r a c t i o n , a r e d i s c u s s e d . Various a s p e c t s of f l u i d b e h a v i o r i n a low-g environment a r e t h e n presented. T h i s i n c l u d e s t h e f o r m a t i o n of s t a t i c i n t e r f a c e s h a p e s , o s c i l l a t i o n and r o t a t i o n o f d r o p s , c o a l e s c e n c e , t h e f o r m a t i o n of foams, tendency f o r c a v i t a t i o n , and d i f f u s i o n i n l i q u i d s which were observed d u r i n g t h e Skylab f l u i d mechanics s c i e n c e d e m o n s t r a t i o n s . L i q u i d r e o r i e n t a t i o n and c a p i l l a r y pumping t o e s t a b l i s h e q u i l i b r i u m c o n f i g u r a t i o n s f o r v a r i o u s system geome t r i e s , observed d u r i n g v a r i o u s f r e e - f a l l (drop-tower) low-g t e s t s , a r e a l s o p r e s e n t e d . S e v e r a l p a s s i v e low-g f l u i d s t o r a g e and t r a n s f e r systems a r e d i s c u s s e d . These systems u s e s u r f a c e t e n s i o n f o r c e s t o c o n t r o l t h e l i q u i d / v a p o r i n t e r f a c e and p r o v i d e g a s - f r e e l i q u i d t r a n s f e r and l i q u i d - f r e e vapor v e n t i n g . INTRODUCTION F l u i d behavior i n s p a c e i s c o n t r o l l e d by s u r f a c e t e n s i o n f o r c e s which a r e g e n e r a l l y i n s i g n i f i c a n t i n a g r a v i t y dominated environment. B a s i c phenomena such a s buoyancy, s e t l i n g , c o n v e c t i o n , mixing and d i f f u s i o n a r e c o n s i d e r a b l y d i f f e r e n t when g r a v i t y i s n o t p r e s e n t . The r e a c t i o n r a t e s f o r many p r o c e s s e s may be a l t e r e d . These, and o t h e r d i f f e r e n c e s i n b e h a v i o r , may o f f e r advantages i n conducting b i o l o g i c a l experiments. For example, l o n g e r r e s i d e n c e t i m e s a r e a v a i l a b l e f o r oxygen bubbles i n c o n t a c t w i t h immiscible subs t r a t e s , and s t r o n g s h e a r f o r c e s induced by a g i t a t i o n i n l i q u i d media can be e l i m i n a t e d . The l a c k of s i g n i f i c a n t buoyancy f o r c e s , on t h e o t h e r hand, may i n t r o d u c e c e r t a i n h a n d l i n g concerns such a s t h e s e p a r a t i o n and c o n t r o l of l i q u i d and v a p o r . An u n d e r s t a n d i n g o f how s u r f a c e t e n s i o n c o n t r o l s f l u i d b e h a v i o r i n low-g i s t h u s needed i n p l a n n i n g any e x p e r i m e n t s where l i q u i d s w i l l be i n t e r a c t i n g w i t h g a s e s and s u r f a c e s .

A d i s c u s s i o n of f l u i d and system c h a r a c t e r i s t i c s t h a t d e t e r m i n e t h e magnitude of t h e s u r f a c e t e n s i o n f o r c e s i s p r e s e n t e d i n t h i s paper. Examples of s u r f a c e t e n s i o n dominated b e h a v i o r i l l u s t r a t i n g some of t h e b a s i c phenomena a r e a v a i l a b l e from b o t h drop tower and Skylab f l u i d mechanics d e m o n s t r a t i o n s . The s e p a r a t i o n of l i q u i d and vapor by s u r f a c e t e n s i o n f o r c e s t o accomplish low-g s t o r a g e and t r a n s f e r i s p r e s e n t e d a s a p r a c t i c a l a p p l i c a t i o n of t h e b a s i c p r i n c i p l e s t o a ' r e a l system. FLUID CHARACTERISTICS The shape of a g a s - l i q u i d i n t e r f a c e i n l o w - g r a v i t y ( g r a v i t y f o r c e s a r e n e g l i g i b l e ) i s determined s o l e l y by c a p i l l a r y f o r c e s . The Young-Laplace e q u a t i o n r e l a t e s l i q u i d s u r f a c e t e n s i o n and t h e c u r v a t u r e of t h e i n t e r f a c e t o t h e p r e s s u r e d i f f e r e n t i a l between t h e g a s and liquid,

where t h e s u b s c r i p t s L and G r e f e r t o t h e l i q u i d and gas r e s p e c t i v e l y , g i s t h e s u r f a c e

t e n s i o n , and R and R a r e t h e r a d i i of c u r v a t u r e of t h e i n t e r f a c e . The r a d i i of curva2 t u r e a r e consiAered p o s i t i v e when t h e i r c e n t e r of c u r v a t u r e i s w i t h i n t h e l i q u i d . The sum of t h e r e c i p r o c a l s of t h e r a d i i , (l/R1 + 1 / ~ ~ i)s , r e f e r r e d t o a s t h e c u r v a t u r e of t h e interface1, Under s t a t i c c o n d i t i o n s , t h e p r e s s u r e of t h e gas i n c o n t a c t w i t h t h e l i q u i d i s u n i form over t h e e n t i r e l i q u i d s u r f a c e . An e q u i l i b r i u m i n t e r f a c e w i l l be e s t a b l i s h e d when t h e p r e s s u r e i n t h e l i q u i d i s a l s o uniform and t h e p r e s s u r e d i f f e r e n t i a l d e f i n e d by equaI f t h e p r e s s u r e d i f f e r e n c e i s c o n s t a n t , t h e n t h e c u r v a t u r e must t i o n (1) i s a constant. a l s o be uniform over t h e e n t i r e s u r f a c e . Considering a g l o b u l e of l i q u i d i n l o w - g r a v i t y , n o t i n c o n t a c t w i t h any s u r f a c e , t h e requirement of uniform c u r v a t u r e can o n l y be s a t i s f i e d i f both r a d i i of c u r v a t u r e a r e e q u a l . The g l o b u l e t h e n h a s t h e form of a s p h e r e , w i t h t h e r a d i i of c u r v a t u r e e q u a l t o i t s r a d i u s . Equ,ation (1) s i m p l i f i e s t o :

where r i s t h e r a d i u s o f t h e s p h e r e . The p r e s s u r e w i t h i n t h e drop of l i q u i d i s g r e a t e r t h a n t h a t o f t h e s u r r o u n d i n g g a s by an amount t h a t i s d i r e c t l y p r o p o r t i o n a l t o t h e s u r f a c e t e n s i o n and i n v e r s e l y p r o p o r t i o n a l t o t h e r a d i u s of t h e drop. The same h o l d s t r u e f o r a bubble surrounded by l i q u i d e x c e p t t h a t t h e p r e s s u r e w i t h i n t h e gas i s g r e a t e r t h a n t h a t i n the liquid. When t h e l i q u i d i s i n c o n t a c t w i t h a s u r f a c e , t h e d e t e r m i n a t i o n of t h e i n t e r f a c e becomes more complex. The c u r v a t u r e of t h e s u r f a c e w i l l s t i l l be a c o n s t a n t , b u t t h e two r a d i i of c u r v a t u r e w i l l v a r y o v e r t h e s u r f a c e . The l i q u i d - t o - s o l i d c o n t a c t a n g l e 0 i s a boundary c o n d i t i o n t h a t must be s a t i s f i e d . T h i s i s t h e a n g l e formed between t h e l i q u i d and t h e s u r f a c e , measured w i t h i n t h e l i q u i d . V a r i a t i o n s i n 0 due t o t e m p e r a t u r e and i n 0 and 0 due t o c o n t a m i n a t i o n , d i s s o l v e d p r e s s u r a n t , and l i q u i d p u r i t y w i l l a f f e c t i n t e r f a c e shape. The v a l u e of c o n t a c t a n g l e p r i m a r i l y depends on t h e l i q u i d s u r f a c e t e n s i o n and t h e s o l i d boundary s u r f a c e energy. The l a t t e r c a n be e x p r e s s e d a s a s o - c a l l e d " c r i t i c a l s u r f a c e t e n s i o n . " I f the liquid surf a c e tension i s l e s s than the c r i t i c a l value, the contact angle i s zero. I f t h e surface t e n s i o n i s a g r e a t e r t h a n t h e c r i t i c a l v a l u e , t h e c o s i n e of t h e c o n t a c t a n g l e i s a l i n e a r p r o p o r t i o n t o t h e d i f f e r e n c e between t h e l i q u i d and t h e c r i t i c a l s u r f a c e t e n s i o n s . Clean m e t a l s u r f a c e s have h i g h c r i t i c a l s u r f a c e t e n s i o n s and most l i q u i d s w i l l completely wet them. Maintaining a c o n t a m i n a n t - f r e e s u r f a c e i s d i f f i c u l t t o a c h i e v e i n p r a c t i c e , however. Most monolayer contaminant f i l m s ( e x c e p t f l u o r o c a r b o n s ) have c r i t i c a l s u r f a c e t e n s i o n s between 20 and 4 5 dynes/cm2y 3. T h i s i s a l s o t r u e of p l a s t i c s . Water, w i t h i t s h i g h s u r f a c e t e n s i o n of 72 dynes/cm, w i l l have h i g h c o n t a c t a n g l e s on t h e s e s u r f a c e s . A c o n t a c t a n g l e of 33.5O i s shown i n F i g u r e 1. Very few l i q u i d s ( o t h e r t h a n l i q u i d m e t a l s ) have a h i g h e r s u r f a c e t e n s i o n t h a n w a t e r . S i n c e w a t e r h a s such a h i g h s u r f a c e e n e r g y , i t i s r e a d i l y contaminated and a c o n s i d e r a b l e lowering of t h e s u r f a c e t e n s i o n t a k e s place4. Contaminants t h a t lower t h e s u r f a c e t e n s i o n o f a l i q u i d a r e r e f e r r e d t o a s s u r f a c e a c t i v e a g e n t s . A s m a l l amount of a s u r f a c e a c t i v e a g e n t ' w i l l impose i t s low s u r f a c e t e n s i o n on a l i q u i d of much h i g h e r s u r f a c e t e n s i o n . A s t h e c o n c e n t r a t i o n of t h e i m p u r i t y i s i n c r e a s e d t h e s u r f a c e t e n s i o n of t h e s o l u t i o n d e c r e a s e s u n t i l i t becomeg s a t u r a t e d . F u r t h e r a d d i t i o n o f t h e i m p u r i t y does not cause any change i n s u r f a c e tension'. For example, adding soap of t h e t y p e used on Skylab t o w a t e r , i n approxi m a t e l y t h e c o n c e n t r a t i o n s used i n t h e d e m o n s t r a t i o n s , reduced t h e s u r f a c e t e n s i o n t o 20 dynes/cm6.

While t h e Young-Laplace e q u a t i o n can d e f i n e more t h a n one i n t e r f a c e f o r any g i v e n s e t o f c o n d i t i o n s , t h e e q u a t i o n f o r s u r f a c e energy d e f i n e s t h e p r e f e r r e d c o n f i g u r a t i o n . A s t a b l e i n t e r f a c e shape i s achieved when t h e s u r f a c e energy i s a minimum. I n s i m p l i f i e d f o r m , s u r f a c e energy i s d e f i n e d by S.E.

=

QLV (ALV

-

ASL

where A i s a r e a and LV d e n o t e s l i q u i d - v a p o r and SL d e n o t e s s o l i d l i q u i d . can be d e f i n e d a s

Ac =

=

=LV

A capillary area

COS 8

Pt" -

f o r any g i v e n i n t e r f a c e . S.E.

(3)

COSS )

*c

Then s u r f a c e energy can be f u r t h e r s i m p l i f i e d t o

(5)

Again c o n s i d e r a f r e e - f l o a t i n g g l o b u l e o f l i q u i d . The s u r f a c e energy w i l l be a m i n i mum when t h e c a p i l l a r y a r e a , which i s t h e a r e a of t h e l i q u i d - v a p o r i n t e r f a c e i n t h i s c a s e , i s a minimum. This o c c u r s when t h e g l o b u l e assumes a s p h e r i c a l s h a p e , a g a i n confirming t h e s t a t i c shape f o r a l i q u i d drop. The c a p i l l a r y a r e a i s reduced when a l i q u i d drop c o n t a c t s a s o l i d s u r f a c e . T h e r e f o r e , c o n t a c t w i t h a s u r f a c e i s a p r e f e r r e d e q u i l i b r i u m c o n f i g u r a t i o n f o r a l i q u i d drop. T h i s d e c r e a s e i n s u r f a c e energy a c c o u n t s f o r t h e a d h e s i v e n e s s o f a l i q u i d on a s u r f a c e . Energy must be added t o remove a drop from a s u r f a c e . The e q u i l i b r i u m i n t e r f a c e c o n f i g u r a t i o n i s e s t a b l i s h e d by t h e mechanism of s u r f a c e t e n s i o n - d r i v e n flow, termed c a p i l l a r y pumping. Liquid w i l l p r e f e r e n t i a l l y o r i e n t within a c o n t a i n e r by c a p i l l a r y pumping i f t h e system i s i n a low-g environment7. The geometry can be m o d i f i e d by compartmentation i n one a r e a of t h e t a n k such t h a t a lower c h a r a c t e r i s t i c dimension r e s u l t s . T h i s reduced l e n g t h c a n be provided by a vane s t r u c t u r e ( s u r f a c e t e n s i o n d e v i c e ) . The d e v i c e can reduce t h e p r e s s u r e of l i q u i d a d j a c e n t t o and w i t h i n t h e d e v i c e t o a v a l u e lower t h a n t h e p r e s s u r e of l i q u i d l o c a t e d away from t h e d e v i c e . The low-pressure r e g i o n w i l l be c r e a t e d when t h e d e v i c e c a u s e s t h e c u r v a t u r e of t h e i n t e r f a c e about t h e d e v i c e t o be l a r g e ( s m a l l r a d i u s of c u r v a t u r e ) i n comparison t o t h e c u r v a t u r e of t h e l i q u i d e l s e w h e r e i n t h e tank. The p r e s s u r e d i f f e r e n c e w i l l have an e f f e c t o n l y i f t h e two l i q u i d volumes a r e i n communication. Under near-zero-g c o n d i t i o n s , s p r e a d i n g of t h e l i q u i d a s i t wets t h e tank w a l l s w i l l u s u a l l y b r i n g t h e l i q u i d i n t o communication w i t h t h e s u r f a c e t e n s i o n d e v i c e . I f t h i s i s not p o s s i b l e , some s o r t of communication channel must be provided. With a communication p a t h provided, l i q u i d w i l l be t r a n s f e r r e d , a s shown i n F i g u r e 2, u n t i l t h e c u r v a t u r e o f t h e i n t e r f a c e throughout t h e t a n k i s t h e same, i . e . , p r e s s u r e i s uniform. The s u r f a c e t e n s i o n d e v i c e i s designed s o t h e c u r v a t u r e of t h e i n t e r f a c e remains h i g h u n t i l t h e d e v i c e h a s f i l l e d w i t h l i q u i d . I n comparison, l i q u i d i n c o n t a c t w i t h o n l y t h e tank w a l l h a s a r e l a t i v e l y low c u r v a t u r e . For a s p h e r i c a l g a s - l i q u i d i n t e r f a c e w i t h l i q u i d i n c o n t a c t w i t h a s u r f a c e , e q u a t i o n ( 2 ) becomes

T h i s p r e s s u r e d i f f e r e n t i a l can be r e l a t e d t o a dimension ( o t h e r t h a n t h e r a d i u s of c u r v a t u r e ) such a s t h e p o r e r a d i u s R and a second parameter, t h e l i q u i d - t o - s o l i d c o n t a c t a n g l e 63. T h i s i s done by i n t r o d u c i n g t h e r e l a t i o n s h i p between R , 0, and r a s shown i n F i g u r e 3 . Then

A f i n a l c r i t e r i o n f o r d e t e r m i n i n g i n t e r f a c e s t a b i l i t y i s t h e Bond number (Bo), a d i m e n s i o n l e s s r a t i o o f a c c e l e r a t i o n f o r c e s t o c a p i l l a r y f o r c e s8

where L i s t h e c h a r a c t e r i s t i c system dimension. The l i q u i d l g a s i n t e r f a c e i s s t a b l e i n a The c r i t i c a l Bo f o r s q u a r e weave s c r e e n c y l i n d r i c a l t a n k o r c i r c u l a r pore when B O G 0.84. i s 0 . 4 5 ~ . S u r f a c e t e n s i o n f o r c e s become s i g n i f i c a n t , producing h i g h l y curved i n t e r f a c e s , f o r Bo i n t h e range of f i v e o r below. The i n t e r f a c e i s e s s e n t i a l l y f l a t f o r Bo 2 509. O t h e r s c a l i n g p a r a m e t e r s , such a s t h e Weber, Froude, and Reynolds numbers, a r e a p p l i e d when l i q u i d flow i s involved8. LOW-G FLUID MECKANICS The above d e s c r i b e d c h a r a c t e r i s t i c s o f . f l u i d b e h a v i o r i n a low-g environment have been demonstrated i n space and i n s i m u l a t e d low-g environments on e a r t h . These experiments i l l u s t r a t e t h e b a s i c phenomena of low-g f l u i d mechanics. A number of f l u i d mechanics s c i e n c e d e m o n s t r a t i o n s were performed on Skylab. While t h e s e d e m o n s t r a t i o n s d i d n o t f o l l o w r i g o r o u s e x p e r i m e n t a l p r o t o c o l s , t h e y d i d p r o v i d e i n t e r e s t i n g d e m o n s t r a t i o n s of b a s i c phenomena, some of which had n o t been p r e v i o u s l y observed6. Skylab F l u i d Mechanics Demonstrations The f o l l o w i n g a r e examples of f l u i d mechanics d e m o n s t r a t i o n s performed aboard Skylab t o i l l u s t r a t e low-g f l u i d b e h a v i o r . S t a t i c I n t e r f a c e Shape.- S u r f a c e t e n s i o n and c o n t a c t a n g l e work t o g e t h e r , a s d i s c u s s e d It was demonp r e v i o u s l y , t o y i e l d t h e s t a t i c shape of a g a s l l i q u i d i n t e r f a c e i n low-g. s t r a t e d t h a t a f r e e f l o a t i n g drop of w a t e r assumes a s p h e r i c a l shape, a s shown i n F i g u r e 4 . Due t o d i s t u r b a n c e s induced i n forming t h e drop and t h e r e l a t i v e a c c e l e r a t i o n of t h e Skyl a b and t h e d r o p , a completely s t a t i c i n t e r f a c e was d i f f i c u l t t o form. Before t h e l i g h t l y damped o s c i l l a t i o n s c e a s e d , t h e drop impacted a s u r f a c e . The a s t r o n a u t s found t h a t t h e s e d i f f i c u l t i e s could be overcome by p l a c i n g t h e drop on a t h r e a d . For t h e drop s i z e s used (30 t o 100 c c ) , t h e t h r e a d r e t a i n e d and c e n t e r e d t h e drop. An e v a l u a t i o n of t h e d a t a showed t h a t t h e t h r e a d had a s i g n i f i c a n t e f f e c t on t h e 5% e l o n g a damping o f t h e drop o s c i l l a t i o n s and caused a d i s t o r t i o n of t h e drop shape ( t i o n a l o n g t h e t h r e a d a x i s ) 6.

-

When t h e drop was i n c o n t a c t w i t h a l a r g e r s u r f a c e l i k e a s t r a w , t h e s u r f a c e i n f l u e n c e was s t r o n g e r and t h e e q u i l i b r i u m i n t e r f a c e shape p o s i t i o n e d t h e drop t a n g e n t t o s u r f a c e . On a l a r g e f l a t s u r f a c e , t h e c o n t a c t a n g l e became a s i g n i f i c a n t f a c t o r i n e s t a b l i s h i n g t h e i n t e r f a c e s h a p e , a s i l l u s t r a t e d i n F i g u r e 5. Water was t h e l i q u i d f o r a l l t h e s e demonstrat i o n s , g i v i n g c o n t a c t a n g l e s r a n g i n g from 30 t o 90 d e g r e e s , depending on t h e s u r f a c e mater i a l . A l i q u i d w i t h a lower s u r f a c e t e n s i o n would wet t h e s e s u r f a c e s and g i v e somewhat d i f f e r e n t i n t e r f a c e shapes6.

.-

Drops were o s c i l l a t e d i n t h e i r b a s i c f i r s t and second modes. O p p o s i t e s i d e s of Surface tension is t h e restoring force t h a t sustains t h e o s c i l l a t i o n . t h e d r o p were p u l l e d by r o d s t o i n d u c e o s c i l l a t i o n , A t h r e a d was a g a i n used t o i n i t i a l l y s t a b i l i z e t h e drop. Measured o s c i l l a t i o n f r e q u e n c i e s w e r e found t o c o r r e l a t e w e l l w i t h t h e o r y . Damping of t h e o s c i l l a t i o n was found t o b e a t l e a s t a n o r d e r of m a g n i t u d e g r e a t e r t h a n p r e d i c t e d by t h e o r y d u e t o t h e p r e s e n c e of h i g h e r modes of o s c i l l a t i n n (a r e s u l t of t h e method of i n d u c i n g o s c i l l a t i o n ) and t h e damping e f f e c t of t h e t h r e a d 6 . C o a l e s c e n c e . - C o a l e s c e n c e depends upon t h e i n t e r a c t i o n of two l i q u i d i n t e r f a c e s when they meet. Depending upon t h e a n g l e of i n c i d e n c e and r e l a t i v e v e l o c i t i e s , t h e d r o p s c a n bounce o f f one a n o t h e r o r combine. Momentum e f f e c t s c a n c a u s e them t o s e p a r a t e a g a i n . Drops were impacted by h o l d i n g one d r o p s t a t i o n a r y on a t h r e a d and maneuvering t h e second d r o p o n t o a c o l l i s i o n c o u r s e . Drops r a n g i n g from 1.8- t o 5.2-cm d i a m e t e r c o u l d b e o b s e r v e d a s they c o a l e s c e d . The d r o p s w e r e c o l o r e d d i f f e r e n t l y s o t h e r a t e of m i x i n g (found t o b e f a i r l y slow) c o u l d b e o b s e r v e d . The c o a l e s c e n c e observed i n Skylab was cons i st e n t w i t h a v a i l a b l e theory6. R o t a t i n g Drop.- When a l i q u i d d r o p i s r o t a t e d , c e n t r i f u g a l and s u r f a c e t e n s i o n f o r c e s b a l a n c e t o p r o d u c e t h e r e s u l t i n g i n t e r f a c e shape. T h i s was t h e most u n i q u e d e m o n s t r a t i o n performed on Skylab. Such a t e s t was n o t p r e v i o u s l y f e a s i b l e b e c a u s e of t h e l o n g low-g p e r i o d r e q u i r e d and t h e need t o m a n i p u l a t e t h e d r o p . A v a i l a b l e t h e o r y p r e d i c t s a comp l e t e l y d i f f e r e n t r e s u l t , b u t d o e s h i n t t h a t o t h e r r e s u l t s may b e p o s s i b l e . When r o t a t e d a t low r a t e s , t h e d r o p h a s a watermelon shape. A t h i g h e r rates, i t p i n c h e s o f f , assuming a p e a n u t s h a p e , a s shown i n F i g u r e 6. I f t h e r a t e i s i n c r e a s e d f u r t h e r , a n e q u i l i b r i u m s h a p e c a n n o t b e a c h i e v e d and t h e d r o p d i v i d e s i n t o two d r o p s 6 . I m m i s c i b l e L i q u i d s . - A d i s p e r s i o n of two i m m i s c i b l e l i q u i d s c a n b e formed i f t h e y a r e s t r o n g l y mixed. I f t h e d e n s i t i e s of t h e two l i q u i d s a r e d i f f e r e n t , t h e d i s p e r s i o n w i l l q u i c k l y s e p a r a t e i n one-g. When g r a v i t y f o r c e s a r e s m a l l , t h e mechanism f o r s e p a r a t i o n of a d i s p e r s i o n i s v e r y d i f f e r e n t . One l i q u i d c a n s e p a r a t e from t h e o t h e r o n l y by c o a l e s c e n c e of t h e f i n e l y d i v i d e d d r o p s . I f d r o p s of o n e l i q u i d d o come i n t o c o n t a c t and do c o a l e s c e , s e p a r a t i o n c a n proceed a t some slow r a t e . An e x p e r i m e n t u s i n g v a r i o u s p r o p o r t i o n s of o i l and w a t e r was performed t o examine t h i s phenomena. The two l i q u i d s w e r e s e p a r a t e d c e n t r i f u g a l l y and t h e n mixed by s h a k i n g . They w e r e o b s e r v e d f o r a p e r i o d of 1 0 h o u r s t o s e e i f any s e p a r a t i o n c o u l d b e o b s e r v e d . Only a " c e l l u l a r s t r u c t u r e t h a t grew c o a r s e " c o u l d b e observed6. On E a r t h , t h e two l i q u i d s completely s e p a r a t e i n l e s s than 10 secondslO. I c e M e l t i n g . - T h i s d e m o n s t r a t i o n p r o v i d e d a n i n d i c a t i o n of t h e i n f l u e n c e of a low-g environment on t h e mechanisms of h e a t t r a n s f e r 6 . I n s t e a d of d r a i n i n g away a s i t d o e s i n It one-g, t h e l i q u i d s u r r o u n d e d t h e i c e , a c t i n g t o i n s u l a t e i t from t h e s u r r o u n d i n g a i r . t o o k 190 m i n u t e s f o r t h e i c e t o c o m p l e t e l y m e l t t o a l i q u i d d r o p i n low-g. The i c e m e l t e d i n 130 m i n u t e s i n t h e same e x p e r i m e n t on earth1'. S i n c e c o n v e c t i o n i s t h o u g h t of a s b e i n g d r i v e n by buoyant f o r c e s , c o n d u c t i o n and r a d i a t i o n h e a t t r a n s f e r a r e u s u a l l y presumed t o b e t h e means o f h e a t t r a n s f e r i n low-g. While n o t e s t a b l i s h e d from t h i s e x p e r i m e n t , c o n v e c t i o n c o u l d s t i l l b e a mechanism of h e a t t r a n s f e r i n low-g. C o n v e c t i o n c a n b e d r i v e n by s u r f a c e t e n s i o n f o r c e s (Marangoni f l o w ) s i n c e g r a d i e n t s i n temperature along an i n t e r f a c e a l s o produce g r a d i e n t s i n s u r f a c e t e n s i o n l 2 . Thermoacoustic e f f e c t s , m e c h a n i c a l v i b r a t i o n s , e l e c t r i c and m a g n e t i c f i e l d s , c o n c e n t r a t i o n g r a d i e n t s and c h e m i c a l p o t e n t i a l s a r e a l s o p o s s i b l e mechanisms f o r c o n v e c t i v e h e a t t r a n s f e r i n 10w-~l3.

C a v i t a t i o n . - A bubble w i t h i n a l i q u i d can o s c i l l a t e i n much t h e same manner a s a l i q u i d drop o s c i l l a t e s . Both hydrodynamic and s u r f a c e t e n s i o n f o r c e s a c t a t t h e s u r f a c e I f t h e magnitude of t h e hydrodynamic f o r c e s exceeds t h e s u r f a c e t e n s i o n of t h e bubble. f o r c e s a t some p o i n t on t h e s u r f a c e , t h e bubble can become u n s t a b l e and c o l l a p s e upon i t s e l f . Each time an u n s t a b l e d r o p c o l l a p s e s a j e t of l i q u i d forms t h a t s h o o t s a c r o s s t h e bubble. T h i s phenomena was demonstrated on Skylab i n t h e f o l l o w i n g manner. A bubble was formed i n s i d e a d r o p such t h a t only a t h i n f i l m s e p a r a t e d them a t one a r e a on t h e s u r f a c e . When t h e s u r f a c e was touched w i t h a p l u n g e r , t h e bubble r u p t u r e d . While t h e s o u r c e of t h e i n s t a b i l i t y of t h e bubble was n o t a hydrodynamic f o r c e , t h e r e s u l t i n g c o l l a p s e was t h e same a s c a v i t a t i o n . A j e t of l i q u i d s h o t o u t of t h e d r o p a t t h e p o i n t t h e bubble had been rupt u r e d , a s shown i n F i g u r e 7. As t h e v e l o c i t y of t h e j e t d e c r e a s e d , s u r f a c e t e n s i o n f o r c e s a c t e d t o r e t r a c t p a r t of t h e j e t back i n t o t h e d r o p , w h i l e some of t h e j e t pinched o f f i n t o a d d i t i o n a l drops6. Drop Tower T e s t i n g Drop tower t e s t f a c i l i t i e s have been e x t e n s i v e l y employed t o i n v e s t i g a t e some of t h e b a s i c low-g f l u i d b e h a v i o r , Although l i m i t e d by r e l a t i v e l y s h o r t t e s t t i m e s , on t h e o r d e r of 2 t o 5 s e c , they do p r o v i d e a q u i t e a c c u r a t e l y c o n t r o l l e d a c c e l e r a t i o n environment Examples of i n t e r f a c e shapes w i t h i n con( a c c e l e r a t i o n r a n g e between 10-5 and 10-I g)14. t a i n e r s and l i q u i d motion r e s u l t i n g i n r e o r i e n t a t i o n w i t h i n a c o n t a i n e r a r e p r e s e n t e d b e low. C a p i l l a r y Pumping and I n t e r f a c e Shapes.- L i q u i d s a r e u s u a l l y s t o r e d i n a c o n t a i n e r and t h e shape assumed by t h e l i q u i d / v a p o r i n t e r f a c e i s i m p o r t a n t t o t h e d r a i n i n g o r f i l l i n g of t h e c o n t a i n e r i n low-g. The t a n k shape and any i n t e r n a l s t r u c t u r e s i n f l u e n c e t h e i n t e r f a c e shape. D i f f e r e n c e s i n c a p i l l a r y p r e s s u r e w i l l c a u s e l i q u i d t o b e t r a n s f e r r e d from a r e g i o n of low c u r v a t u r e t o a r e g i o n of h i g h e r c u r v a t u r e , a s d i s c u s s e d p r e v i o u s l y . T h i s c a p i l l a r y pumping e s t a b l i s h e s t h e e q u i l i b r i u m i n t e r f a c e s h a p e s . I n t e r f a c e s h a p e s and pumping r a t e s h a v e been e s t a b l i s h e d f o r numerous geometries7. As a n example, o r i e n t a t i o n of s m a l l l i q u i d volumes by v a r i o u s vane c o n f i g u r a t i o n s i s shown i n F i g u r e 8. O r i e n t a t i o n of t h e l i q u i d i n a b a r e t a n k i s shown i n t h e upper l e f t hand c o r n e r . L i q u i d p o s i t i o n i n g w i t h each of t h e f i v e d i f f e r e n t vane d e v i c e s , however, i s such t h a t g a s - f r e e l i q u i d could be s u p p l i e d t o a n o u t l e t located a t the 6 o'clock position. L i q u i d Motion.- D i s t u r b i n g f o r c e s a c f i n g on a c o n t a i n e r c a n c a u s e t h e l i q u i d w i t h i n t o r e o r i e n t . A s m a l l l a t e r a l a c c e l e r a t i o n component w i l l make t h e l i q u i d flow a l o n g one s i d e of t h e t a n k a s i t r e o r i e n t s . I n a t a n k w i t h a smooth i n t e r i o r w a l l t h e flow a d h e r e s t o t h e t a n k w a l l c o n t i n u i n g p a s t i t s f i n a l e q u i l i b r i u m p o s i t i o n . A t y p i c a l example i s shown i n F i g u r e 915. A s t h e l i q u i d began t o move, t h e l i q u i d i n t e r f a c e remained r e l a t i v e l y f l a t s o t h e motion appeared a s a r o t a t i o n of t h e i n t e r f a c e a b o u t i t s c e n t e r . Very l i t t l e s p l a s h i n g of t h e l i q u i d o c c u r r e d d u r i n g t h e r e o r i e n t a t i o n . Once t h e l e a d i n g edge of t h e flow reached t h e t a n k dome, t h e l i q u i d i n t e r f a c e began t o a c q u i r e some c u r v a t u r e . The l i q u i d o v e r s h o t i t s f i n a l e q u i l i b r i u m p o s i t i o n , c o n t i n u i n g around t h e tank and r e c i r c u l a t i n g some of t h e l i q u i d . B a f f l e s o r s u r f a c e t e n s i o n d e v i c e s t r u c t u r e w i l l breakup t h i s B a f f l e s do i n d u c e r e c i r c u l a t i o n and speed t h e achievement of i t s new s t a t i c p o s i t i o n . t u r b u l e n c e and produce g a s b u b b l e s w i t h i n t h e b u l k l i q u i d however. STORAGE AND TRANSFER O p e r a t i o n of p a s s i v e d e v i c e s f o r t h e s t o r a g e and t r a n s f e r of l i q u i d i n low-g i l l u s t r a t e s a p r a c t i c a l a p p l i c a t i o n of t h e p r e v i o u s l y described, s u r f a c e t e n s i o n dominated, b e h a v i o r . Devices a r e a v a i l a b l e t o h a n d l e a wide range of f l u i d s such a s a l c o h o l s , f r e o n s , p r o p e l l a n t s and cryogens16,17. C o n f i g u r a t i o n s d i f f e r because of v a r i e d f u n c t i o n a l r e q u i r e m e n t s ; however, t h e o p e r a t i o n a l p r i n c i p l e f o r each system r e l i e s on t h e r e l a t i v e l y s m a l l

p r e s s u r e d i f f e r e n t i a l t h a t e x i s t s a c r o s s any curved g a s l l i q u i d i n t e r f a c e due t o i n t e r m o l e c u l a r f o r c e s . Liquid s u r f a c e t e n s i o n and u l l a g e p r e s s u r e s u p p o r t a r e used t o p a s s i v e l y p r o v i d e n e a r - i n s t a n t a n e o u s , g a s - f r e e l i q u i d e x p u l s i o n on demand. I n g e n e r a l t h e s u r f a c e t e n s i o n d e v i c e s a r e d i v i d e d i n t o two c a t e g o r i e s : d e v i c e s t h a t use fine-mesh s c r e e n f o r l i q u i d o r i e n t a t i o n and c o n t r o l and t h o s e t h a t use s h e e t o r vanetype s t r u c t u r e s . The c h a r a c t e r i s t i c dimension, p o r e s i z e , i s t h e s i g n i f i c a n t parameter t h a t d i f f e r e n t i a t e s between t h e two c a t e g o r i e s . The vane d e v i c e s w i t h l a r g e r c h a r a c t e r i s t i c p o r e dimensions o p e r a t e only i n low a c c e l e r a t i o n environments on t h e o r d e r of 1 0 " g~ o r l e s s , depending on t h e s i z e of t h e t a n k o r c o n t a i n e r . Fine-mesh s c r e e n d e v i c e s , by v i r t u e o f very s m a l l p o r e s i z e s and s m a l l r a d i i of c u r v a t u r e , can p r o v i d e r e t e n t i o n and c o n t r o l of l a r g e l i q u i d masses o v e r a wide range of s p a c e c r a f t a c c e l e r a t i o n s . The s m a l l c a p i l l a r y p r e s s u r e d i f f e r e n c e s t h a t e x i s t s a t t h e s c r e e n p o r e s must b a l a n c e o r exceed t h e sum of o t h e r p r e s s u r e d i f f e r e n c e s t e n d i n g t o breakdown t h e p a s s i v e l y - c o n t r o l l e d l i q u i d l g a s i n t e r f a c e . Premature i n t e r f a c e breakdown reduces t h e q u a n t i t y of g a s - f r e e l i q u i d t h a t can be e x p e l l e d from t h e tank. During s t o r a g e w i t h no l i q u i d outflow t h e c a p i l l a r y p r e s s u r e d i f f e r e n c e must exceed t h e h y d r o s t a t i c head supported by t h e s c r e e n . A d d i t i o n a l l o s s e s which must a l s o be balanced by t h e c a p i l l a r y p r e s s u r e d i f f e r e n c e a r e i n t r o d u c e d w i t h a flowing system. For a p p l i c a t i o n s r e q u i r i n g a s c r e e n system, t h e r e a r e two c a t e g o r i e s of d e v i c e s : t o t a l communication systems and p a r t i a l r e t e n t i o n o r t r a p systems18. Trap d e v i c e s a r e s c r e e n r e s e r v o i r s which p o s i t i o n only a s m a l l p e r c e n t a g e of t h e t o t a l l i q u i d l o a d o v e r the outlet. T o t a l communication d e v i c e s a r e composed of s c r e e n l i n e r s o r i n d i v i d u a l l i q u i d supply c h a n n e l s which a r e i n c o n t i n u o u s communication w i t h t h e bulk l i q u i d . An example o f a t o t a l communication channel d e v i c e i s shown i n F i g u r e 10. I f t h e t a n k i s l a r g e , on t h e o r d e r of 3 f e e t and g r e a t e r , o r t h e a c c e l e r a t i o n i s r e l a t i v e l y l a r g e , t h e t a n k can be compartmented s o t h a t t h e h y d r o s t a t i c head t o which any segment of t h e d e v i c e i s exposed i s reduced. A schematic of such a d e v i c e i s shown i n F i g u r e 11. An example of a d e v i c e which u s e s vanes t o o r i e n t p r o p e l l a n t s f o r g a s - f r e e l i q u i d e x p u l s i o n i n a v e r low-g environment i s provided by M a r t i n M a r i e t t a Viking 1975 O r b i t e r p r o p e l l a n t tankage1 9 The s u r f a c e t e n s i o n p r o p e l l a n t management d e v i c e (PMD) o r i e n t s t h e l i q u i d o v e r t h e t a n k o u t l e t s o t h a t a b u b b l e - f r e e supply of p r o p e l l a n t i s a v a i l a b l e t o t h e r o c k e t e n g i n e s . Ullage c o n t r o l i s provided by t h e PMD f o r l i q u i d - f r e e gas v e n t i n g o f t h e t a n k d u r i n g a l l maneuvers of t h e v e h i c l e f o l l o w i n g s e p a r a t i o n from t h e C e n t a u r upper s t a g e . During s p a c e c r a f t c r u i s e and immediately b e f o r e e n g i n e b u r n s , t h e PMD o r i e n t s t h e p r o p e l l a n t s y m m e t r i c a l l y about t h e t a n k c e n t e r l i n e t o enhance s p a c e c r a f t p o i n t i n g a c c u r a c y d u r i n g engine f i r i n g s .

.

A schematic of t h e p r o p e l l a n t management d e v i c e i s shown i n F i g u r e 12. The primary elements of t h e PMD were t h e vane assembly, t h e communication channel assembly, and t h e mounting cap assembly. 'Ihe vane assembly, shown i n F i g u r e 13, c o n s i s t e d of a h o l l o w c e n t r a l t u b e o r s t a n d p i p e t o which twelve 6A1-4V t i t a n i u m s h e e t vanes were a t t a c h e d . A communication c h a n n e l was p o s i t i o n e d a l o n g t h e w a l l t o p r o v i d e f o r t h e c a p i l l a r y pumping of t h e p r o p e l l a n t s from t h e t o p t o t h e bottom of t h e t a n k d u r i n g low-g. C l e a n i n g of t h e PMD and t h e i n s i d e of t h e tank was important t o a s s u r e n e a r - z e r o c o n t a c t a n g l e s between t h e p r o p e l l a n t s and m e t a l l i c s u r f a c e s . The e f f i c i e n t t r a n s f e r of l i q u i d from one t a n k t o a n o t h e r i n a w e i g h t l e s s environment i s p r i m a r i l y dependent on t h e i n i t i a l l i q u i d l v a p o r i n t e r f a c e shapes and t h e i r l o c a t i o n s i n both t h e supply and r e c e i v e r c o n t a i n e r s , and t h e f l o w r a t e of t h e t r a n s f e r . ~ i q u i d l v a p o r s e p a r a t i o n and c o n t r o l i s r e q u i r e d i n t h e supply c o n t a i n e r t o accomplish g a s - f r e e l i q u i d flow from t h e c o n t a i n e r . S e v e r a l k i n d s of d e v i c e s a r e a v a i l a b l e f o r accomplishing expuls i o n from t h e supply c o n t a i n e r i n c l u d i n g t h e p r e v i o u s l y d i s c u s s e d p a s s i v e s u r f a c e t e n s i o n d e v i c e s , p o s i t i v e e x p u l s i o n d e v i c e s such as b l a d d e r s and m e t a l diaphragms, and bellows.

The p a r t i c u l a r d e v i c e s e l e c t e d depends upon f a c t o r s such a s f l u i d c o m p a t i b i l i t y , c y c l e l i f e , expulsion efficiency, cost. etc. P r e s s u r e c o n t r o l i n t h e r e c e i v e r t a n k d u r i n g f i l l i n g i s a key d e s i g n c o n s i d e r a t i o n . When s m a l l q u a n t i t i e s of l i q u i d and s m a l l c o n t a i n e r s a r e b e i n g u s e d , e v a c u a t i n g t h e c o n t a i n e r t o a c h i e v e t r a n s f e r i s a r a t h e r s i m p l e a p p r o a c h . On a l a r g e r s c a l e , s t r u c t u r a l c o n s i d e r a t i o n s a n d / o r v a p o r i z a t i o n o f t h e l i q u i d u s u a l l y r u l e o u t t h i s method o f t r a n s f e r . F o r a h i g h v a p o r p r e s s u r e f l u i d o r a c r y o g e n t h e r e c e i v e r c o n t a i n e r must be v e n t e d d u r i n g t h e t r a n s f e r , and t h e l o c a t i o n of t h e g a s must b e c a r e f u l l y c o n t r o l l e d . An example of e v a c u a t i n g one c o n t a i n e r t o a c h i e v e t r a n s f e r i n a low-g environment was d e m o n s t r a t e d d u r i n g t h e S k y l a b m i s s i o n s 6 . A s a m p l e o f blood was drawn i n a l a r g e s y r i n g e and t r a n s f e r r e d t o a n e v a c u a t e d b o t t l e . The p r e s s u r e w i t h i n t h e b o t t l e was s e l e c t e d s o t h a t t h e a d d i t i o n o f a g i v e n volume o f blood r e d u c e d t h e p r e s s u r e d i f f e r e n t i a l between t h e s y r i n g e and b o t t l e t o z e r o . The n e e d l e of t h e s y r i n g e was i n s e r t e d i n t o t h e b o t t l e , p i e r c i n g a diaphragm. I t e x t e n d e d p a r t way i n t o t h e b o t t l e . Blood i m m e d i a t e l y began t o t r a n s f e r from t h e s y r i n g e t o t h e b o t t l e , s i n c e t h e b o t t l e was a t a p r e s s u r e somewhat below a m b i e n t . A s t h e f l o w o f l i q u i d b e g a n , a d r o p c o u l d be o b s e r v e d f o r m i n g a t t h e t i p of t h e s y r i n g e . No t u r b u l e n c e o r g e y s e r i n g o f t h e blood was o b s e r v e d . The d r o p c o n t i n u e d t o expand u n t i l i t c o n t a c t e d t h e w a l l o f t h e b o t t l e . A t t h i s p o i n t t h e r e was a volume o f l i q u i d , l o c a t e d n e a r one end o f t h e b o t t l e , d i v i d i n g t h e g a s i n t o two s e p a r a t e volumes. The i n t e r f a c e on e a c h s i d e o f t h e l i q u i d volume moved toward t h e e n d s o f t h e b o t t l e c o m p r e s s i n g t h e two g a s volumes a s t h e t r a n s f e r c o n t i n u e d . The volume o f g a s n e a r t h e d i a p h r a g m of t h e b o t t l e was t h e s m a l l e r o f t h e two. Each volume was a t t h e same p r e s s u r e w i t h t h e l i q u i d a c t i n g as a p i s t o n between them. Some p r e s s u r e was a p p l i e d t o t h e p l u n g e r o f t h e s y r i n g e t o a c h i e v e c o m p l e t e t r a n s f e r of t h e liquid. The t r a n s f e r of l i q u i d from one t a n k t o a second v e n t e d t a n k i n a w e i g h t l e s s e n v i r o n . s u r f a c e - t e n s i o n b a f f l e d e s i g n s were ment was d e m o n s t r a t e d by t h e crew of A p o l l o 1 4 ~ ~ Two i n c o r p o r a t e d i n s e p a r a t e t a n k s of a s c a l e - m o d e l l i q u i d - t r a n s f e r s y s t e m w i t h e a c h t a n k b e i n g used a l t e r n a t i v e l y as t h e supply and r e c i e v e r t a n k . A s k e t c h o f t h e t a n k s i s p r e s e n t e d i n F i g u r e 14. One t a n k c o n t a i n e d a s t a n d p i p e - l i n e r b a f f l e s t r u c t u r e . c o n s i s t i n g o f a p e r f o r a t e d s t a n d p i p e l o c a t e d o v e r t h e d r a i n l f i l l p o r t and a w a l l - l i n e r s p a c e d a f i x e d d i s t a n c e away from t h e t a n k w a l l . The second t a n k c o n t a i n e d a curved-web b a f f l e s t r u c t u r e c o n s i s t i n g o f t h r e e c i r c u l a r p e r f o r a t e d p l a t e s n e s t e d around a s m a l l f e e d e r c a p i l l a r y s e c tion. The c u r v e d web b a f f l e s are a r r a n g e d o f f - c e n t e r such t h a t t h e c r o s s - s e c t i o n a l a r e a between b a f f l e s i n c r e a s e s g r a d u a l l y from t h e f e e d e r s e c t i o n t o w a r d s t h e o p p o s i t e end of t h e tank. This arrangement tends t o r e t a i n t h e bulk l i q u i d a d j a c e n t t o t h e f e e d e r . T e s t i n g was performed t o d e t e r m i n e t h e a b i l i t y t o a c h i e v e g a s - f r e e o u t f l o w from t h e s u p p l y t a n k and o r d e r l y i n f l o w i n t o t h e r e c e i v e r t a n k w i t h g a s l o c a t e d a t t h e t a n k v e n t G a s - f r e e l i q u i d was t r a n s f e r r e d t o and from e i t h e r b a f f l e d and l i q u i d a t t h e f i l l p o r t . t a n k t o w i t h i n 2 p e r c e n t o f t h e l i q u i d a v a i l a b l e f o r t r a n s f e r and t h e r e c e i v e r t a n k v e n t remained i n c o n t a c t w i t h g a s . T r a n s f e r between u n b a f f l e d t a n k s was i n c l u d e d f o r comparis o n and g a s i n g e s t i o n o c c u r r e d when l e s s t h a n 1 2 p e r c e n t o f t h e s u p p l y t a n k volume had b e e n d e l i v e r e d . A t t h e t e r m i n a t i o n of t r a n s f e r l i q u i d had i n g e s t e d i n t o t h e r e c e i v e r t a n k v e n t . CONCLUDING REMARKS The p a r a m e t e r s t h a t g o v e r n f l u i d b e h a v i o r i n a w e i g h t l e s s e n v i r o n m e n t h a v e been p a r t i a l l y c h a r a c t e r i z e d and v e r i f i e d by ground and o r b i t a l t e s t i n g . Because t h e e n v i r o n m e n t , geometry, and f l u i d s of i n t e r e s t i n f l u e n c e t h i s b e h a v i o r and a l s o d i f f e r from s y s t e m t o s y s t e m , e a c h new a p p l i c a t i o n must be e v a l u a t e d t o a s s u r e t h a t t h e d e s i r e d p e r f o r m a n c e i s a c h i e v e d . E a r l y a s s e s s m e n t of f l u i d b e h a v i o r and c o n t r o l f o r each e x p e r i m e n t i s r e q u i r e d . One-g bench a n d / o r d r o p tower t e s t i n g c a n p r o v i d e v e r i f i c a t i o n p r i o r t o o r b i t a t o p e r a t i o n .

REFERENCES

1.

H. M. P r i n c e n , "The E q u i l i b r i u m Shape of I n t e r f a c e s , Drops, and Bubbles,'' S u r f a c e and C o l l o i d S c i e n c e , Vol. 2 , E. M a t i j e v i c , E d i t o r , John Wiley & Sons, New York, 1969.

2.

H. W . Fox and W . A.

3.

E . G. S h a f r i n and W. A. Zisman, "Upper L i m i t s t o t h e Contact Angles of L i q u i d s on S o l i d s , " C o n t a c t Angle, W e t t a b i l i t y and Adhesion, Advances i n Chemistry S e r i e s 4 3 , American Chemical S o c i e t y , Washington, D. C . , 1965, pp. 145-157.

4.

W a l t e r Drost-Hansen, "Aqueous I n t e r f a c e s . " P a r t s I and I1 i n Chemistry and P h y s i c s of I n t e r f a c e s , American Chemical S o c i e t y , Washington, D. C . , 1965.

5.

J . J . Bikerman, S u r f a c e Chemistry, Theory and A p p l i c a t i o n s , Academic P r e s s , I n c . ,

Zisman, J o u r n a l of C o l l o i d S c i e n c e s , Vol. 5 , 1950, p. 514.

New York, 1958. 6.

J. R. T e g a r t and J. R. Butz, "Analysis of Skylab F l u i d Mechanics S c i e n c e Demonstrat i o n s , " MCR-75-151-1, Martin M a r i e t t a C o r p o r a t i o n , Denver, Colorado, May 1975.

7.

J . R. T e g a r t and D. A. F e s t e r , "Space S t o r a b l e P r o p e l l a n t A c q u i s i t i o n System," J . S p a c e c r a f t , Vol. 12, No. 9, September 1975, pp. 544-551.

8.

H. L. P a y n t e r and T. R. B a r k s d a l e , " C r i t e r i a f o r P a s s i v e P r o p e l l a n t C o n t r o l Schemes. J . S p a c e c r a f t , Vol. 7, No. 6 , J u n e 1970, pp. 702-706.

9.

L. J . H a s t i n g s and R. R u t h e r f o r d , "Low G r a v i t y Liquid-Vapor I n t e r f a c e Shapes i n Axisymmetric C o n t a i n e r s and a Computer S o l u t i o n , " NASA TMX-53790, George C. M a r s h a l l Space F l i g h t C e n t e r , H u n t s v i l l e , Alabama, October 7, 1968.

10.

L. Lacy and H. O t t o , "The S t a b i l i t y of Liquid D i s p e r s i o n s i n Low-Gravity," A I A A Paper No. 74-1242, AIAAIAGU Conference on S c i e n t i f i c Experiments of Skylab, H u n t s v i l l e , Alabama, November 1974.

11.

G. H. O t t o and L. L. Lacy, "Observations of t h e ~ i q u i d l ~ o l iI n d t e r f a c e i n Low-Gravity Melting," AIAA Paper No. 74-1243, AIAAIAGU Conference on S c i e n t i f i c Experiments of Skylab, H u n t s v i l l e , Alabama, November 1974.

12.

J. L. McGrew, e t a l . , "The E f f e c t of Temperature Induced S u r f a c e Tension G r a d i e n t s on Bubble Mechanics , I 1 Applied S c i e n c e Research, Vol. 29, June 1974, pp 195-210.

13.

F. T. Dodge, e t a l . , " F l u i d P h y s i c s , Thermodynamics, and Heat T r a n s f e r Experiments i n Space: F i n a l Report of t h e Overstudy Cormnittee," NASA CR-134742, Southwest Research I n s t i t u t e , San Antonio, Texas, January 1975.

14.

H. L. P a y n t e r , "The M a r t i n Company's Low-g Experimental F a c i l i t y , " Proceedings of t h e Symposium on F l u i d Mechanics and Heat T r a n s f e r Under Low G r a v i t y , United S t a t e s A i r Force O f f i c e of S c i e n t i f i c Research and Lockheed M i s s i l e s and Space Company, June 1965, pp. 15-1 t o 15-17.

15.

J . R. T e g a r t , " P r o p e l l a n t R e o r i e n t a t i o n w i t h Of f - a x i s A c c e l e r a t i o n s , " AIAA Paper No. 75-1195, P r e s e n t e d a t t h e AIAA/SAE 1 1 t h P r o p u l s i o n Conference, Anaheim, C a l i f o r n i a , September 29-October 1, 1975.

16.

D. A. F e s t e r , e t a l . , " A c q u i s i t i o n / E x p u l s i o n System f o r E a r t h O r b i t a l P r o p u l s i o n System S t u d y , F i n a l R e p o r t , Volume V - E a r t h S t o r a b l e D e s i g n , " MCR-73-97 (Vol. V), M a r t i n M a r i e t t a C o r p o r a t i o n , Denver, C o l o r a d o , O c t o b e r 1973.

17.

G. R. P a g e , e t a l . , " k c q u i s i t i o n / E x p u l s i o n System f o r E a r t h O r b i t a l P r o p u l s i o n System S t u d y , F i n a l R e p o r t , Volume I11 - C r y o g e n i c T e s t s , " MCR-73-97 (Vol. 1 1 1 ) , M a r t i n M a r i e t t a C o r p o r a t i o n , Denver, C o l o r a d o , O c t o b e r 1973.

18.

D. A. F e s t e r , R. N. E b e r h a r d t , a n d J . R. T e g a r t , "Space S h u t t l e R e a c t i o n C o n t r o l Subs y s t e m P r o p e l l a n t A c q u i s i t i o n Technology," AIAA P a p e r No. 74-1106, P r e s e n t e d a t t h e AIAA/SAE 1 0 t h P r o p u l s i o n C o n f e r e n c e , San ~ i e ~ C o a,l i f o r n i a , O c t o b e r 21-22, 1974.

19.

C . D . Brown, e t a l . , "Viking O r b i t e r 1975 P r o p e l l a n t Management D e v i c e , " SE 010-47-01, M a r t i n M a r i e t t a C o r p o r a t i o n , Denver, C o l o r a d o , May 1973.

20.

K. L. A b d a l l a , e t a l . , " L i q u i d T r a n s f e r D e m o n s t r a t i o n on Board A p o l l o 1 4 D u r i n g T r a n s e a r t h C o a s t , " NASA TMX-2410, Lewis R e s e a r c h C e n t e r , C l e v e l a n d , O h i o , November 1971.

ORIGBsra PAGE IS OF POOR QUALITY

Figure 1.- 33.5' c o n t a c t angle of a drop on a contaminated s u r f a c e .

COMMUNICATION

OUTLET Figure 2.-

C a p i l l a r y pumping with a vane surface-tension device.

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Figure 10.- Total communication screen device.

Liquid Flow Annulus

Figure 11.- Schematic of a compartmentali z e d screen device.

Figure 12.- Schematic of t h e Viking o r b i t e r 1975 p r o p e l l a n t management device. Figure 13.- Photograph of t h e Viking o r b i k e r 1975 vane s t r u c t u r e .

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(a)

F i l l i n g of standpipe.

D i r e c t i o n of Flow

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Standpipe s e c t i o n n e a r l y empty.

14.- Apollo 14 f l u i d t r a n s f e r with b a f f l e d tanks ( r e f .

20).

-

SPACE PROCESSING ON SKYLAB AND A S P R , S. Snyder Marshall Space Flight Center

Marshall Space Flight Center, AL 35812

ABSTRACT The Skylab and Apollo-Soyuz Test Project (ASTP) missions provided the opportunity to examine the influence of micro-gravity on the processing of various materials. The majority of the experiments dealt with the solidification of alloys, semiconductors, and composite materials o r basic liquid-liquid and liquid- solid interactions necessary to understand complex processing. The space results have yielded basic data and increased knowledge of fundamental materials behavior. Potential advantages of space processing to several materials disciplines have been identified. INTRODUCTION In the following paragraphs the results of physical and engineering experiments in metallurgy, fluids handling and crystal growth on two space missions, Skylab and the Apollo- Soyuz Test Project (ASTP), will be discussed. The presentation is incorporated into a colloquium concerned with bioprocessing in space because there a r e similarities in process objectives in the two fields and the results may trigger some ideas in biological applications even though the metallurgists and solid state physicists were concerned with metals and semiconductors. Some of the properties explored in space should have utility in all scientific fields. For example, many disciplines a r e now expressing an interest in freely suspending their material and then performing manipulations o r measurments with no contamination o r uncontrolled disturbance. Homogeneous isotropic materials a s well a s ordered highly anisotropic material have been proposed for space investigation. The unique advantage of the space environment for modifying o r improving the properties of materials is the long term weightlessness. The extended weightlessness in space is created by the motion of the spacecraft under the influence of i t s high orbital velocity and the gravitational pull of earth. Objects located at the center of mass within the spacecraft a r e not accelerated by any additional outside force and they will "float". Objects displaced from this point will follow trajectories prescribed by well known equations of motion. These motions have been frequently photographed in space. Space is also characterized by a vast vacuum capacity and the possibility of attaining a vacuum significantly below the capability of any vacuum pump system on earth. The Skylab and ASTP experiments, however, were designed to utilize primarily the microgravity advantage of space.

PRECEDING PAGE BLANK NOT PII,

2

These two missions encompassed a wide variety of experiments intended to explore the capabilities of the space environment and the details of each of them cannot be given in this b ief overview paper. Several review articles of the experiments a r e already avail&le F in addition to the individual reports of the principal investigators.

A group of experiments that could be done in a common facility on Skylab, shown in Figure 1, were selected in 1969. An electron beam was incorporated to melt metals typically used in welding and to form freely floating metal spheres. The brazing of two tubes with an exothermic chemical reaction was proposed to investigate the capillary flow of the melt around the tube ends. The growth of a gallium arsenide single crystal in a solution of liquid gallium was designed to investigate an improved method to grow this important semiconductor material. All control functions, battery power and storage containers were included in this facility. By 1971, interest in space processing had developed considerably. The Apollo 14 mission had flown three simple demonstrations of high interest to the program: electrophoresis; melting and solidification of composite materials; and fluid behavior under various thermal conditions. These experiments, done during the return of the Apollo from the moon, showed both the advantages and some of the difficulties of processing materials in space. In 1972, an additional set of eleven experiments were proposed to use the furnace concept of the gallium arsenide experiment but modified for multiple purpose use. (Figure 2) These a r e listed in Table 1 to show the variety of different experiments that were planned for this versatile facility. As is shown, several experiments were done on Skylab 111 and repeated with additional samples on Skylab IV. Since the furnace processed three sample cartridges each time, a significant amount of material was returned to Earth for analysis. The "Vapor Growth of IV-VI Compounds" experiment investigated a technique for growing crystals of electronic materials by condensing vapor evolved from a heated source of the same material. Turbulence in the vapor due to the imposed temperature gradient limits the results on earth but swirling convection currents should not occur in weightlessness. The results of this experiment were unanticipated since up to ten times more crystalline material was produced in space. Dr. Wiedemeier proposes that this is primarily due to an inadequate theory used to determine growth on the ground a s well a s in space and the Skylab results will modify the fundamental understanding of this technology on Earth. Additionally, the structural perfection of the space grown crystals was clearly superior and the size of one crystal, shown in Figure 3, was six times larger than ever achieved. Two experiments with different semiconductor materials, germanium and indium antimonide, were done to give a direct comparison of ground and space characteristics. A single crystal, contained in a cylin'drical ampoule, was partially remelted and then solidified in space. The principal objective was to achieve improved homogeneity of a specific impurity by removing thermal convection at the solidification interface. The availability of precise electrical measurements systems and high resolution techniques to study segregation inhomogeneities on the order of fractions of a micrometer make semiconductor materials excellent candidates for space processing. Figure 4 shows a comparison of Earth and space-grown crystal of indium antimonide from Dr. Gato's experiment.

Dr. Hans Walter made crystals in a way that is impossible on Earth. A containerless melt was formed on the end of a seed crystal and then directionally solidified to form single crystals typified by Figure 5. The initial samples were cylinders of single crystal indium antimonide, one end fastened in a heat sink and the other in a cavity that was heated above the sample melting point. The cylinder was partially melted in space forming a sphericai melt in contact with the solid seed material. The temperature of the cavity was then slowly lowered. The melt solidified first near the seed and finally at the tip in contact with the inner wall of the cavity, Although the melt was surely spherical, the final oblong crystal shape was determined by the forces intrinsic to the solidification process itself. These crystals were all characterized by flat surface facets and decreased internal imperfections.

The measurement of diffusion of one particle species through another in liquids is difficult on Earth because gravity will act on any small difference in particle density to produce convection currents. Dr. Ukanwa designed an experiment to detect and measure any disturbances to pure diffusion caused by any source. In this experiment, cylindrical rods of zinc containing radioactive zinc-65 atoms confined to a zone at the end of the cylinder were melted and solidified in space. By slicing the cylinder on Earth, the diffusion of radioactive zinc atoms was measured, the results show agreement with theory, again confirming the absence of convection currents driven by a temperature gradient. APOLLO SOYUZ TEST PROJECT (ASTP) The success of the Skylab experiments led to the proposal of over 30 experiments that could be accommodated in the Skylab furnace with minimum modification. The ASTP timeline allowed only seven experiments to be done by utilizing almost the entire mission in the heat-up, soak at high temperature and controlled cool-down required to fulfill the experiment requirements. The experiments selected for ASTP a r e listed in Table 11. Three proposals were follow-on to Skylab experiments in which the same scientists wanted to obtain additional data. The USSR proposed an experiment to melt several different material combinations during the joint Russian American part of the mission but their analysis has not been completed. '

The ASTP experiment on "Surface Tension Induced Convection" further investigated the elimination of convection currents in liquids metals. The Skylab experiment on diffusion in zinc indicated that gravity-induced thermal convection is effectively eliminated in space processing. However, a s a result of this condition, it was hypothesized that convection effects caused by surface tension gradients may become significant in space processing, Since surface tension gradients occur due to thermal o r concentration differences which a r e not gravity dependent factors, the objective of this experiment was to determine if similar surface tension induced convection effects would result from concentration gradients. The experiment concept consisted of melting three samples of bi-metallic materials (lead and lead-gold alloy) in wetting and non-wetting capsules in the multipurpose furnace, allowing inter-diffusion of the two components, and then resolidification. If no convective stirring effects due to the surface tension difference between the materials exist, then a normal concentration - distance profile for the gold would result. The preliminary examination of the flight samples indicates that a normal concentration-distance profile of gold in the sample was obtained. Autoradiographs compare favorably with distance predictions based on the zinc self-diffusion data obtained on Skylab indicating that the diffusion of gold in lead was in the typical liquid diffusion range. "Monotectic and Syntectic Alloys", investigated the effects of weightlessness on the melting and solidification of two material systems, lead-zinc and aluminum antimonide. The objectives were to investigate phase segregation effects in low gravity for the 55

immiscible binary lead-zinc and to determine the influences of low-gravity solidification on the microstructural homogeneity and Stoichiometry of semiconducting compound aluminum antimonide. The large density difference between the two metals makes it difficult to avoid severe gravity separation of the phases during solidification of earthprepared systems. The comparison of the microstructure of the ground and space processed material, Figure 6, clearly shows an improvement of homogeneity obtained in space. The experiment, "Interface Marking in Crystals", was developed to study quantitatively the basic solidification behavior of high temperature melts under near zerogravity conditions and answer questions raised by the earlier experiment by Professor Gatos of MIT on Skylab. The experimental hardware on ASTP differed from the related Skylab experiment in that an electrical pulsing unit was utilized to provide interface demarcation during solidification from the melt, Evaluation of the demarcation lines which were clearly established throughout the samples show that the microscopic growth rate was subject to an initial transient which did not stabilize immediately. The observed r a t e behavior in space was comparable to the ground-based samples and at variance with the behavior predicted by theory. Segregation analysis again showed striking differences between one-gravity and zero- gravity conditions. ASTP experiment "Processing of Magnets" was conducted to determine the potential advantage of space processing to modify critical magnetic properties by improving chemical homogeneity, morphological and crystalline perfection of the magnetic substructure. Preliminary results now indicate that a magnetic alloy of manganese and bismuth resolidified directionally in space has attained a significantly higher coercive strength. The objectives of "Crystal Growth from the Vapor Phase" were to continue the Skylab investigation into the effects of micro-gravity on the structure of single crystals of mixed systems and to determine the mass transport rates of these systems using the chemical transport technique, The materials, germanium selenide and germanium telluride were chosen to be similar to Dr. Wiedemeier's Skylab experiment. The ASTP experiment gave close agreement of data which confirms predictions that the vapor transport technique is suitable for the growth of crystals of significantly better quality in a micro-gravity environment. Crystals of high structural uniformity were produced and the vapor transport rate in space has consistently been greater than predicted by earth models resulting in much larger single crystals than can be produced on earth by identical techniques. Experiment MA- 131, "Halide Eutectic Growth", was developed to investigate production in space of a eutectic mixture consisting of continuous fibers of LiF embedded in a NaCl matrix. When grown on earth, this material has not realized its full potential for optical transmission due to discontinuous fibers resulting from convection currents in the melt. Dr. Yue's related Skylab experiment demonstrated that continuous fibers of NaF embedded in a NaCl matrix could be grown in space by the directional solidification technique resulting in a material that exhibited improved optical properties in comparison to earth-grown samples. This technique was again successfully utilized in the ASTP experiment and improved optical transmission was obtained. Figure 7 shows a comparison of the optical transparency for the best Earth-grown material and the sample from space. Demonstrations of fluid banding in space and two electrophoresis experiments done on A S P a r e described elsewhere in this Colloquium. There a r e other bioprocessing

experiments that could be done advantageously in space and they could be related to the solidification experiments just discussed. The ease with which liquids can be immobilized and manipulated should have relevance to bioprocessing. Ideas for new experiments should be developed and proposed to NASA f o r the Spacelab missions of the 1980's.

REFERENCES: 1.

Proceedings of the Third Space Processing Symposium Space Flight Center, April 30 - May 1, 1974 Apollo- Soyuz T e s t Project February 1976

- Skylab Results, Marshall

- Preliminary Science Report, NASA TMX- 58173,

SKYLAB EXPERIMENTS

S KYLAB MISSION I1 111

MATERIALS PROCESSING FACILITY M551: METALS-MELTING EXPERIMENT, R. R. POORMAN, MSFC ASTRONAUTICS LAB.................. M552: EXOTHERMIC BRAZING EXPERIMENT, J. R. WILLIAMS, MSFC PRODUCT ENG. UB.............X M553: SPHeRE-FORMING EXPERIMENT, E. A. HASEMEYER, MSFC ENG. LAB............. M555: *GALLIUM ARSENIDE CRYSTAL-GROWTH EXPERIMENT, R. S . SEIDENSTICKER, WESTINGHOUSE RESEARCH UB........................................................

IV

X

...........X

MLT'ITIPURPOSE FURNACE SYSTEM M556: VAPOR GROWTH OF IV-VI COMPOUNDS, H. WIEDEMEIER, RENSSELAER POLYTECHNIC INSTITUTE................................................................ x M557: IMMISCIBLE ALLOY COMPOSITIONS, J. L. REGER, TRW SYSTEMS.. X M558: RADIOACTIVE-TRACER DIFFUSION, A. 0. UKANWA, MSFC SPACE SCIENCES LAB,............. X M559: MICROSECRATION I N GERMANIUM, F. A. PADOVANI, TEXAS INSTRUMENTS........... X ~ 5 6 0 : GROWTH OF SPHERICAL CRYSTALS, H. U. WALTER, UNIV. OF ALABAMA..................... X M561: WHISKER-REINFORCED COMPOSITES, T. KAWADA, NATIONAL INSTITUTE FOR METALS RESEARCH........................................................ X M562: INDIUM ANTIMONIDE CRYSTALS, H. C. GATOS, MIT..................................... X M563: MIXED 1 1 1 - V CRYSTAL GROWTH, W. R. WILCOX, USC.................................... . X M564: ALKALI HALIDE EUTECTICS, A.S. YUE, UCLA.......................................... X M565: SILVER GRIDS MELTED I N SPACE, A. DERUYTHERRE, KATHOLIEKE UNIV., X LEUVEN, BELGIUM..............................................o................... M566: COPPER-ALUMINUM EUTECTIC, E. A. HASEMEYER, MSFC PRODUCT ENG. LAB................. X

........................ ........

..........

3

EXPERIMENTS PERFORMED ON EACH MISSION:

* NOT

FLOWN BECAUSE STORAGE AREA PREEMPTED BY SKYLAB REPAIR KIT.

TABLE I

F':

11

X X

X

x X X

X

7

ASTP MULTIPURPOSE FURNACE EXPERIMENTS MA-010: MA-041: MA-044: MA-060: MA-070: MA-085: MA-131: MA-150:

MULTIPURPOSE FURNACE - J. McHUGH, WESTINGHOUSE. ................ SURFACE TENSION INDUCED CONVECTION - R. E. REED, OAK RIDGE.. .. MONOTECTIC AND SYNTECTIC ALLOYS - C. Y. ANG, USRA/MSFC.. ...... INTERFACE MARKING IN CRYSTALS - H. C. GATOS, MIT.. ............... PROCESSING O F MAGNETS - D. J. LARSON, GRUMMAN.. ................. CRYSTAL GROWTH FROM T H E VAPOR PHASE - H. WIEDEMEIER, RPI.. ... HALIDE EUTECTIC GROWTH - A. S. YUE, UCLA. ........................ MULTIPLE MATERIALS MELTING - I. IVANOV, USSR.. ...................

W 4 '3

TABLE I1

% r-i

h x m C 0

bD

.C

[I) [I)

.rl

HEAT HEAT

Figure 2.- ~ 5 1 8multipurpose electric furnace.

L n

rnw

1 ".

Figure 4.- Segment of the initial regrowth interface of tellurium-doped InSb crystal (B-1) ; dopant inhomogeneities are seen in the Earth-grown (upper) section; no dopant inhomogeneities are present in the space-grown (lower) section; 125X magnification.

Figure 5 .

- Space-grown

( c o n t a i n e r l e s s ) s i n g l e c r y s t a l o f semiconductor indium antimonide; Skylab experiment M560 ( s p e c i a l i l l u m i n a t i o n ) .

B

64

(a) Low g ( ~ 1 8 5 ) .

( b ) One g

Figure 6.-

(~141).

Comparison of ASTP and GBT-2 AlSb microstructure.

( a ) Space-grown.

Figure 7.- Optical transparency of samples from h a l i d e e u t e c t i c s experiment ~564.

THREE MODEL SPACE EXPERIMENTS ON CHEMICAL REACTIONS By Philomena Grodzka, Lockheed Missiles and Space Go. , P . 0. Box 1103, Huntsville, Alabama 35807, and Barbara Facemire, NASA- Marshall Space Flight Center, Alabama 35812 ABSTRACT Three simple science demonstrations conducted aboard Skylab IV and Apollo-Soyuz involved phenomena that a r e of interest to the biochemistry community. The three experiments a r e identified h e r e a s the Formaldehyde Clock Reaction, the Equilibrium Shlft Reaction, and the Electrodeposition Reaction. The Formaldehyde Clock Reaction and the Equilibrium Shift Reaction experiments conducted aboard Apollo- S oyuz demonstrated the effect of low-g foams o r air/liquid dispersions on reaction r a t e and chemical equilibrium. The Electrodeposition Reaction experiment conducted aboard Skylab IV demonstrated the effect of a low-g environment on an electrochemical displacement reaction. In a formaldehyde clock reaction, a number of chemical reactions occur simultaneously and at such r a t e s that the end of the reaction, signaled by a change of color from colorless to r e d , does not occur until about 20 seconds after the reactant solutions have been mixed. A clock reaction embodies some of the features of periodic chemical reactions which a r e of great interest at present because they suggest a relevancy to mechanisms controlling biological rhythms. In exploratory ground experiments, the purpose of which was to identify a good space demonstration experiment, it was discovered that a formaldehyde clock reaction displays effects that can be attributed to Gibbs (or van d e r Waal) adsorption of polymeric formaldehyde solution species. Also discovered were internal effects that a r e caused either by internal s h e a r a s the result of residual fluid flow o r by formation of three- dimensional formaldehyde species networks. The various noted behaviors of the Formaldehyde Clock Reaction in ground tests and in the low-g t e s t s a r e described. It i s concluded that the unique behaviors observed in low-g a r e the result of the presence of many more small a i r bubbles than were present in the one-g cases. In the Chemical Shift Reaction a reversible chemical equilibrium i s caused to shift by means of foam formation. Evidence of the chemical shift i s given by the color of the foam (pink) which i s different from the color of the bulk solution (amber brown). In low-g the pink foam was many times more stable than under one-g conditions. In the Skylab Electrodeposition Experiment, a chemical displacement reaction caused silver crystals to be deposited. The silver crystals obtained in low-g were quite dlfferent than those obtained in one-g because of the differing convection c u r r e n t s generated in the two situations. This experiment i s not discussed in detail. Only the implications of the conclusions f o r biochemical type reactions a r e considered. The implications of the three space experiments f o r various applications a r e considered. THE FORMALDEHYDE CLOCK REACTION It i s well known that the rates of many, if not most, chemical reactions a r e heavily dependent on the concentrations of the reacting species. Thus, if reacting chemical species a r e not uniformly distributed throughout the solution, a reaction can occur f a s t e r in one p a r t of the solution than in another, At constant temperarure and p r e s s u r e , a nonuniform distribution of solute species in a well mixed solution can occur a s the result either of adsorption at a liquid/gas o r a liquid/solid interface o r of a hydrostatic p r e s s u r e 67

effect on chemical potential. The f i r s t of these effects i s the one of interest h e r e . F o r the sake of clarity, it may be well to note that the type of adsorption we a r e considering h e r e i s Gibbs adsorption, i. e . , a solution becomes more o r l e s s concentrated (positive and negative adsorption) in solute species in the liquid- gas o r liquid/solid interface zones but no change in phase occurs nor i s there any chemical reaction between solute species and species in the gas o r solid phases. It i s generally well known that Gibbs adsorption can cause the surface tension of a solvent to be either increased o r decreased when solute i s added. The effect of Gibbs adsorption on r a t e s of chemical reaction, however, has not, to the best of our knowledge, been directly demonstrated. A number of previous investigators have conducted chemical reaction experiments, however, in which either insoluble monomolecular films were involved o r chemisorption had occurred. In some cases where Gibbs adsorption was undoubtedly involved, the evidence was indirect. These various cases a s well a s some p r i o r , reported speculations a r e briefly reviewed in the following paragraphs.

E. X. Rideal was a very active investigator in this a r e a and published a number of works on chemical reactions involving monolayers of one reactant (Refs. 1-4). Among some of the systems investigated by Rideal and others a r e (Refs. 1-9): Type Reaction oxidation hydrolysis hydrolysis hydrolysis photolysis

polymerization chemical reaction chemical reaction chemical reaction

lactonization complex formation

Monolayer Film

Bulk Reactant

oleic acid ethyl butyrate ethyl palmitate lecithin stearanilide benzyls tearylamine 6 -phenylethylstearyl- amine proteins barium stearate- stearic acid maleic anhydride compound of eleostearin amines and aldehydes egg albumin caseinogen sterol carcinogenic hydrocarbons and sterols y-hydroxystearic acid s t e a r i c acid

permanganat e pancreatin enzyme aqueous alkali snake venom enzymes

trypsin enzyme sodium c etyl sulfate

heavy metal ions

The r a t e s of chemical reaction were shown to be strongly affected by species adsorption at charcoal interfaces in the hydrolysis of bromoethylamine and in the conversion of dimethyleneimine hydrobromide into bromoethylamine in hydrobromic acid solution (Ref. 10). Foaming was found to promote the lipolysis of milk (Ref. 11). The cited study concluded that a foam promotes lipolysis by providing optimum conditions a s follows (i) greatly increased liquid surface, (ii) selective concentration of enzyme at the air-liquid interface, (iii) activation of the substrate by surface denaturation of the membrane materials surrounding fat globules, and (iv) intimate contact of enzyme and activated substrate. ln ground studies conducted to identify a good space demonstration experiment, the discovery was made that a formaldehyde clock reaction can display both a Gibbs adsorption and an internal structure o r a s h e a r effect on the r a t e of the reaction. A report of the observed evidences of Gibbs adsorption and internal s h e a r o r three-dimensional structure

effects in a formaldehyde clock reaction has not yet appeared in the open literature although a submission i s currently being reviewed f o r publication (Ref. 12). A brief summary, theref o r e , i s given h e r e .

The surface and internal effects to be described were observed with solutions and procedures which a r e a s follows : Stock solutions : (a) 3.3%formaldehyde (9.0 ml 37.7% reagent grade formaldehyde diluted to 100 ml with distilled water), (b) 1 gm phenolphthalein dissolved and diluted to 500 ml with 50% ethanol- water, and (c) 10.0 gms sodium metabisulfite (Na2S205) and 1.5 gms of sodium sulfite (Na2S03), reagent o r certified grades, diluted to 100 ml with distilled water. Procedures : One ml of formaldehyde stock and 0 . 5 ml of phenolphthalein stock a r e added to 8 ml of distilled water in a t e s t tube. A 0 . 5 ml portion of the bisulfitel sulfite stock solution i s then added rapidly, the t e s t tube capped, and shaken vigorously f o r about 5 sec. The mixed solution remains colorless f o r about 20 sec at the end of which time a sudden appearance of a red color occurs. The time interval between the time of bisulfite/sulfite addition to the time of red color appearance can be varied by adjustment of solution concentrations (Ref. 13). Plastic syringes bought i n drugstores f o r a few cents make handy devices f o r measuring and adding the small amounts of reagents involved. The stock solutions of formaldehyde and phenolphthalein a r e stable indefinitely. The sulfitel bisulfite solution, however, deteriorates. The deterioration r a t e , however, i s f a i r l y slow if oxygen and light exposure a r e kept to a minimum. The chemical reactions involved a r e a s follows (Ref. 13): Rate Constants 2 . 8 llmol s e c

HCHO + H S O ~ --+ CH~OHSO~-

0 . 1 4 llmol sec

H20

+ HCHO + so3=--+ C H ~ O H S O +~ -OH-

instantaneous

OH-

+

HSO3-

-+

SO3

--

+ H20

Thus, excess hydroxide ion becomes available to react with phenolphthalein indicator only when all of the bisulfite ion i s used up. The surface and internal structure effects observed a r e briefly a s follows : In plastic (Lexan) t e s t tubes the color change i s most frequently seen to occur f i r s t in the small drops that cling to the sides of the tube. O r the color change will s t a r t at a point in the liquid/vapor/solid interface o r in the bulk of the solution and then spread out a s a wave into the remainder of the solution. If the reaction i s allowed to occur in contact with a polystyrene surface, color spots appear a t the solid/liquid interface s i t e s , the color change then proceeding into the bulk of the solution. In chilled solutions the surface effects a r e greatly enhanced. In addition to the described surface effects, internal structure effects, a s evidenced by complex colored shapes in the bulk of the solution, a r e also seen. The internal structure effects may be caused either by s h e a r a s the result of residual fluid flow o r by a three-dimensional formaldehyde species network. 1n s t i r r e d solutions the color changes outline vividly fluid flow phenomena such a s vortices. Various size drops of reacting solution placed on various surfaces change color most frequently in the o r d e r of large drops f i r s t , medium- sized drops next, and small drops last. Further evidences of the Gibbs and internal structure effects a r e given in the not-yet-published report (Ref, 12) and in film strips s f the various ground t e s t s . In the Apollo- Soyuz experiment Astronaut D . X. Slayton performed in earth orbit the formaldehyde clock reaction in capped Eexan sentrbfuge tubes. The objective of the space experiment was to determine the effect of a

69

low- g gas/liquid dispersion o r "foam" structure on the reaction. The low- g experiment showed that in low-g "foams" the red color f i r s t appears at the gas/liquid/solid interface and then spreads out rather evenly from this interface. A homogeneous light pink color appearing about the same time a s the f i r s t red color i s also noted. Further results such a s reaction times and velocity of color wave advance await a complete analysis of the flight data. THE EQUILIBRIUM SHIFT REACTION Only a few workers have delved into the a r e a of chemical equilibrium shift induced by foam o r emulsion formation. The following indicator equilibria a r e reported by Freundlich to be shifted by emulsion formation (Ref. 10) :

Violet (2.0)

Green (1 .O)

Methyl- violet

Green (1.5)

Yellow (0.5)

Malachite- green

Blue (7.4)

Yellow (6.2)

Brom- thymol blue

The numbers in the brackets a r e pH values, Thus, if an aqueous solution of bromothymol blue of pH of 7 . 4 i s shaken with benzene, the color changes from blue to yellow. The yellow color remains a s long a s fine drops of benzene remain. Similarly a solution of thymol blue of pH of about 2.8 will show a change of color from brown-amber to pink when foamed by shaking. The foam under one- g conditions disappears in a few seconds, however. The preceding data would indicate that the interface regions favor the undissociated forms of the indicators while the bulk phases favor the dissociated forms, although to the best of o u r knowledge, the matter i s still not conclusively settled. Other reactions along these same lines a r e : Rhodarnine-0 (colorless base of) benzene solution + water colorless

emulsion red

benzene solution colorless

on filter paper o r quartz powder red

-

colorless form probably lactoid form colored form probably betaine form Rhodaime 6G extra and Rhodamine 3G extra organic liquid + water emulsion another color

organic liquids one color Rases of the preceding dyes benzene solution

benzene red

-

70

+

water emulsion

water solution red

-

benzene solution

on quartz powder red P

Silver Eosinate aqueous solution containing silver nitrate pinkish-yellow with green fluorescence

----

adsorbed on surface of s i l v e r halide red

-

- solution + benzene emulsion red precipitate at interface

-shaken in a i r

r e d precipitate at interface

Probably not all of the preceding reactions a r e strictly reversible. however, i s not affected.

The illustrative point,

In the Apollo-Soyuz experiment an aqueous solution of thymol blue was shaken. The pink foam lasted a great deal longer than one generated on the ground. Times f o r foam collapse, however, were not measured. THE ELECTRODEPOSITION REACTION T h e Electrodeposition Reaction experiment conducted aboard Skylab IV was designed primarily to study metal c r y s t a l growth in a low-g environment. However, because the reaction involved an electrochemical displacement reaction, certain features of it should be of interest to the biochemical community. The experiment consisted of the astronaut inserting a copper wire into a 5 wt % silver nitrate solution. The following electrochemical displacement reaction occurred : Cu

+

2 ~ ~ +CU++ + + 2Ag

The silver crystals grown in space a r e quite different from those grown on the ground because of the differing convection currents generated in each case. Full details will be found in a report which will appear shortly (Ref. 14) and in a paper which i s currently being reviewed f o r publication (Ref. 15). The experiment, while not immediately relevant to biochemistry, does r a i s e some common questions. F o r example, what a r e the gravity effects on e.m.f. and what i s the nature of low-g convection in cases where electric fields a r e involved. These common aspects a r e covered in more detail in the following discussions. Gravity effects on e. m. f . : The fact that gravity can affect e. m. f. values h a s been long known (Ref. 16). The gravity dependency of e m. f i n ordinary cells i s the result of the change of chemical potential of the ions with change in gravity level. This dependency i s given by:

. .

(M, -

-

>

Vi,u gdh d w h e r e h i i s the chemical potential of the ion in question, Mi i t s molecular weight, Vi the partial molal volume, p the density of the solution, g the gravitational acceleration, and h the height measured fro? some reference height. One early worker found that e. m.f. chaaged about 0.510 x 10- volt p e r cm of height f o r a cell employing a 2.71 molal potassium chloride solution. Thus, on earth an e. m. f . may be generated just by having one half cell higher than another identical half cell. In living systems gravity has been shown to electrically polanize an organ placed horizontally. F o r example, after shoots and leaf =

stalks had been placed horizontally, their upper sides assumed a potential several millivolts more negative than the lower (Ref. 17). The explanation given for this effect i s that the diffusion potentials already existing a c r o s s membranes becomes modified a s the result of gravity. The diffusion potentials can be modified by a change of ion mobilities due to gravity and by displacement of growth hormone which r a i s e s the ionic selectivity of the membranes concerned in the region of its enrichment (Ref. 17). Low- Q, electro- and other types of convection : Various types of relatively ill-understood convections undoubtedly play important roles in biochemical processes. F o r example, D r . W . Dorst, Amsterdam, believes that convective processes within a millipore membrane a r e the major factors in his experiments dealing with the effect of gravity on the permeability of a synthetic membrane (Ref. 18). Adding substance to this belief i s the demonstration of f r e e convection in electrolysis cells whose total volume was 0 . 2 5 ml and which contained solution of depths of l e s s than 2 mm (Ref. 19). Convective processes have undoubtedly also played roles in experiments on anomalous and thermo- osmosis which gave puzzling r e s u l t s (Ref. 20). Recently D r . J. R. Melcher of MIT conducted some preliminary tests on electrically-driven convections in aqueous solutions (Ref. 21). It might be added that the electroconvection D r . Melcher was concerned with had to do with electrical forces on zones of concentration inhomogeneities within the bulk of the solution and not with electro-osmosis type convection. It i s interesting to speculate what role electro-convection might play in various electrolysis and electrophoretic processes, but speculations would be fruitless a t present when s o little i s known about the basic phenomenon. The literature i s s o scanty on convective phenomenain biological processes and processing that we expect that a whole new exciting e r a will be initiated once serious attention is turned in this direction. It may be well to add that gravity and electric fields a r e not the only forces that can drive fluid flow. Surface and interfacial tensions, phase changes, thermally induced volume changes, vibrations o r g- jitter, magnetic fields a r e also possible driving forces. In addition, c a r e must be taken when analyzing data from low-g environments not to tacitly assume that gravity-driven convection was absent. Even in low-g, gravity can b e a major driving force. It all depends on the particulars of a given situation. The general nature of convection in low-g environments was recently reviewed (Ref. 21). POSSIBLE APPLICATIONS The implications of the space experiments with regard to possible applications f a l l into two a r e a s . One a r e a i s concerned with directions f o r basic r e s e a r c h on biophysical processes and the other with directions f o r processing applications in low-g. In the a r e a of basic r e s e a r c h , the Formaldehyde Clock Reaction would appear to have relevancy to pheilomena such a s the clotting of blood and the formation of cataracts. F o r example, the following description of how a lobster's blood clots on a glass slide sounds much like a description of how a formaldehyde clock reaction occurs in a plastic tube: "A wave of changes must s t a r t at the interface between the glass and blood, and prog r e s s through the l a t t e r , involving these sensitive corpuscles in i t s path. . . The two impressive features of this phenomenon a r e (1) that a chemical change of catastrophic character can b e started at an interface, .and (2) that the change can be propagated apparently indefinitely through one of the phases. " (Ref. 22) A description of the processes involved in the formation of cataracts (Ref. 23) also sounds a s if processes similar to those found in the Formaldehyde Clock Reaction a r e involved, F o r example, the formation of regions of cortical opacities within a cataractous lens can be compared to the formation of red spots within the solution in the Formaldehyde Clock Reaction because an opacity i s caused by an abrupt o r i r r e g u l a r change in protein soncentration. Also iriteresting i s the fact that s h e a r o r internal s t m c m r e effects appear to play a role both in the Formaldehyde Clock Reaction and in the formation ~f c a t a r a c t s .

Also solution size o r volume effects a r e notable in both phenomena. The hints that the two phenomena may be related a r e tantalizing and a number of experiments immediately suggest themselves, With regard to electrical phenomena in low- g , it would appear that space experiments could help a great deal in explaining the role of convection in a number of membrane transport phenomena. In the a r e a of processing applications, the demonstration of a formaldehyde clock reaction and an equilibrium shift reaction in low- g indicates that low-g forms can be unique environments f o r conducting biochemical reactions. Also the demonstration of a longer lasting foam in low- g indicates foam separation processes that cannot be done on earth because of the long times required f o r adsorption, i. e . , the foam on earth does not l a s t long enough f o r adsorption to occur. The demonstration of an electrodeposition i n space points towards organic syntheses and separations utilizing electrolysis. ADDENDUM In the discussion that occurred a f t e r this paper was given at the Bioprocessing Colloquium, a couple of questions were raised on which we should like to elaborate. The f i r s t concerns the role of oxygen in the surface effects noted in the Formaldehyde Clock Reaction. Oxygen may affect significantly only the sulfite o r bisulfite species through the reaction

The reaction i s slow i n the absence of catalysts. Even if it did occur to any appreciable extent a t the liquidlair interface, however, the effect would be to make the interface solution more acid. Thus the onset of red color would be expected to be considerably delayed a t air/liquid interfaces, not accelerated a s i s actually observed. It may, therefore, be concluded that oxygen i s not a signrficant variable in the reaction. T o verrfy this conclusion, a test was run which included a nitrogen purge in a closed test tube p r i o r to performance of the reaction. The reaction was observed to proceed in a l l respects the same a s it does when no purging i s employed. It was also mentioned after the talk that the red color i s seen to form f i r s t a t a negatively charged platinum electrode. The question was raised whether the red color was due to the clock reaction o r to electrolysis of hydrogen ions. A check of our notes verified that no red color develops at the negative electrode if formaldehyde i s left out of the reaction mixture, i. e. , no o r insufficient electrolysis occurs with the 13 volt system used to change the color of the phenolphthalein indicator. The accelerating effect of the negative electrode on the Formaldehyde Clock Reaction, therefore, is real.

REFERENCES

1.

Davis, J . T , , and E. X . Rideal, Interfacial Phenomena, Chapter 6 , Academic P r e s s , New York and London, 1963,

2. Rideal, E. X . , "Film Reactions a s a New Approach to Biology," Nature, Vol. 144, 1939, pp. 693-7.

.

3.

Rideal, E. X , "Reactions with Monolayers and Their Biological Analogies, " Nature, Vol. 144, 1939, pp. 100-2.

4.

Rideal , E. K pp. 83-90.

5.

Gaines , Jr., G. L. , Insoluble Monolayers at Liquid-Gas Interfaces, Interscience Publishers, New York, London, Sydney.

6.

Monomolecular L a y e r s , Editor: H. Sobotka, American Association f o r the Advancement of Science, Washington, D. C . , 1954.

7.

Schulman, J. H. , and E. K Rideal, "On Monolayers of Proteolytic Enzymes and P r o teins," Biochem Journ. , Vol. 27, 1933, pp. 1581-97.

8.

Clowes, G. H. A . , "Interactions of Biologically Significant Substances in Surface Films, with Especial Reference to Two-Dimensional Solutions and Association Complexes formed by Carcenogenic Hydrocarbons and S t e r o l s , " Surface Chemistry, Editor: F R. Moulton, American Association f o r the Advancement of Science, Publication No. 21, 1943, pp. 1-26.

. , "Surface Chemistry in Relation to Biology, " Endeavor,

Vol. 4, 1945,

.

.

9.

Davis, W. W. , M. E. Krahl, and G. H. A. Clowes, "Interactions between Polycyclic Hydrocarbons and S t e r o l s in Mixed Surface Films at the Air-Water Surface, Journ. Am. Chem. S o c . , Vol. 62, 1940, pp. 3080.

10.

Freundlich, H. , "Surface F o r c e s and Chemical Equilibrium, " Journ. Chemical S o c . 1930, pp. 164-179.

11.

Tarassuk, N . P . , and E. N . Frankel, "On the Mechanism of Activation of Lipolysis and the Stability of Lipose Systems of Normal Milk," Journ. Dairy Sci. , Vol. 39, 1955, pp. 438-9.

12.

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,

S U R m Y O F CELL BIOLOGY E X P E R I m N T S

By G e r a l d R. T a y l o r Lyndon B. J o h n s o n Space C e n t e r Houston, T e x a s

ABSTRACT

A l t h o u g h s i n g l e c e l l s are g e n e r a l l y c o n s i d e r e d t o be l e s s v u l n e r a b l e t h a n h i g h e r o r g a n i s m s t o v a r i a t i o n s i n g r a v i t a t i o n a l f o r c e s , many c e l l e x p e r i m e n t s have b e e n c o n d u c t e d i n t h e r e d u c e d gra;ity o f s p a c e . S t u d i e s involving isolated viruses, b a c t e r i a , yeasts, filamertous fungi, protozoans, and c e l l s i n s m a l l g r o u p s ( s u c h a s t i s s u e c u l t u r e s and e a r l y embryos) a r e reviewed t o i l l u s t r a t e t h e v a r i e t y of s p e c i e s examined. E a r l y s t u d i e s , c o n d u c t e d w i t h h i g h a l t i t u d e b a l l o o n s , s o u n d i n g r o c k e t s , and p r i m i t i v e o r b i t a l s a t e l l i t e s , demonstrated t h e c a p a b i l i t y o f c e l l s t o s u r v i v e t h e s p a c e f l i g h t e n v i r o n m e n t . These r e s u l t s r e v i v e d i n t e r e s t i n P a n s p e r m i a and d e m o n s t r a t e d t h e p o s s i b l e r e q u i r e m e n t f o r s t e r i l i z a t i o n o f a l l s p a c e c r a f t l a n d i n g on f o r e i g n h e a v e n l y b o d i e s . Because s p a c e v e h i c l e s , e q u i p m e n t , and p a s s e n g e r s a r e n o t s t e r i l i z e d b e f o r e f l i g h t , it h a s b e e n i m p o r t a n t t o s t u d y t h e e f f e c t s , i f a n y , o f s p a c e f l i g h t on t e r r e s t r i a l c e l l s y s t e m s . With some i m p o r t a n t e x c e p t i o n s , which a r e d i s c u s s e d , s t a t i c c e l l s y s t e m s c a r r i e d a b o a r d USA and USSR s p a c e f l i g h t s have f a i l e d t o r e v e a l s p a c e - r e l a t e d a n o m a l i e s . Some s o p h i s t i c a t e d d e v i c e s which have b e e n d e v e l o p e d f o r v i e w i n g d i r e c t l y , o r c o n t i n u o u s l y r e c o r d i n g , t h e growth o f c e l l s , t i s s u e c u l t u r e s and e g g s i n f l i g h t , a r e d e s c r i b e d and t h e r e s u l t s summarized. The u n i q u e p r e s e n c e o f h i g h e n e r g y , m u l t i c h a r g e d (HZE) p a r t i c l e s and f u l l - r a n g e u l t r a v i o l e t i r r a d i a t i o n i n s p a c e h a s prompted s e v e r a l i n v e s t i g a t o r s t o e v a l u a t e t h e r e s p o n s e o f s i n g l e cells t o t h e s e f a c t o r s . Summary r e s u l t s and g e n e r a l c o n c l u s i o n s a r e p r e s e n t e d . a r e a s of research i n f u t u r e space f l i g h t s a r e i d e n t i f i e d .

Potential

INTRODUCTION E x p e r i m e n t a l e v i d e n c e t h a t o r g a n i s m s are a f f e c t e d by g r a v i t a t i o n a l f o r c e s was f i r s t o b t a i n e d i n 1806 b y K n i g h t who d e m o n s t r a t e d , w i t h t h e

PRECEDING PAGE BLANK NOT P E

a i d o f a w a t e r - d r i v e r c e n t r i f u g e , t h a t o r i e n t a t i o n i n p l a n t s was d e t e m i n e d b y t h e g r a v i t a t i o n a l v e c t o r ( 1 ) . The dependence o f a n i m a l s upon g r a v i t y was f i r s t observed i n 1883 b y P f l u g e r w i t h d e m o n s t r a t i o n s t h a t t h e d e v e l o p ment o f f r o g e g g s i n a n i n v e r t e d p o s i t i o n r e s u l t e d i n a h i g h r a t e o f a b n o r m a l i t i e s ( 2 ) . Many s t u d i e s , d e s i g n e d t o e v a l u a t e t h e e f f e c t s o f r e s u l t a n t f o r c e s i n e x c e s s o f one g r a v i t a t i o n a l u n i t , i s s u e d from t h e s e b e g i n n i n g s . I n a d d i t i o n , some i n v e s t i g a t o r s have o b s e r v e d o r g a n i s m s i n d e v i c e s which compensate f o r , o r o p p o s e , t h e E a r t h ' s g r a v i t a t i o n a l a t t r a c t i o n . The neut r a l bouyancy t a n k which p r o v i d e s a f l e x i b l e l i f t e q u a l t o t h e mass o f t h e t e s t o b j e c t , and t h e r o t a t i n g c l i n o s t a t which c o n t i n u a l l y a l t e r s t h e d i r e c t i o n o f t h e g r a v i t a t i o n a l v e c t o r , a r e t h e two most w i d e l y used d e v i c e s (3) With t h e a d v e n t o f t h e s p a c e age i t became p o s s i b l e t o r e d u c e t h e t o t a l f o r c e upon t e s t s y s t e m s t o l e s s t h a n one g r a v i t a t i o n a l u n i t b y removing t h e m . f r o m t h e E a r t h . T h i s o p p o r t u n i t y was r e c o g n i z e d b y many i n v e s t i g a t o r s who conducted e x p e r i m e n t s on a l a r g e v a r i e t y o f d i f f e r e n t t y p e s o f l i v i n g t e s t s y s t e m s . Those s y s t e m s which i n v o l v e s i n g l e c e l l s o r s m a l l g r o u p s o f c e l l s ( s u c h a s b l a s t u l a s o r t i s s u e c u l t u r e ) a r e - r e v i e w e d and summarized i n Tables I through V I , i n an e f f o r t t o demonstrate t h e v a r i e t y o f tests t h a t have b e e n conducted i n s p a c e . I n a d d i t i o n , g e n s r a l c o n c l u s i o n s a r e p r e s e n t e d and a r e a s p o t e n t i a l l y w o r t h y o f f u t u r e s p a c e r e s e a r c h a r e i d e n t i f ied

.

S u r v i v a l Of C e l l s I n S p a c e

P r e p a t o r y t o s t u d i e s on o r b i t a l s p a c e f l i g h t s , s e v e r a l m i c r o b i a l s p e c i e s w e r e exposed t o a l t i t u d e s up t o 1900 Km i n b a l l o o n and s o u n d i n g r o c k e t f l i g h t s ( T a b l e s I , 11, 111, and I V ) . T h e s e e x p o s u r e s , which were i n i t i a t e d i n 1935 ( 4 ) , were c o n d u c t e d t o d e t e r m i n e i f m i c r o o r g a n i s m s c o u l d s u r v i v e h i g h a l t i t u d e f l i g h t and have b e e n t h o r o u g h l y reviewed (5, 6, 7, 8). Although rudimentary, t h e s e s t u d i e s p e r m i t t e d t h e i n v e s t i g a t o r s t o observe t h a t a l a r g e p e r c e n t a g e o f f u n g a l s p o r e s and dormant v e g e t a t i v e c e l l s c o u l d s u r v i v e s h o r t - d u r a t i o n d i r e c t exposure t o t h e s p a c e environment a t t h e s e a l t i t u d e s (9, L O )

.

B e g i n n i n g w i t h t h e U S S R r e c o v e r a b l e S p u t n i k 5 f l i g h t i n 1960 ( 1 1 ) and t h e USA Gemini/Agena m i s s i o n s i n 1 9 6 3 , t h e r e q u i r e m e n t t o s t e r i l i z e s p a c e v e h i c l e s d e s t i n e d t o l a n d on o t h e r h e a v e n l y b o d i e s h a s b e e n s t u d i e d ( 8 ) . I n a t y p i c a : example, a v a r i e t y o f m i c r o b i a l s p e c i e s ( P e n i c i l l i u m , B a c i l l u s s u b t i l i s s p o r e s , Tobacco Mosiac V i r u s , and Tl c o l i p h a g e ) w e r e c a r r i e d a b o a r d t h e Gemini 9A and Gemini 12 s p a c e c r a f t ( 9 ) . V i a b l e r e p r e s e n t a t i v e s o f a l l s p e c i e s w e r e recovered f o l l o w i n g n e a r l y 17 h o u r s o f " d i r e c t e x p o s u r e " t o s p a c e c o n d i t i o n s . These same s p e c i e s , when p r o t e c t e d from d i r e c t s o l a r i r r a d i a t i o n , s u r v i v e d 4 months s f e x p o s u r e on

.

t h e Agena 8 o r b i t e r ( 10) S i m i l a r t e s t s on t h e S o v i e t Cosmos 368 E a r t h o r b i t a l s a t e l l i t e , and t h e Zond 8 a u t o m a t i c l u n a r s t a t i o n , r e v e a l e d t h a t I

I

b a i l i . , and E s c h e r i c h i a c o l i , c e l l s were a l l a b l e t o s u r v i v e s p a c e f l i g h t (124,13) . I n t h e e n s u i n g y e a r s , v i a b i l i t y measurements have g e n e r a l l y b e e n i n cluded i n a l l space c e l l b i o l o g y s t u d i e s . A s a r e s u l t it has been e s t a b l i s h e d t h a t microorganisms i n and on i n t e r p l a n e t a r y s p a c e c r a f t may b e capable of surviving t o contaminate e x t r a t e r r e s t r i a l bodies (8, 9, 10, 12, 3 4 1 5 6 , 7 The r e c o r d f o r v i a b i l i t y i n s p a c e was r e p o r t e d f o r S t r e p t o c o c c u s m i t i s which was r e c o v e r e d from i n t e r n a l components o f a S u r v e y o r I11 t e l e v i s i o n camera t h a t had r e s i d e d on t h e s u r f a c e o f t h e Moon f o r 2 . 5 y e a r s ( 1 8 ) . Even t h o u g h t h e p o s s i b i l i t y o f s u r v i v a l i n s p a c e h a s b e e n r e p e a t e d l y p r o v e d , it was c o n s i d e r e d o p e r a t i o n a l l y n o n - f e a s i b l e t o s t e r i l i z e s p a c e v e h i c l e s , e q u i p m e n t , and p a s s e n g e r s b e f o r e f l i g h t . Accordi n g l y it became i m p o r t a n t t o e v a l u a t e t h e e f f e c t s , i f a n y , o f s p a c e f l i g h t on t e r r e s t r i a l c e l l s y s t e m s . A l t h o u g h i n t e r e s t e d i n t h e same o b j e c t i v e , t h e American and S o v i e t s p a c e programs p r o c e e d e d d i f f e r e n t l y t o e v a l u a t e t h e s e e f f e c t s . T h i s d i f f e r e n c e i s o u t l i n e d b y J e n k i n s ( 6 ) who d e m o n s t r a t e d t h a t i n t h e f i r s t d e c a d e o f o r b i t a l f l i g h t , S o v i e t s c i e n t i s t s e v a l u a t e d 56 d i f f e r e n t s p e c i e s ( o r preparations) including v i r u s e s , b a c t e r i a , y e a s t s , fungi, p l a n t s , a n i m a l s , and t i s s u e c u l t u r e s . D u r i n g t h e same p e r i o d t h e USA e v a l u a t e d More i m p o r t a n t l y , o n l y 35 d i f f e r e n t s p e c i e s and c e l l u l a r p r e p a r a t i o n s . s e v e r a l o f t h e S o v i e t s a t e l l i t e s w e r e flown p r i m a r i l y t o o b t a i n b i o l o g i c a l d a t a t o q u a l i f y man f o r s p a c e f l i g h t . I n c o n t r a s t , t h e e a r l y American b i o l o g y s t u d i e s w e r e o p e r a t e d on a n o n - i n t e r f e r e n c e b a s i s and n o s u c c e s s f u l , d e d i c a t e d b i o l o g y s a t e l l i t e was flown u n t i l t h e l a u n c h o f B i o s a t e l l i t e I1 i n September 1967 ( 6 ) . E f f e c t Of S p a c e f l i g h t On Growing C u l t u r e s

I n a d d i t i o n t o t h e p r e v i o u s l y mentioned v i a b i l i t y t e s t s which i n v o l v e d s t a t i c o r dormant c e l l s , s p o r e s , o r c y s t s , some i m p o r t a n t s t u d i e s , o u t l i n e d i n T a b l e V I I , have b e e n c o n d u c t e d on growing c e l l s . I n f l i g h t microbial growth was f i r s t m o n i t o r e d d u r i n g t h e f l i g h t s o f S p u t n i k 5 ( 1 9 ) and o t h e r , non-recovered S o v i e t s a t e l l i t e s ( 2 0 ) , w i t h t h e a i d o f a n a u t o m a t e d d e v i c e known a s " B i o e l e m e n t s " . T h i s d e v i c e was d e s i g n e d t o measure t h e r a t e o f g a s p r o d u c t i o n i n a c t i v e l y growing C l o s t r i d i u m b u t y r i c u m c u l t u r e s and t o r e l a y t h e s e d a t a t o e a r t h . D a t a from t h i s t e s t , and from Vostok 1 and 2 where a m o d i f i e d " B i o e l e m e n t s " was u s e d , showed g a s p r o d u c t i o n r a t e s i n d i s t i n g u i s h a b l e from ground c o n t r o l s . Growing and r e p r o d u c i n g p r o t o z o a n s have b e e n v a r i o u s l y s t u d i e d . P l a n e l e t al. ( 2 1 ) have r e p o r t e d a n i n c r e a s e i n c e l l u l a r g r o w t h r a t e f o r Paramecium a u r e l i a exposed t o h i g h - a l t i t u d e b a l l o o n f l i g h t f o r 6 h o u r s . A d d i t i o n a l l y , amoebae w e r e o b s e r v e d fobleswing t h e 45 h o u r f l i g h t sf B i o s a t e l l i t e I%, T h e r e were n o s i g n i f i c a n t d i f f e r e n c e s between f l i g h t c e l l s

79

and ground c o n t r o l s , b u t Ekberg e t a l . ( 2 2 ) r e p o r t e d a " t r e n d " t o w a r d s a h i g h e r d i v i s i o n r a t e d u r i n g f l i g h t . I t i s w e l l known t h a t amoebae r e q u i r e g r a v i t y ( o r some f o r c e v e c t o r ) t o a t t a c h t o s u b s t r a t e s . A l t h o u g h t h i s a t t a c h m e n t i s g e n e r a l l y c o n s i d e r e d t o b e r e q u i r e d f o r l o c o m o t i o n and f e e d i n g , t h e s e o r g a n i s m s s u r v i v e d t h e f l i g h t and f e d , a s s i m i l a t e d f o o d , grew, and performed a l l o t h e r measured f u n c t i o n s i n a manner i n d i s t i n g u i s h a b l e from t h e ground c o n t r o l s ( 2 3 ) . These r e s u l t s g e n e r a l l y c o n f i r m d a t a o b t a i n e d from e a r l i e r s i m u l a t i o n s t u d i e s a b o a r d C-131 a i r c r a f t i n Keplerian t r a j e c t o r y (24). A n o t h e r t e s t s y s t e m , which was u n u s u a l l y r e f i n e d f o r a u t o m a t e d s a t e l l i t e s t u d i e s , was d e s i g n e d t o s t u d y t h e d e v e l o p i n g f r o g e g g u n d e r reduced g r a v i t y c o n d i t i o n s . T h i s s e r i e s , flown a b o a r d t h e Gemini 8 , Gemini 1 2 , and B i o s a t e l l i t e I1 s p a c e c r a f t , p r o v i d e d f o r i n f l i g h t g r o w t h and Developing f r o g d i f f e r e n t i a t i o n of f e r t i l e e g g s from t h e 2 c e l l s t a g e . e g g s on E a r t h e x h i b i t a marked s e n s i t i v i t y t o d i s o r i e n t a t i o n w i t h r e s p e c t t o t h e normal g r a v i t y v e c t o r , w i t h t h e e a r l y embryo ( u p t o t h e e i g h t - c e l l s t a g e ) b e i n g t h e most s e n s i t i v e ( 2 5 ) . I n s p i t e o f t h i s known s e n s i t i v i t y n o d i f f e r e n c e s c o u l d b e d e t e r m i n e d between f l i g h t and ground c o n t r o l s . The a u t h o r s p o i n t o u t t h a t , t o c o m p l e t e t h i s l i n e o f r e s e a r c h , f r o g e g g s s h o u l d b e f e r t i l i z e d a f t e r l a u n c h and m a i n t a i n e d f o r a l o n g e r t i m e i n t h e r e d u c e d g r a v i t y s t a t e ( 2 5 , 26)

.

I n a s i m i l a r manner, young K i l l i f i s h e g g s ( F u n d u l u s h e t e r o c l i t u s ) , w e r e a l l o w e d t o d e v e l o p and h a t c h d u r i n g t h e 56-day S k y l a b , t h e 20-day Cosmos 782, and t h e 10-day A p o l l o Soyuz T e s t P r o j e c t (ASTP) f l i g h t s . In a l l c a s e s s p a c e - h a t c h e d f r y e x h i b i t e d n o o b s e r v a b l e t e n d e n c y toward d i s o r i e n t e d swimming a c t i v i t y ( 2 7 , 28) a l t h o u g h dependence on v i s u a l o r i e n t a t i o n c u e s a b o a r d t h e S k y l a b and f o l l o w i n g r e t u r n t o E a r t h s u g g e s t e d t h e p o s s i b l e absence o f v e s t i b u l a r i n p u t (28). One o f t h e most e l e g a n t and complex growth s t u d i e s t o d a t e w a s cond u c t e d w i t h W i s t a r - 3 8 human embryonic l u n g c e l l s i n t i s s u e c u l t u r e a b o a r d t h e m i d d l e S k y l a b f l i g h t ( S k y l a b 3 ) . C o n t i n u o u s c u l t u r e s w e r e maintained a t 36O C and p h o t o g r a p h e d w i t h t i m e - l a p s e m o t i o n p i c t u r e c a m e r a s , t h r o u g h p h a s e - c o n t r a s t m i c r o s c o p e s a t 20X and 40X m a g n i f i c a t i o n , f o r 28 d a y s ( 2 9 ) . Many p a r a m e t e r s were e v a l u a t e d , i n c l u d i n g growth c u r v e s , m i t o t i c i n d i c e s , c e l l m i g r a t i o n r a t e s , v a c u o l e f o r m a t i o n , c e l l s i z e , n u c l e a r s i z e and l o c a t i o n , n u c l e o l a r s i z e and number, and G- a n d C-band p a t t e r n s i n chromosomes. A l t h o u g h t h e e x p e r i m e n t o p e r a t e d a c c o r d i n g t o p l a n , n o d i f f e r e n c e s w e r e d e t e c t e d between f l i g h t c e l l s and s u i t a b l e ground c o n t r o l s ( 3 0 ) . More r e c e n t l y , growing c o l o n i e s o f S t r e p t o m y c e s l a v o r i s w e r e f l o w n a b o a r d t h e Soyuz 16 and t h e A p o l l o Soyuz T e s t P r o j e c t f l i g h t s ( 3 1 ) . The f o r m a t i o n o f a l t e r n a t i n g r i n g s o f s p o r e - b e a r i n g and s t e r i l e mycelium a l l o w e d c o n t i n u o u s a n a l y s i s o f c h a n g e s i n c y c l i c g r o w t h and p r o v i d e d a method f o r k e e p i n g t r a c k o f c e r t a i n i n f l i g h t m u t a t i o n s . A c o r r e l a t i o n between t h e c y c l i c s p o r e f o r m a t i o n and s p a c e f l i g h t was rise d e m o n s t r a t e d . should a l s o be noted A l t h o u g h a n a l y t i c a l d a t a a r e n o t y e t a v a i l a b l e i% $ h a t S o v i e t i n v e s k i g a % o r s have r e p o r t e d active o b s e r v a t i o n of c u l t u r e s of

c o l i f o m b a c i l i , f e r t i l i z e d f r o g e g g s and S e r i a n Hamster c e l l t i s s u e c u l t u r e s i n t h e " f l y i n g o a s i s " o f Soylz 17 - S a l y u t 4 (32)

.

Genetic Studies

B a c t e r i o p h a g e i n d u c t i o n h a s been e x t e n s i v e l y employed, b y S o v i e t i n v e s t i g a t o r s , a s a model s y s t e m f o r v i s u a l i z i n g t h e e f f e c t s o f s p a c e f l i g h t on t h e g e n e t i c a p p a r a t u s o f m i c r o o r g a n i s m s ( T a b l e V I I I ) . Escherichia c o l i K-12 ( A ) b a c t e r i o p h a g e have b e e n c a r r i e d a b o a r d most o f t h e f l i g h t s o f t h e S p u t n i k s e r i e s , a l l s i x o f t h e manned Vostok f l i g h t s , Voskhod 1 and 2 , t h e unmanned b i o s a t e l l i t e Cosmos 110, and Zond 5 and 7 , b o t h o f which c i r c l e d t h e Moon ( 1 9 , 33, 3 4 ) . T h i s s y s t e m was used a s a r a d i a t i o n d o s i m e t e r b e c a u s e i n c r e a s e s i n phage p r o d u c t i o n c o u l d b e s t i m u l a t e d b y a s l i t t l e a s 0.3 r a d o f gamma r a d i a t i o n o r b y s m a l l d o s e s o f p r o t o n s o r r a p i d n e u t r o n s ( 3 3 , 3 5 ) . Because phage i n d u c t i o n i n v o l v e s i n j u r y t o t h e g e n e t i c a p p a r a t u s , t h e l y s o g e n i c b a c t e r i a s y s t e m was u s e d t o p r o v i d e i n f o r m a t i o n I t was rea b o u t t h e p o t e n t i a l m u t a g e n i c a c t i v i t y o f cosmic r a d i a t i o n . p o r t e d t h a t t h e s p a c e f l i g h t e f f e c t (measured i n t e b s o f i n c r e a s e d phage p r o d u c t i o n i n s p a c e a s compared t o t h e magnitude o f s p o n t a n e o u s phage p r o d u c t i o n i n t h e ground c o n t r o l s ) i n c r e a s e d w i t h m i s s i o n d u r a t i o n t h r o u g h o u t t h e Vostok s e r i e s ( 7 , 3 5 ) . T h i s r e l a t i o n s h i p i s summarized i n f i g u r e 1. L a b o r a t o r y s t u d i e s d e m o n s t r a t e d t h a t s i m u l a t e d l a u n c h v i b r a t i o n f o l l o w e d b y e x p o s u r e t o 6 0 ~ ogamma r a d i a t i o n r e s u l t e d i n a n i n c r e a s e d m u t a t i o n r a t e which was h i g h e r t h a n t h a t o b t a i n e d b y gamma r a d i a t i o n o r s i m u l a t e d l a u n c h v i b r a t i o n a l o n e ( 3 3 , 3 5 ) . T h i s was i n t e r p r e t e d a s i n d i c a t i n g t h a t t h e Vostok l a u n c h v i b r a t i o n s " s e n s i t i z e d " t h e c e l l s s o t h a t t h e y were n o t s u s c e p t i b l e t o i n f l i g h t i r r a d i a t i o n . Two d i f f e r e n t b a c t e r i o p h a g e s y s t e m s were t e s t e d a s p a r t o f t h e 45-hour E a r t h - o r b i t a l f l i g h t o f t h e American B i o s a t e l l i t e I1 ( 3 6 , 3 7 ) . S a l m o n e l l a were t e s t e d f o r typhimurium BS-5 (P-22)/P-22, and E. c o l i C-60 ( X ) / l a l t e r a t i o n s i n b a c t e r i a l c e l l g r o w t h and b a c t e r i a l p r o p h a g e i n d u c k i o n following s p a c e f l i g h t (Table V I I I ) . During t h e f l i g h t , d i f f e r e n t a l i q u o t s o f c e l l s were exposed t o a t o t a l d o s e o f from 265 t o 1648 r a d o f 8 5 ~ r gamma r a d i a t i o n w i t h t h e r e s u l t i n g r a d i a t i o n r e s p o n s e c u r v e s b e i n g compared w i t h a p p r o p r i a t e ground c o n t r o l c u r v e s . Neither u l t r a s t r u c t u r a l nor v i a b i l i t y d i f f e r e n c e s w e r e n o t e d between f l i g h t and g r o u n d - c o n t r o l E . c o l i systems. However, w i t h t h e 2. tvphimurium s y s t e m t h e a u t h o r s r e p o r t e d a n i n c r e a s e d c e l l d e n s i t y i n t h e s p a c e - f lown c u l t u r e f l u i d i n d i c a t i n g i n c r e a s e d growth a c t i v i t y . T h i s same r e s u l t was l a t e r d u p l i c a t e d i n c l i n o s t a t s t u d i e s which s u p p l i e d a c o n t i n u a l l y s h i f t i n g g r a v i t a t i o n v e c t o r , d i d n o t a l l o w s e t t l i n g o f c e l l s , and k e p t t h e g r o w t h medium c o n t i n u a l l y a g i t a t e d . Even though t h e r e s u l t a n t i n c r e a s e i n g r o w t h c o u l d be s i m u l a t e d i n t h e c l i n o s t a t t h e a u t h o r s s p e c u l a t e d t h a t t h e mechanism was p r o b a b l y d i f f e r e n t (36, 37).

T e s t a b l e numbers o f p h a g e w e r e n o t p r o d u c e d w i t h t h e g. coli s y s t e m b e c a u s e t h e f l i g h t was s h o r t e r t h a n had b e e n p l a n n e d . I n t h e 5. s y s t e m t h e r e was n o d i f f e r e n c e s i n t h e f r e e P--22 d e n s i t y o f t h e f l i g h t and ground c u l t u r e s , a l t h o u g h t h e s p a c e - f l o w n c e l l s w e r e more r e s i s t a n t t o gamma r a d i a t i o n , a s i n d i c a t e d b y a d e c r e a s e i n p h a g e p r o d u c t i o n . E f f o r t s t o reproduce these r e s u l t s with a c c e l e r a t i o n , v i b r a t i o n , and c l i n o s t a t t e s t s w e r e u n s u c c e s s f u l . T h i s d e c r e a s e i n phage i n d u c t i o n s u p p o r t s t h e r e s u l t s r e p o r t e d f o r t h e E. coli s y s t e m f l o w n on Cosmos 110 b u t i s counter t o t h e r e s u l t s reported f o r a l l of t h e o t h e r Russian coliphage s t u d i e s (34)

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A d d i t i o n a l s p a c e f l i g h t i r r a d i a t i o n s t u d i e s have been conducted which d i d n o t i n v o l v e phage i n d u c t i o n systems (Table I X ) . A v a r i e t y o f microo r g a n i s m s , c a r r i e d a b o a r d t h e Cosmos 368 e a r t h - o r b i t a l s a t e l l i t e , w e r e i r r a d i a t e d w i t h 6 0 ~ ogamma i r r a d i a t i o n b e f o r e f l i g h t a n d / o r a f t e r r e t u r n t o e a r t h . T h e r e was n o e v i d e n c e t h a t t h e s p a c e f l i g h t had s e n s i t i z e d t h e s e s p e c i e s i n a way t h a t a l t e r e d t h e i r v i a b i l i t y o r m u t a b i l i t y ( 1 5 ) . D u r i n g t h e f l i g h t o f Gemini X I , c o n i d i a o f N e u r o s p o r a c r a s s a w e r e e x p o s e d t o a 3 2 b~ e t a s o u r c e , and c e l l s o f t h e same s p e c i e s w e r e e x p o s e d r s o u r c e d u r i n g t h e 45-hour B i o s a t e l l i t e I1 f l i g h t ( 3 8 , 39) t o a 8 5 ~gamma F o r b o t h e x p e r i m e n t s t h e a s s a y e d s y s t e m w a s a g e n e t i c a l l y marked twocomponent h e t e r o k a r y o n w h i c h was h e t e r o z y g o u s f o r t w o d i f f e r e n t g e n e s t h a t control sequential steps i n purine biosynthesis. The e x p o s u r e o f g r o u n d c o n t r o l and i n f l i g h t c e l l s t o a r a n g e o f r a d i a t i o n i n b o t h t e s t s a l l o w e d f o r comparative a n a l y s e s o f dose-response curves.

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A n a l y s e s o f t h e Gemini X I samples i n d i c a t e d t h a t n e i t h e r t h e s u r v i v a l r a t e n o r t h e m u t a t i o n f r e q u e n c y o f c o n i d i a d e p o s i t e d o n membrane f i l t e r s w a s a l t e r e d b y 7 1 h o u r s o f o r b i t a l f l i g h t . However, t h e f l i g h t c e l l s suspended i n a g a r d e m o n s t r a t e d h i g h e r l e v e l s o f s u r v i v a l and l o w e r f r e quencies o f induction, i n d i c a t i n g t h a t t h e s p a c e f l i g h t a f f e c t e d a prot e c t i v e i n f l u e n c e ( 3 9 ) . The a u t h o r s p o i n t o u t t h a t t h e s e d a t a m u s t be c o n s i d e r e d e q u i v o c a l s i n c e t h e y c o u l d have b e e n t h e r e s u l t o f a n o x i a However, when t h e e x p e r i caused by high temperatures i n t h e s p a c e c r a f t . m e n t w a s r e p e a t e d 12 m o n t h s l a t e r i n t h e B i o s a t e l l i t e I1 unmanned o r b i t o r a g a r s u s p e n s i o n s w e r e n o t u s e d and t h i s p o r t i o n o f t h e t e s t was n e v e r r e p e a t e d . As i n t h e G e m i n i X I t e s t , t h e r e w e r e n o d i f f e r e n c e s b e t w e e n t h e f l i g h t and ground c o n t r o l r a d i a t i o n s u r v i v a l c u r v e s o r o v e r a l l i n d u c t i o n . I n a d d i t i o n t o t h e s t u d i e s w i t h ionizing r a d i a t i o n , p o s s i b l e synerg i s t i c r e l a t i o n s h i p s between s p a c e f l i g h t and s o l a r u l t r a v i o l e t l i g h t have a l s o been t e s t e d . The d a t a p r e s e n t e d i n T a b l e X i l l u s t r a t e t h a t t h e T 1 c o l i p h a g e , q. r o q u e f o r t i , a n d t o b a c c o m o s i a c v i r u s (TMV) p a r t i c l e s h a v e b e e n f l o w n o n v a r i o u s s p a c e v e h i c l e s . From t h e s e s t u d i e s , L o r e n z e t a l . (40) concluded t h a t s o l a r u l t r a v i o l e t i r r a d i a t i o n w i t h w a v e l e n g t h s between 200 a n d 300 nm was t h e m a i n c a u s e o f i n f l i g h t i n a c t i v a t i o n o f t h e s e microo r g a n i s m s . T h e s e d a t a d o n o t d i f f e r from t h e r e s u l t s o f ' t h e many l a b o r a t o r y W - r e s p o n s e e x p e r i m e n t s , s u g g e s t i n g t h a t g r o u n d - b a s e d s t u d i e s may be u s e d a s model s y s t e m s f o r p r e p a r a t i o n of i n f l i g h t e x p e r i m e n t s .

En a n o t h e r s t u d y , prepared by a group o f American and European i n v e s t i g a t o r s , e i g h t m i c r o b i a l s p e c i e s were exposed t o s o l a r W and space vacuum o u t s i d e of t h e ApoZlo 3.6 command module d u r i n g i t s r e t u r n from t h e Moon ( 4 1 , 4 2 ) . The use o f v a r i o u s combinations of o p t i c a l f i l t e r s t o p r o v i d e exposure o f d i f f e r e n t t e s t a l i q u o t s t o v a r y i n g amounts of s o l a r i r r a d i a t i o n a t peak wavelengths o f 254, 280, and 300 run, allowed f o r a d i f f e r e n t dose-response curve a t e a c h o f t h e s e t h r e e w a v e l e n g t h s ( 4 3 ) . The T-7 b a c t e r i o p h a g e p r e p a r a t i o n s of E . c o l i which were exposed t o i n f l i g h t i r r a d i a t i o n were found t o b e more s e n s i t i v e t o W l i g h t t h a n were i r r a d i a t e d ground c o n t r o l s ( 4 4 ) . There were no s i g n i f i c a n t d i f f e r e n c e s r e p o r t e d between p o s t f l i g h t s u r v i v a l r a t e s of n o n - i r r a d i a t e d f u n g a l c e l l s when compared w i t h a p p r o p r i a t e ground c o n t r o l s ( 4 5 ) a l t h o u g h t h e s u r v i v a l r a t e o f space-flown Chaetomium qlobosum, Rhodotorula r u b r a , and Saccharomyces c e r e v i s i a e was s l i g h t l y d e p r e s s e d and samples o f Trichophyton t e r r e s t r e , and 2. c e r e v i s i a e demonstrated some s e n s i t i v i t y t o i n f l i g h t s o l a r W when measured i n terms o f a l o s s o f c e l l v i a b i l i t y ( c o r r e s p o n d i n g ground c o n t r o l d a t a were n o t r e p o r t e d ) . No changes i n s u r v i v a l r a t e , m u t a t i o n r a t e , o r t o x i n p r o d u c t i o n could be d e t e c t e d w i t h p o s t f l i g h t a n a l y s e s of B z c i l l u s t h u r i n q i e n s i s and Aeromonas p r o t e o l y t i c a ( 4 6 ) . However, it was r e p o r t e d t h a t t h e combination o f s o l a r W and s p a c e vacuum r e s u l t e d i n a g r e a t e r l o s s of v i a b i l i t y i n d r i e d B a c i l l u s s u b t i l i s c u l t u r e s t h a n w i t h W a l o n e , i n d i c a t i n g t h a t t h e s p o r e s were s e n s i t i z e d t o W by t h e vacuum ( 17)

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C e l l S t u d i e s With M u l t i c h a r g e d , High Energy (HZE)

,

Cosmic P a r t i c l e s

Experiments designed t o s t u d y t h e b i o l o g i c a l e f f e c t s of i n d i v i d u a l heavy n u c l e i of cosmic r a d i a t i o n d u r i n g s p a c e f l i g h t o u t s i d e t h e magnetos p h e r e o f t h e E a r t h have been r e p e a t e d l y conducted b y a c o n s o r t i u m o f European i n v e s t i g a t o r s ( 4 7 , 4 8 ) . These e x p e r i m e n t s were housed i n t h e BIOSTACK, a complex package c o n s i s t i n g o f a l t e r n a t i n g l a y e r s o f n u c l e a r t r a c k d e t e c t o r s , and b i o l o g i c a l o b j e c t s imbedded i n p o l y v i n y l a l c o h o l ( P V A ) . Among o t h e r s p e c i e s , s p o r e s o f B a c i l l u s s u b t i l i s and c y s t s o f A r t e m i a s a l i n a were exposed t o HZE p a r t i c l e s d u r i n g t h e f l i g h t s o f A p o l l o 1 6 , 1 7 , and t h e Apollo-Soyuz T e s t P r o j e c t (Table X I ) Individual c e l l s o r cysts i n the p a t h of HZE p a r t i c l e s were e v a l u a t e d f o r g e r m i n a t i o n , o u t g r o w t h , and p r o d u c t i o n of abnormals. The f i r s t v e g e t a t i v e c e l l s i s s u i n g from b a c t e r i a l s p o r e s l y i n g i n t h e p a t h of high e n e r g y , m u l t i c h a r g e d p a r t i c l e s were f r e q u e n t l y found t o b e abnormally s w o l l e n . Artemia s a l i n a c y s t s , l y i n g a l o n g n u c l e a r t r a c k s , showed reduced h a t c h i n g and l a r v a l emergence and an i n c r e a s e i n t h e i n c i d e n c e o f developmental anomalies.

.

I n a f u r t h e r a t t e m p t t o u n d e r s t a n d t h e e f f e c t o f g a l a c t i c HZE p a r t i c l e s upon b i o l o g i c a l o b j e c t s , S o v i e t i n v e s t i g a t o r s i n c l u d e d t h e y e a s t Saccharomyces c e r e v i s i a e i n t h e "Bioblock" which was aboard t h e 2 month Cosmos 613 e a r t h o r b i t a l f l i g h t . Although many o f t h e c o l o n i e s d i d n o t s u r v i v e t h e long s t o r a g e , a t e n - f o l d i n c r e a s e i n t h e i n c i d e n c e o f " r a d i a t b damaged c e l l s " w a s r e p o r t e d ( 4 9 ) .

CONCLUSIONS

The a b o v e r e v i e w h a s i l l u s t r a t e d t h a t , w h e r e a s a l a r g e v a r i e t y o f cell b i c l o g y s t u d i e s h a v e b e e n c o n d u c t e d i n s p a c e , c o n s i s t e n t s p a c e m e d i a t e d a l t e r a t i o n s have n o t b e e n i d e n t i f i e d . A l t h o u g h i n d i v i d u a l s k u d i e s o f t e n produced e q u i v o c a l d a t a , e v a l u a t i o n o f t h e a g g r e g a t e r e s u l t s i n d i c a t e s t h a t c e l l systems a r e g e n e r a l l y no l e s s s t a b l e i n space t h a n t h e y a r e i n t h e E a r t h - b a s e d l a b o r a t o r y . Of c o u r s e t h e c o n d i t i o n s t o which c e l l systems a r e exposed i n s p a c e a r e u s u a l l y l e s s w e l l c o n t r o l e d (and less c o n t r o l a b l e ) , o f t e n l e a d i n g t o more v a r i a b l e a n d e r r a t i c r e s u l t s . It has n o t y e t been demonstrated t h a t t h e s p a c e f l i g h t environment On t h e c o u l d b e u s e d t o a f f e c t u n i q u e o r h i t h e r t o unknown c e l l c h a n g e s . c o n t r a r y , c e l l systems appear t o remain s u f f i c i e n t l y stable t o permit e x p e r i m e n t a t i o n w i t h models which r e q u i r e a f i x e d c e l l l i n e . T h e r e f o r e , t a k e n as a u n i t , t h e c e l l biology s t u d i e s conducted d u r i n g t h e preceding I t i s now p o s s i b l e two d e c a d e s s h o u l d d e f i n i t e l y b e c o n s i d e r e d a s u c c e s s . t o p r e p a r e c e l l b i o l o g y e x p e r i m e n t s f o r t h e Space S h u t t l e era w i t h a r e a s o n a b l e p r o b a b i l i t y t h a t t h e c e l l s w i l l n o t react e n g i m a t i c a l l y t o t h e unique environment encountered w i t h i n t h e s p a c e c r a f t .

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Some Zhukov-Verezhnikov, N . N . : Mayskiy, I . N . ; e t a l . R e s u l t s and P r o s p e c t s o f S t u d y i n g t h e B i o l o g i c a l A c t i o n o f S p a c e R a d i a t i o n and Dynamic F l i g h t F a c t o r s w i t h t h e H e l p o f M i c r o b i o l o g i c a l and C y t o L o g i c a l Models. Problemy Kosmicheskay M e d i t s i n y , V. V . P a r i n , e d . , 1966, pp. 172173. M a t t o n i , R. H. T.: S p a c e - F l i g h t E f f e c t s and Gamma R a d i a t i o n I n t e r a c t i o n on Growth and I n d u c t i o n o f L y s o g e n i c B a c t e r i a . B i o S c i e n c e , v o l . 1 8 , 1968, pp. 602-608. M a t t o n i , R. H. T.; K e l l e r , E. C . , Jr., E b e r s o l d , W. T.; E i s e r l i n g , I?. A , ; and Romig, W . R.: Induction of Lysogenic B a c t e r i a i n t h e Space Environment. The ~ x p e r i m e n t so f B i o s a t e l l i t e I I . , J. S a u n d e r s , e d . , NASA SP-204, 1971, pp. 309-324.

de S e r r e s , F. J.: Mutagenic E f f e c t i v e n e s s o f Known Doses of R a d i a t i o n i n Combination w i t h Zero G r a v i t y on crassa. The Experiments o f B i o s a t e L l i t e II., J, S a u n d e r s , e d . , NASA SP-204, 1971, pp. 325-331. de S e r r e s , F . J . ; M i l l e r , I . R . ; Smith, D. B . ; Kondo, S . ; and' Bender, M. A . : The Gemini-XI S-4 Space F l i g h t R a d i a t i o n I n t e r a c t i o n Experiment. I I . A n a l y s i s o f S u r v i v a l L e v e l s and Forward-Mutation F r e q u e n c i e s i n Neurospora c r a s s a . R a d i a t i o n Res., v o l . 39, 1969, pp. 436-444. Survival Lorenz, P. R . , O r l o b , G . B . ; and Hemenway, C. L.: of Microorganisms i n Space: Comparison o f S u r v i v a l Data Obtained i n F i f t e e n Balloon-Rocket-, and S a t e l l i t e - B o r n e Exposure Experiments w i t h I n c i d e n t S o l a r Photons. Space L i f e S c i e n c e s , v o l . 1, 1969, pp. 491-500. T a y l o r , G . R . ; S p i z i z e n , J . ; F o s t e r , B . G . ; Volz, P. A . ; ~ i i c k e r ,H . ; Simmonds, R. C . ; Heimpel, A. M . ; and Benton, E. V . : A D e s c r i p t i v e A n a l y s i s o f t h e A p o l l o 16 M i c r o b i a l Response t o Space Environment Experiment. - B i o S c i e n c e , v o l . 24, 1974, pp. 505-511. T a y l o r , G . R.: Background and G e n e r a l Design o f t h e M i c r o b i a l Response t o Space Environment Experiment (M191) System. P r o c e e d i n g s of t h e M i c r o b i a l Response t o Space Environment Symposium, G . T a y l o r , e d . , NASA TMX-58103, 1973, pp. 3-19. Physical T a y l o r , G. R . ; B a i l e y , J . V . ; and Benton, E. V . : D o s i m e t r i c E v a l u a t i o n s i n t h e A p o l l o 16 M i c r o b i a l Response Experiment. L i f e S c i e n c e s and Space Research, v o l . 13, Akademie-Verlag, B e r l i n , 1975, pp. 135-141. Effects S p i z i z e n , J . ; Isherwood, J . E . ; and T a y l o r , G. R.: o f S o l a r U l t r a v i o l e t R a d i a t i o n on B a c i l l u s s u b t i l l i s s p o r e s and T-7 B a c t e r i o p h a g e . L i f e S c i e n c e s and Space Research, v o l , 13, Akademie-Verlag, B e r l i n , 1975, pp. 143-149. Volz, P. A . : Mycological S t u d i e s Housed i n t h e A p o l l o 16 M i c r o b i a l Ecology E v a l u a t i o n Device. Proceedings of t h e M i c r o b i a l Response t o Space Environment Symposium, G. T a y l o r , ed., NASA TMX-58103, 1973, pp. 121-135.

Simmonds, R . C . ; Wrenn, R . T . ; Heimpel, A . M.; and T a y l o r , 6 . 8. ; P o s t f l i g h t A n a l y s e s o f B a c i l l u s Organisms Exposed t o S p a c e f l i g h t C o n d i t i o n s on A p o l l o 1 6 , Aerospace M e d i c i n e , v o l . 4 5 , #11, 1 9 7 4 , pp. 1244-1247. B b c k e r , H.: The B i o s t a c k E x p e r i m e n t s I and I1 Aboard A p o l l o 16 and 17. Life S c i e n c e s and Space R e s e a r c h X I , 1 9 7 4 , p p , 43-50. BEcker, H , ; F a c i u s , R . ; H i l d e b r a n d , D . ; H o r n e c k , G . ; R e i t z , G ; et al.: B i o s t a c k 111: Experiment MA-107. A p o l l o Soyuz T e s t P r o j e c t P r e l i m i n a r y S c i e n c e s R e p o r t , NASA Document TM X-58173, 1976, pp. 14-1 t h r o u g h 14-27. Benevolensky, V . N . ; Marenny, A . . M . ; S o l y a n o v , B. I . ; Abromova, V. M.; V a k s k i n a , L . K . ; S a k o v i t c h , I . S . ; e t a l . : R a d i o b i o l o g i c a l Experiment To S t u d y t h e E f f e c t o f t h e Heavy N u c l e i o f t h e G a l a c t i c Cosmic R a d i a t i o n on Board a n A r t i f i c i a l E a r t h S a t e l l i t e "Cosmos-613". Kosmicheskia B i o l o g i i a i M e d i t s i n a , i n p r e s s , 1976. Antipov, V. V . ; B i o l o g i c a l S t u d i e s Aboard t h e S p a c e c r a f t "Vostok" and "Voskhod". In: P r o b l e m s o f S p a c e B i o l o g y , N . M. S i s a k y n , e d . , Nauka P r e s s , M o s c o w , 1967, pp. 67-83. E f f e c t s o f S o l a r I r r a d i a t i o n on E x t r a c e l l u l a r F o s t e r , B. G . ; In: Proceedings of t h e Enzymes o f Aeromonas p r o t e o l v t i c a . M i c r o b i a l Response t o Space Environment Symposium. G . T a y l o r , e d . , NASA, L. B. J o h n s o n Space C e n t e r , TM X-58103, 1973, pp. 139-151. G l e m b o t s k i y , Ya. L . ; Prokof'yeva-Belgovskaya, A. A . ; Sharnina, Z . B . ; Khovstova, V. V . ; V a l e v a , S. A . ; E y g e s , N. S . ; and I n f l u e n c e o f S p a c e - f l i g h t F a c t o r s on Nevzgodina, L. V . : H e r e d i t y and Development i n Actinomyces and Higher-Order N . M. S i s a k y a n , Plants. In: Problems o f Space B i o l o g y . e d . , U.S.S.R. Academy o f S c i e n c e s P u b l i s h i n g House, Moscow, 1962, pp. 259-271. The Khvostova, V. V . ; S i d o r o v , B. N . ; and S o k o l o v , N . N.: E f f e c t o f Space F l i g h t C o n d i t i o n s on t h e S e e d s o f H i g h e r P l a n t s and A c t i n o m y c e t e s . In: Problems o f Space B i o l o g y , N. M. S i s a k y a n and V. I. Yazdovskiy, e d s . , 1962, pp. 161178.

.

54.

K o v y a z i n , N. V ; L u k i n , A . A . ; a n d P a r f e n o v , G . P : The E f f e c t o f Cosmic F l i g h t F a c t o r s o f "Vostok-2" o n M i c r o o r g a n i s m s : S t u d i e s on Y e a s t s o f D i f f e r e n t P l o i d y . Pskusstvennye Sputnik: Z e m l i , A k a d , Mauk. U . S , S . R . , 1 9 6 2 , v o l , 1 3 , pp, 1 2 3 - 1 2 9 .

55,

von B o r s t e l , R . C.; S m i t h , R . H.; W h i t i n g , A . R . ; a n d G r o s c h , D. S.: M u t a t i o n a l and P h y s i o l o g i c Responses o f Habrobracon i n B i o s a t e l l i t e 11. I n : The E x p e r i m e n t s o f B i o s a t e l l i t e 11, J. S a u n d e r s , e d . , NASA SP-204, 1 9 7 1 , pp. 17-39.

56.

P l a n e l , H. ; S o l e i l h a v o u p , J . P . ; B l a n q u e t , Y . ; a n d K a i s e r , R. : S t u d y o f C o s m i c Ray E f f e c t s on A r t e m i a s a l i n a E g g s D u r i n g T h e Apollo 16 and 17 F l i g h t s . L i f e S c i e n c e s And S p a c e R e s e a r c h , 1 9 7 4 , v o l . 1 2 , p p . 85-89.

TABLE I ,

-

SPACE-FLOWN ISOLATED VIRUSES

Gemini IXA Tobacco m o s a i c v i r u s Gemini X/ Aqena V I I I Gemini X I 1 Poliomyelitis virus

Vaccinia v i r u s

U.S.

I

I

Lorenz 1968

Dry

10

H o t c h i n 1969

9

H o t c h i n 1969

9

Balloon

-

Gemini X I 1 I

Influenza virus

U.S.S.R.

Unknown

J e n k i n s 1968

Influenza (PR-8 s t r a i n ) Canine h e p a t i t i s I n f e c t i o u s bovine Rhinotracheitis

Gemini XI1

I

H o t c h i n 1969

9

TABLE 11,

Escherichia K-12/K-12

Escherichia

&

&T

-

SPACE-FLOWN BACTERIAPHAGE AND HOST

Antipov 1967

50

Jenkins 1968

6

Hotchin 1969

Jenkins 1968

Salmonella

Aerobacter

6

, TABLE

Escherichia (

111.

-

SPACE-FLOWN BACTERIA

coli K-12

1

Escherichia

coli B

Parfenov 1973

7

Agar c u l t u r e s Aerobacter aeroqenes 1321 Staphylococcus aureus

Bacillus subtilis ATCC 6 0 5 2

Verezhnikov 1966

Parefenov 1973

7

Hotchin 1969

9

Bccker 1976

lembotskiy 1962 S t r e p t o r n ~ c e se r y t h r a e u s 8594

35

48

52

TABLE I V .

-

SPACE-FLOWN YEASTS AND FILAMENTOUS FUNGI

Flight Z yqosaccharomyces

Cosmos 368

I

/ (Zyqosaccharomyces) 40-2587 ( h a p l o i d )

1 I

Cosmos 368 Saccharomyces ( d i p l o i d ) 139-B

Saccharomyces cerevisiae

C e l l s on Aqar Suspensions both unsensitized and sensitized with o l i c acid

, On a g a r and i n aqueous suspension

I

Kovyazin 1962

54

G r i g o r y e v 1972

15

Cosmos 613

C o l o n i e s on agar (0.5 1 . 0 rnm d . )

B e n e v o l e n s k y 1976

49

Voskhod 1

Suspensions both unsensitized and sensitized with o l i c acid

Kovyazin 1962

54

Apollo 16

W Exposure

Volz 1973

45

U.S.

Dry s p o r e s ( 3 4 km a l t i tude f o r 6 h r s .) Dry s p o r e s

1

Penicillium roqueforti

Neurospora c r a s s a

Balloon

Gemini X I 1 Gemini IXA

P a r f e n o v 197 3

7

H o t c h i n 1969 I l o t c h i n 1969 H o t c h i n 1969

9 9 9

Dry s p o r e s Gemini X/ Agena V I I I Biosatellite 11 (p.-1037)

Gemini X I Neurospora s p e c i e s

Nerv I

Discoverer XVIII

Dry s p o r e s '5srll~lk~~own Dry s p o r e s phosphorus32 (32 ) - a and mefaboli z i n g spore suspension 32p-b 1900 Km altitude for 28 min Dry S p o r e s

DeSerres 1971 1Iotchin 1969

D e S e r r e s 1969

' J e n k i n s 1968

38 9

39

6

Chaetomium qlobosum Trichophyton t e r r e s t r e A p o l l o 16

W Exposure

V o l z 1973

45

Zond 8

On Agar

Romanova 1 9 7 1

13

Rhodotorula r u b r a Cand i d a t r o p i c a l i s SK-4

TABLE V.

-

SPACE-FLOWN PROTOZOANS

Species Colpoda c u c u l l u s

A p o l l o 17 (Biostack 11)

C y s t s i n monolayers of polyvinyl I alcohol

I

,

Pelomyxa c a r o l i n e n s i s (giant multinucleate Amoeba)

B i o s a t e l l i t e I1

Amoeba

C-131 A i r c r a f t i n Keplerian trajectory

Paramecium aurelia

USSR B a l l o o n

I

Dividing, Free feeding c e l l s

47

I

1

Abel 1971 Ekberg 1971

23 22

Growing c e l l s

McKinney 1 9 6 3

24

Growing

P l a n e 1 1975

21

TABLE V I .

-

SPACE-FLOWN CELLS I N SMALI GROUPS

Young 1971

25

D e v e l o p i n g egg: from f i r s t cleavage

Young 1968

26

F e r t i l e Frog Eggs

Apenchenko 1975

32

Dry B l a s t o c y s t s

voi Borstel

55

I

Gemini 8 Frog Eggs Gemini 12

Frog Eggs 1

Soyuz 17/ Salyut 4 B i o s a t e l l i t e I1

197 1

Apollo 16 (Biostack I ) A p o l l o 17 B i o s t a c k 11) ASTP ( B i o s t a c k 111)

Artemia s a l i n a ( B r i n e shrimp)

Carausius

Encysted blastula i n monolayers o f polyvinyl alcohol

A p o l l o 17 (Biostack 11)

morosus (grasshopper)

Eggs i n monolayers of polyvinyl alcohol 32-336 h r embryos i n sea water

ASTP Fundu l u s heteroclitus (killifish)

C

Skylab 3 Cosmos 782

Danio r e r i o -(fish)

Soyuz 1 6

WI-38 d i p l o i d human embryonic lung c e l l s

Skylab 3

5-day o l d f e r t i l e eggs i n sea water 32-i28 h r embryos i n flea w a t e r Fertilized eggs

Izvestiya 8 Dec. 1974

Growing c u l t u r e s from single c e l l s

Montgomery 1974

S c h e l d 1976

30

-

S e r i a n Hamster cells Carrot Tissue culture

Soyuz 17/ Salyut 4

II

Cosmos 7 8 2

1I

Tissue culture

Apenchenko

Crown g a l l and proembryonic

S c h e l d 1976

28

TABLE VII.

- WJOR

SPACEFLIGHT STUDIES WITH GROWING CELLS \

Biosatell i te

No biological indications of HZE damage.

TABLE VIII.

-

BACTERIOPHAGE

Voskhod 1 & 2

INDUCTION SYSTEMS TESTED IN SPACE

Simulated launch vibration plus 6 0 ~ o7 irradiation gave increases higher than irradiation alone.

ZOND 5 and 7

Biosatell ite

B5sr

Y

inflight.

Flight terminated early, no

TABLE I X .

-

ADDITIONAL SPACEFLIGHT STUDIES WITH RADIATION SOURCES

Zygosaccharomyces

3amna ( p r e f l i g h t and

radiosensi t i v i t y

baili (haploid)

postflight) 3 2 ~ beta

Neurospora

:inflight)

conidia

N e i t h e r s u r v i v a l r a t e o r mutation frequency

crassa

Gemini X I

altered f o r dry cells.

B e t t e r s u r v i v a l and

lower mutation frequency f o r agar-suspended cells

85~r gamna

Neurospora crassa

:inflight)

Biosatellite

coni d i a

TABLE X.

- INFLIGHT

FLIGHT

6 sounding rockets 6 balloon f l i g h t s

I

CELL STUDIES WITH ULTRAVIOLET IRRADIATION

EVENT

II

No i n f l i g h t e f f e c t on d r y c e l l s

II

TEST SYSTEM

Exposed To Direct

RESULTS

T 1 Coliphage

Confirms t h a t UV between 200 and

UV

Escherichia

coli

F l i g h t specimens more s e n s i t i v e t o UV

T-7 bacteriophage

than ground c o n t r o l s although shape o f

I Exposed To D i r e c t UV plus Apollo 16

Components

dose response curves s i m i l a r .

No evidence o f synergism between i n f l i g h t Chaetomi uw globosum

UV i r r a d i a t i o n and reduced g r a v i t y

T r i chophyton t e r r e s t r e

at

1

NO change i n s u r v i v a l r a t e a t 1 atm.

254, 280, and 300 nm

Baci 11us s u b t i 1i s -

Combined UV and vacuum r e s u l t e d i n g r e a t e r l o s s o f v i a b i l i t y than UV alone. (Spores s e n s i t i z e d t o UV by vacuum) No change i n s u r v i v a l r a t e s . i n a b i l i t y t o produce t o x i n s

99

No change

II

TABLE X I .

-

CELL STUDIES WITH COSMIC HZE* PARTICLES

Bacillus subtilis -

S w e l l i n g d u r i n g growth o f f i r s t v e g e t a t i v e c e l l s from " h i t "

Apollo 16 and 17 Those ' ] h i t u by HZE showed r e d u c t i o n Artemiasal i n a -

i n l a r v a l emergence and hatching. Incidence o f developmental anomali

Z A 8 and .I2 h i t s w i t h 1.3% o f c e l l s d e m n s t r a t e d " r a d i a t i o n damage" compared w i t h 0.15% normally. 4 2x10 c e l l s damaged p e r p a r t i c l e .

*HE=

Heavy (high atomic number) high-energy p a r t i c l e s

I

0

20

60 80 Duration of flight, hr

40

100

120

Figure 1.- E f f e c t of d u r a t i o n of Vostok space missions on K-12 ( A ) bacteriophage i n d u c t i o n i n Escherichia from d a t a compiled i n r e f e r e n c e 6. V t o V denote 1 6 Vostok f l i g h t number. Space-flight-effect f a c t o r = number of bacteriophage p a r t i c l e s p e r ground c o n t r o l c e l l .

GRAVITY AND THE CELL:

-

V

--&'

-p

INTRACELLULAR STRUCTURES AND STOKES SEDIMENTATION

By P a u l Todd, The Pennsylvania S t a t e U n i v e r s i t y , U n i v e r s i t y P a r k , P e n n s y l v a n i a

ABSTRACT P l a n t and c e r t a i n animal embryos a p p e a r t o b e r e s p o n s i v e t o t h e g r a v i t y v e c t o r d u r i n g e a r l y s t a g e s of development. The s e n s i n g of g r a v i t y of i n d i v i d u a l c e l l s c o u l d b e b a s e d upon c o n v e c t i o n of p a r t i c l e s e d i m e n t a t i o n . Various i n t r a c e l l u l a r p a r t i c l e s have been proposed a s g r a v i t y s e n s o r s i n t h e c e l l s of d e v e l o p i n g p l a n t s , and t h e p a r t i c i p a t i o n of a m y l o p l a s t s and dictyosomes h a s been s u g g e s t e d b u t n o t proven. An e x p l o r a t i o n of t h e mammalian c e l l f o r s e d i m e n t i n g p a r t i c l e s r e v e a l s t h a t t h e i r e x i s t e n c e i s u n l i k e l y , e s p e c i a l l y i n t h e p r e s e n c e of a network of m i c r o t u b u l e s and m i c r o f i l a m e n t s c o n s i d e r e d t o b e r e s p o n s i b l e f o r i n t r a c e l l u l a r o r g a n i z a t i o n . D e s t r u c t i o n of t h e s e s t r u c t u r e s r e n d e r s t h e c e l l s u s c e p t i b l e t o a c c e l e r a t i o n s s e v e r a l t i m e s g. Large d e n s e p a r t i c l e s , such a s chromosomes, n u c l e o l i , and c y t o p l a s m i c o r g a n e l l e s a r e a c t e d upon by f o r c e s much l a r g e r t h a n t h a t due t o g r a v i t y , and t h e i r p o s i t i o n s i n t h e c e l l a p p e a r t o be insensitive t o gravity. INTRODUCTION Space Biology Research was o r i g i n a l l y d e s i g n e d t o answer t h e q u e s t i o n , Is Space S a f e ? , and t h e n e x t phase of r e s e a r c h i s d e s i g n e d around t h e u s e of t h e c o n d i t i o n s of s p a c e f l i g h t a s a b i o l o g i c a l r e s e a r c h t o o l . The l a t t e r p h a s e i s d e s i g n e d t o answer s u c h q u e s t i o n s a s , Can We Learn Something of Fundamental S i g n i f i c a n c e by Performing Experiments Under Space F l i g h t C o n d i t i o n s and Obtain B i o l o g i c a l I n s i g h t s t h a t Cannot b e Acquired on t h e Ground? At t h e i n c e p t i o n of s p a c e r e s e a r c h some 20 y e a r s ago, t h e r e was c o n c e r n i n b o t h t h e U.S. and t h e S o v i e t Union about t h e e f f e c t s of w e i g h t l e s s n e s s on l i v i n g t h i n g s . It needed t o b e known i n p a r t i c u l a r whether t h e a b s e n c e of g r a v i t y had no e f f e c t o r a c a t a s t r o p h i c e f f e c t on b i o l o g i c a l systems under s p a c e f l i g h t conIt was e a s y t o s o l v e problems i n t r o d u c e d by t h e s p a c e environment by t h e u s e ditions. of e n g i n e e r i n g t o p r o t e c t a g a i n s t t h e l a c k of a n atmosphere and t h e p r e s e n c e of r a d i a t i o n , b u t e n g i n e e r i n g a g a i n s t w e i g h t l e s s n e s s and i t s p o s s i b l e b i o l o g i c a l e f f e c t s proved t o b e e x t r e m e l y d i f f i c u l t . F o r t u n a t e l y , e a r l y experiments i n d i c a t e d t h a t t h e b i o l o g i c a l e f f e c t s of z e r o G was c e r t a i n l y n o t c a t a s t r o p h i c and t h e 84-day S k y l a b m i s s i o n s u f f e r e d no c a t a s t r o p h e s a s a consequence of t h e absence of a g r a v i t a t i o n a l f i e l d . I n view of t h e c o n c l u s i o n t h a t t h e a b s e n c e of g r a v i t y h a s no c a t a s t r o p h i c e f f e c t on man i n s p a c e , f u t u r e r e s e a r c h i s d i r e c t e d a t t h e b a s i c s t u d y of what we presume t o b e g r a v i t y dependent environmental r e s p o n s e s . I n o t h e r words, s p a c e f l i g h t c o n d i t i o n s a r e t o b e made a v a i l a b l e f o r b a s i c s c i e n c e experiments. Due t o volume l i m i t a t i o n s and o t h e r l i m i t a t i o n s on s p a c e c r a f t , i t i s l o g i c a l t o b e g i n w i t h r e s e a r c h a t t h e c e l l u l a r level. Although we know of many b i o l o g i c a l phenomena a f f e c t e d by g r a v i t y , t h e i r connect i o n t o m o l e c u l a r and p h y s i c a l c o n c e p t s a r e e x t r e m e l y p o o r l y understood. In this s e n s e , t h e e f f e c t of g r a v i t y i s p a r a d o x i c a l b e c a u s e t h e c e l l i s t h e b a s i c s t r u c t u r e of l i v i n g t h i n g s , and t h e organisms' p r o p e r t i e s depend upon c e l l s . Yet i t is much e a s i e r t o t h i n k of g r a v i t y a s a c t i n g on l a r g e r systems a s c e l l s a r e a t t h e l i m i t of s i z e and mass which i s i n f l u e n c e d by t h e g r a v i t a t i o n a l f i e l d . DEVELOPING SYSTEMS The e f f e c t of abnormal g r a v i t a t i o n a l exposure upon embryonic development was n o t e d d u r i n g t h e p r e v i o u s c e n t u r y ( 1 ) . The most remarkable gravity-dependent phenomena i n

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developmental b i o l o g y i n c l u d e t h e obvious p o l a r i z a t i o n of amphibian egg c e l l d i v i s i o n a t e a r l y s t a g e s and t h e r e l i a b l e upward growth of c o l e o p t i l e s and downward growth of r o o t s i n germinating p l a n t s e e d l i n g s . It should be no s u r p r i s e t h a t t h e s e phenomena have been t h e f a v o r i t e s u b j e c t s of i n v e s t i g a t i o n s of t h e e f f e c t s of g r a v i t y compens a t i o n and w e i g h t l e s s n e s s ( 2 , 3 ) . Amphibian Embryos The i n v e r s i o n of embryos of Rana sp. b e f o r e t h e y reach t h e 4 - c e l i s t a g e can l e a d t o t h e formation of double embryos ( 1 , 4 ) . G r a v i t y compensat i o n and c e n t r i f u g a t i o n E v i d e n t l y , v e r y soon can l e a d , under a p p r o p r i a t e c o n d i t i o n s , t o s i m i l a r e f f e c t s ( 4 , 5 ) . a f t e r f e r t i l i z a t i o n , e v e n t s occur which o r i e n t t h e egg and e s t a b l i s h t h e p l a n e s of f u r t h e r c e l l d i v i s i o n s and t h e u l t i m a t e symmetry of t h e organism. The g r a v i t y s e n s i n g mechanism i n t h i s system i s thought t o be a s s o c i a t e d w i t h a d e n s i t y g r a d i e n t i n t h e m a t e r i a l s of t h e yolk. Attempts t o induce developmental a b n o r m a l i t i e s i n w e i g h t l e s s n e s s d u r i n g o r b i t a l f l i g h t of Rana eggs y i e l d e d n e g a t i v e r e s u l t s ( 6 , 7 , 8 ) , presumably because exposure t o w e i g h t l e s s n e s s d i d n o t a d e q u a t e l y c o i n c i d e w i t h t h e g r a v i t y - s e n s i t i v e p e r i o d of o r i e n t a t i o n o r p o s s i b l y because Rana p i p i e n s , used i n o r b i t a l experiments, i s n o t a s s e n s i t i v e t o o r i e n t a t i o n a s Rana f u s c a , which was used i n c l a s s i c a l experiments ( 1 ) . There was a l s o no microscopic evidence f o r t h e r e d i s t r i b u t i o n of morphological s t r u c t u r e s d u r i n g o r b i t a l weightlessness (8). P l a n t Geotropism ,Cytological s t u d i e s on t h e d i s t r i b u t i o n of amyloplasts i n wheat s e e d l i n g s flown on B i o s a t e l l i t e I1 l e d t o t h e c o n c l u s i o n t h a t t h e s e g r a n u l e s were d i s t r i b u t e d a t random under w e i g h t l e s s n e s s , a s i n s e e d l i n g s grown on a c l i n o s t a t , r a t h e r t h a n b e i n g clumped on t h e lower c e l l w a l l as i n e r e c t c o n t r o l s e e d l i n g s (9,lO). F i x a t i o n experiments i n d i c a t e d t h a t t h e s e p l a s t i d s r e t u r n t o t h e i r normal p o s i t i o n i n t h e c e l l i n l e s s t h a n 4 h r . These o r g a n e l l e s were observed because t h e y are thought by some (11,12), b u t n o t o t h e r s (13) t o p l a y a r o l e as " s t a t o 1 i t h s " - - i n t r a c e l l u l a r i n d i c a t o r s of t h e g r a v i t y v e c t o r . The i d e n t i f i c a t i o n of " s t a t o l i t h s " , however, depends on t h e a b i l i t y of t h e p l a n t physiol o g i s t t o d i s t i n g u i s h between cause and e f f e c t . It remains t o b e determined whether t h e e l o n g a t i o n p l a n t c e l l responds t o sedimenting amyloplasts o r p o s i t i o n s i t s amylop l a s t s i n response t o a m e t a b o l i c g r a d i e n t formed by a c t i v i t i e s o t h e r t h a n s e d i m e n t a t i o n . Other p l a n t c e l l o r g a n e l l e s have been c o n s i d e r e d w i t h r e s p e c t t o p o s s i b l e r o l e s i n geotropism. These i n c l u d e mitochondria (14) and t h e Golgi a p p a r a t u s (15-18). The d i c t y osomes of t h e Golgi a p p a r a t u s , d e s p i t e t h e i r g e n e r a l l y accepted r e l a t i o n s h i p t o i n t e r n a l membranes, appear t o be p o s i t i o n e d i n a manner s t r o n g l y r e l a t e d t o t h e g r a v i t y v e c t o r 6 , 7 Whether t h e y a r e s e r v i n g a s s t a t o l i t h o r responding t o m e t a b o l i c g r a d i e n t s is unknown, b u t one might c o n s i d e r t h e f o l l o w i n g m e t a b o l i c i n t e r r e l a t i o n s h i p s a s a t e s t a b l e a l t e r n a t i v e t o t h e s t a t o l i t h theory: 1 ) C e l l w a l l compression produces a membrane response. 2 ) T h i s response consumes auxin. 3) Auxin i s t r a n s p o r t e d down i t s g r a d i e n t . 4) C e l l w a l l s y n t h e s i s i s s t i m u l a t e d . 5) New w a l l s y n t h e s i s d e p l e t e s Golgi p r o d u c t s . 6) The c e l l produces more a c t i v e dictyosomes. 7 ) Golgi forms i n d i r e c t i o n of t h e s e c r e t i o n ( a s i n animal systems). ORGANELLES I N MAMMALIAN CELLS Animal c e l l s d i f f e r e x p l i c i t l y from p l a n t c e l l s i n t h e i r l a c k of a need t o synthesi z e a c e l l w a l l i n a p a r t i c u l a r d i r e c t i o n . I f p l a n t c e l l s need t o respond t o g r a v i t y f o r t h i s purpose o n l y , t h e n one would n o t e x p e c t t h e i n t r a c e l l u l a r a c t i v i t i e s of animal c e l l s t o be v e r y r e s p o n s i v e t o g r a v i t y . An a n a l y s i s of t h e c o n s t i t u e n t s of t h e mammalian c e l l should i n d i c a t e whether o r n o t t h e r e e x i s t any o r g a n e l l e s t h a t can sediment under the i n f l u e n c e of g r a v i t y . B i o p h y s i c a l r e s e a r c h i n t h e p a s t decade h a s added c o n s i d e r a b l y

t o our knowledge of t h e s t r u c t u r a l and hydrodynamic p r o p e r t i e s of chromosomes, plasma membranes, n u c l e a r membranes, cytoplasm, nucleoplasm, chromatin, n u c l e o l u s and membranous o r g a n e l l e s . Using r e c e n t measurements, an attempt i s made h e r e t o e s t i m a t e t h e e f f e c t s of t h e g r a v i t a t i o n a l f i e l d upon t h e p o s i t i o n and motion of t h e c e l l s ' d e n s e s t s t r u c t u r e s , The Nucleolus E a r l i e r t h e o r e t i c a l work i n d i c a t e d t h a t t h e n u c l e o l u s might b e a s u f f i c i e n t l y l a r g e dense s t r u c t u r e t o be i n f l u e n c e d by g r a v i t y (19). T h i s would c e r t a i n l y be t h e c a s e i f t h e n u c l e o l u s could b e c o n s i d e r e d a s a s o l i d o b j e c t suspended i n a v i s c o u s l i q u i d medium. However, t h i s i s n o t t h e c a s e . Our c u r r e n t u n d e r s t a n d i n g of t h e nucleol u s ( s e e Fig. 1 ) i s t h a t i t s r o l e i s t h e s y n t h e s i s of ribosomal RNA and t h e assembly of ribosomes ( 2 0 , 2 1 ) . Although i t i s t r u l y a d e n s e l y packed s t r u c t u r e , i t i s n o t i s o l a t e d from t h e surrounding nucleoplasm a s a s o l i t a r y hydrodynamic u n i t . I n s t e a d it has t h r e a d s of chromatin running through it--presumably t h e chromatin which c o n t a i n s ribosomal DNA genes (21). The n u c l e o l u s is t h e r e f o r e suspended i n t h e n u c l e u s by a number of t h r e a d s , and i t s motion i s t h e r e f o r e c o n s t r a i n e d by t h e motion of t h e chromatin w i t h which i t i s a s s o c i a t e d . Hence, a s shown i n t h e e l e c t r o n micrographs of F i g . 1, t h e r e i s l i t t l e o r no e v i d e n c e f o r t h e s e d i m e n t a t i o n of n u c l e o l i t o t h e bottoms of n u c l e i i n c u l t u r e d human c e l l s . On t h e average, t h e n u c l e o l u s i s j u s t about a s c l o s e t o t h e t o p of t h e n u c l e a r membrane a s i t i s t o t h e bottom. It is t o be l e a r n e d from t h i s d i s c u s s i o n t h a t f i b r o u s m a t e r i a l s i n t h e c e l l can g r e a t l y i n f l u e n c e t h e response of i t s o r g a n e l l e s t o g r a v i t y . The C e l l Nucleus Now l e t u s c o n s i d e r t h e n u c l e u s a s a whole. Recent s t u d i e s have i n d i c a t e d t h a t t h e c e l l cytoplasm can b e c o n s i d e r e d a s a network of m i c r o f i l a m e n t s and m i c r o t u b u l e s ( 2 3 ) . The i n c r e a s i n g r a t e a t which c o n t r a c t i l e p r o t e i n s a r e b e i n g d i s c o v e r e d i n s o - c a l l e d nonc o n t r a c t i l e c e l l s i s s o alarming t h a t we wonder why t h e y were n o t p r e v i o u s l y found. Two c l a s s e s of s t r u c t u r e a r e of i n t e r e s t t o o u r d i s c u s s i o n . The main p r o t e i n of m i c r o t u b u l e s i s t u b u l i n (24). The t u b u l i n e x i s t s i n s u b - u n i t s of m i c r o t u b u l e s . The s u b - u n i t s a r e assembled i n t o t u b u l e s f o r such purposes a s t h e g u i d i n g of chromosomes a t m i t o s i s , t h e s t r e n g t h and movement of c i l i a , and f o r axoplasmic flow i n n e r v e axons. The assembly of t h e s e s u b - u n i t s i n t o t u b u l e s i s i n h i b i t e d by c o l c h i c i n e and s i m i l a r v i n c a a l k a l o i d s . M i c r o f i l a m e n t s , on t h e o t h e r hand, a p p e a r t o c o n s i s t of a m i x t u r e of a c t i n , myosin, and o t h e r c o n t r a c t i l e muscle p r o t e i n s ( 2 5 ) . Microfilaments have been c o n s i d e r e d e s s e n t i a l f o r t h e normal m i g r a t o r y behavior of c u l t u r e d f i b r o b l a s t s ( 2 6 ) . C y t o c h a l a s i n B i n t e r F i g u r e 2 i n d i c a t e s t h e p r e s e n c e of f e r e s w i t h t h e normal a c t i o n of m i c r o f i l a m e n t s ( 2 7 ) . b o t h a c t i n and myosin i n t h e m i c r o f i l a m e n t s of c u l t u r e d f i b r o b l a s t s and shows t h a t t h e s e m i c r o f i l a m e n t s envelope t h e c e l l n u c l e u s . It a p p e a r s t h a t t h e n u c l e u s i s p o s i t i o n e d i n t h e cytoplasm under c o n s t r a i n t s imposed by m i c r o f i l a m e n t s a n d / o r m i c r o t u b u l e s . I f c u l t u r e d c e l l s a t t a c h e d t o c o v e r s l i p s a r e c e n t r i f u g e d a t moderate speed, one f i n d s t h a t c e l l s remain i n t a c t w i t h o u t s i g n i f i c a n t displacement of t h e i r n u c l e i . I f , on t h e o t h e r hand, one t r e a t s c u l t u r e d c e l l s a t t a c h e d t o c o v e r s l i p s w i t h c y t o c h a l a s i n B and t h e n s u b j e c t s t h e a t t a c h e d c e l l s t o a c e n t r i f u g a l f i e l d , it i s found t h a t t h e c e n t r i f u g a l a c c e l e r a t i o n is t h e n a d e q u a t e t o e n u c l e a t e t h e c e l l s ( 2 8 ) . I f one were t o assume t h a t t h e n u c l e u s i s a hydrodynamic u n i t approximated a s a s p h e r e 1 2 microns i n diameter w i t h d e n s i t y 1.14 suspended i n a f l u i d w i t h v i s c o s i t y 1 7 c e n t i p o i s e and d e n s i t y 1 . 0 3 , . t h e n one would a n t i c i p a t e a s e d i m e n t a t i o n v e l o c i t y of t h e c e l l n u c l e u s e q u a l t o about 20 micrometers p e r hour. C l e a r l y , a l l n u c l e i would sediment t o t h e bottoms of t h e i r c e l l s i n a few minutes. That t h i s i s n o t t h e c a s e i s o b s e r v a b l e i n mammalian t i s s u e s e c t i o n s i n which t h e n u c l e i a r e always c e n t r a l and i n v e r t i c a l s e c t i o n s of c u l t u r e d c e l l s ( F i g . I ) , where t h e n u c l e i a r e a l s o r a t h e r c e n t r a l l y p o s i t i o n e d . E v i d e n t l y , m i c r o f i l a m e n t s o r o t h e r c e l l u l a r s t r u c t u r e s deny t h e c e l l n u c l e u s any motion induced by g r a v i t y .

The e f f e c t of g r a v i t y on n u c l e a r shape i s now c o n s i d e r e d . It has been noted t h a t i s o l a t e d c e l l n u c l e i a r e more s u s c e p t i b l e t o deforming f o r c e s t h a n a r e n u c l e i w i t h i n

c e l l s . E v i d e n t l y t h e d e f o m i b i l i t y of c e l l n u c l e i i s a l s o i n f l u e n c e d by cytoplasmic materials. I f t h e nucleus were t o b e p i c t u r e d a s a c o l l o i d a l s o l i n s i d e a deformable bag, one would expect n u c l e i t o be broader a t t h e bottom t h a n a t t h e t o p where up and I f c e l l s from s e c t i o n e d t i s s u e e v e r down a r e d e f i n e d by t h e g r a v i t a t i o n a l v e c t o r . i n d i c a t e d such an a n i s o t r o p i c f e a t u r e i t was never r e p o r t e d . Upon examining human c e l l s i n c u l t u r e such a s i n F i g . 1, i n which t h e g r a v i t y v e c t o r is c l e a r l y d e f i n e d , one might s e e k a g r a v i t a t i o n a l a f f e c t i n t h e form of n u c l e i which a r e b r o a d e r i n t h e i r lower h a l v e s t h a n i n t h e i r upper h a l v e s and i n which t h e t o p r a d i u s of c u r v a t u r e i s much l e s s t h a n t h e lower r a d i u s of c u r v a t u r e of t h e n u c l e u s . Indeed, one f i n d s evidence f o r t h i s o c c u r r i n g i n a s i g n i f i c a n t p r o p o r t i o n of c e l l s examined. It should b e c a u t i o n e d , however, t h a t such a n i s o t r o p i c n u c l e a r shape might have n o t h i n g t o do w i t h t h e g r a v i t a t i o n a l f i e l d because t h e n u c l e u s may assume t h i s shape simply because t h e c e l l which surrounds i t i s b r o a d e r a t t h e bottom as a consequence of being a t t a c h e d and s p r e a d a t i t s bottom s u r f a c e and not a t i t s t o p s u r f a c e . There i s , t h e r e f o r e , no c o n c r e t e e v i dence t h a t g r a v i t y i n f l u e n c e s n u c l e a r p o s i t i o n o r n u c l e a r shape. Chromosomes F i n a l l y , l e t us c o n s i d e r t h e p o s s i b i l i t y of a g r a v i t a t i o n a l e f f e c t on chromosomes a t m i t o s i s . Ever s i n c e t h e d i s c o v e r y of chromosomes, s c i e n t i s t s have been f a s c i n a t e d by t h e i r movements d u r i n g c e l l d i v i s i o n . T h e i r k i n e m a t i c s and mechanics have been cons i d e r e d i n d e t a i l e d p h y s i c a l t h e o r i e s of t h e m i t o t i c p r o c e s s , A s we have done w i t h t h e o t h e r o r g a n e l l e s , l e t u s c o n s i d e r t h e s e d i m e n t a t i o n v e l o c i t y of a f r e e chromosome suspended i n t h e f l u i d m a t r i x of t h e m i t o t i c c e l l . The g e n e r a l e q u a t i o n of motion f o r a sedimenting p a r t i c l e i s 2

where t h e f r i c t i o n f a c t o r , f , f o r a long p r o l a t e e l l i p s o i d i s e s t i m a t e d a s

S u b s t i t u t i n g f o r f and s o l v i n g e q u a t i o n (1) f o r t h e t e r m i n a l v e l o c i t y we f i n d t h a t

i n which a l l o f t h e v a l u e s i n e q u a t i o n (3) a r e known. a r e t a b u l a t e d i n Table I ,

These v a l u e s and t h e i r s o u r c e s

We may a l s o u s e t h e e q u a t i o n of motion (equation 1 ) a s a f o r c e b a l a n c e e q u a t i o n . By u s i n g t h e boundary c o n d i t i o n s t h a t v e l o c i t y and a c c e l e r a t i o n e q u a l 0 , we may determine t h e d i f f e r e n c e between t h e g r a v i t a t i o n a l and buoyant f o r c e and t h e r e b y e s t i m a t e t h e f o r c e r e q u i r e d t o p r e v e n t t h e chromosome from s e d i m e n t a t i n g i n t h e cytoplasm. The c o n s t a n t s needed f o r t h i s c a l c u l a t i o n a r e given i n Table I and F i g u r e 3. The c a l c u l a t i o n s a p p l i e d t o a " t y p i c a l " mammalian chromosome and i n d i c a t e t h a t i f t h e chromosome were suspended i n a f r e e s o l u t i o n w i t h cytoplasmic d e n s i t y and v i s c o s i t y i t would sediment w i t h v z 2 x LO-' cm/sec. Assuming v = o i n e q u a t i o n (1) l e a d s t o a b a l a n c i n g f o r c e of about lose dyne, o r l e s s t h a n t h a t of t h e c o v a l e n t bonds which e x i s t w i t h i n t h e c r o s s s e c t i o n of a spindle fibre.

M i t o t i c Spindle One may now q u e s t i o n whether o r not t h e m i t o t i c s p i n d l e can e x e r t t h e f o r c e req u i r e d t o prevent chromosome s e d i m e n t a t i o n i n t h e cytoplasm, A s e t of experiments w a s done i n t h e following way: c u l t u r e d Chinese hamster M3-1 c e l l s (34) and c u l t u r e d human kidney T-l c e l l s (35) were allowed t o a t t a c h and g r o w on t h e s u r f a c e of p l a s t i c t i s s u e c u l t u r e b o t t l e s (Falcon #3012) f o r 24 hours i n t h e h o r i z o n t a l p o s i t i o n , a f t e r which h a l f of t h e sample b o t t l e s were f i l l e d w i t h medium and o r i e n t e d v e r t i c a l l y . A f t e r 18-20 more hours of i n c u b a t i o n a t 37OC, t h e c u l t u r e s were r i n s e d w i t h Hanks' balanced s a l t s s o l u t i o n and f i x e d without changing t h e i r o r i e n t a t i o n . They were s t a i n e d i n t h e h o r i z o n t a l p o s i t i o n w i t h H a r r i s ' hematoxylin and mordanted t a p water. The a n g l e (3 subtended by t h e d i r e c t i o n of t h e s p i n d l e and a v e r t i c a l l i n e (Fig. 4) was e s t i m a t e d w i t h i n 30' i n t e r v a l s m i c r o s c o p i c a l l y , and t h e number of d i v i d i n g c e l l s l y i n g i n each 30- d e g r e e i n t e r v a l was determined (Table 1 1 ) . The f o l l o w i n g r e s u l t s a r e t o be expected: 1 ) I f m i t o s i s i s o r i e n t e d by t h e growth s u r f a c e o n l y , t h e r e w i l l be an e q u a l p r o p o r t i o n of c e l l s i n each 30-degree i n t e r v a l i n both v e r t i c a l and h o r i z o n t a l c u l t u r e s , and a p r e f e r e n c e f o r chromosome motion p a r a l l e l t o t h e growth s u r f a c e . 2) I f m i t o s i s i s o r i e n t e d by g r a v i t y a l o n e , t h e r e w i l l be a p r e f e r r e d o r i e n t a t i o n around 0-90' ( i n t e r v a l 3) i n t h e v e r t i c a l c u l t u r e . 3) I f b o t h g r a v i t y and t h e growth s u r f a c e a c t t o o r i e n t m i t o s i s , t h e r e w i l l b e a p r e f e r r e d o r i e n t a t i o n around 0=90° and a p r e f e r e n c e f o r chromosome motion p a r a l l e l t o t h e growth s u r f a c e i n t h e v e r t i c a l c u l t u r e s . C e l l s were a s s i g n e d t o groups 1 through 6 according t o t h e v a l u e of O. An i s o t r o p i c c u l t u r e should have roughly e q u a l numbers of d i v i d i n g c e l l s i n each group. The e x i s t e n c e of a n i s o t r o p y should b e i n d i c a t e d by an excess of d i v i d i n g c e l l s i n one o r two of t h e a n g u l a r i n t e r v a l s . I n h o r i z o n t a l f l a s k s i s o t r o p i c d i s t r i b u t i o n s were genera l l y found. N e v e r t h e l e s s , t h e p r o p o r t i o n of m i t o s e s o r i e n t e d a t each a n g l e i n h o r i z o n t a l c u l t u r e s was used a s a b a s e l i n e a g a i n s t which t o compare t h e p r o p o r t i o n a t t h e same a n g l e i n v e r t i c a l c u l t u r e s , and t o determine t h e e f f e c t of growth on t h e v e r t i c a l s u r f a c e , t h e f o l l o w i n g v e r t i c a l - t o - h o r i z o n t a l r a t i o was d e f i n e d : V = p r o p o r t i o n of c e l l s i n 0 i n t e r v a l , v e r t i c a l , H p r o p o r t i o n of c e l l s i n O i n t e r v a l , h o r i z o n t a l

t h e n h i s t o g r a m s were prepared of V/H v s . O i n t e r v a l . An example of such a histogram is given i n F i g u r e 5, which s u g g e s t s t h a t t h e prop o r t i o n o f m i t o s e s o r i e n t e d a t each a n g l e d i d not d i f f e r s i g n i f i c a n t l y between h o r i z o n t a l and v e r t i c a l c u l t u r e s i n t h i s experiment. Chinese hamster M3-1 c e l l s grow i n t o c o l o n i e s w i t h a l a r g e a x i a l r a t i o . I f t h e p l a n e of d i v i s i o n o c c u r s w i t h g r e a t e r frequency a t a p a r t i c u l a r p o s i t i o n f o r c e l l s grown on a v e r t i c a l s u r f a c e , t h e n t h e long a x i s of t h e r e s u l t a n t c o l o n i e s should b e p r e f e r e n t i a l l y o r i e n t e d . The a n g l e subtended by t h e . l o n g a x i s of t h e c o l o n i e s and t h e long a x i s o f t h e b o t t l e was e s t i m a t e d f o r v e r t i c a l l y - and h o r i z o n t a l l y - grown c u l t u r e s and t h e corresponding V/H r a t i o determined f o r each a n g l e i n F i g u r e 6. I n o r d e r t o a v e r t t h e a m b i g u i t i e s a s s o c i a t e d w i t h counting s m a l l numbers of d i v i d i n g c e l l s (about 300 c e l l s p e r d i s h were measured), experiments were designed s o t h a t t h e d i r e c t i o n of d i v i s i o n could b e determined f o r a l a r g e number of c e l l s p l a t e d a t r e l a t i v e l y low d e n s i t y . Human kidney T1 c e l l s were p l a t e d and t h e v e r t i c a l b o t t l e s were o r i e n t e d as soon a s t h e c e l l s were f i r m l y a t t a c h e d ; t h u s , t h e f i r s t d i v i s i o n occurred a f t e r t h e b o t t l e s had been o r i e n t e d . The o r i e n t e d b o t t l e s were t h e n incubated f o r e x a c t l y one g e n e r a t i o n time (about 24 hours) and s t a i n e d . The p l a n e of d i v i s i o n was determined f o r 1,000 c e l l s i n two experiments. The V / H r a t i o presumably h a s t h e same meaning a s i n experiments i n which o n l y d i v i d i n g c e l l s were measured, a s t h e a n g l e s were determined o n l y f o r c o l o n i e s c o n t a i n i n g two c e l l s , The d i s t r i b u t i o n of t h e V/H r a t i o i s given i n F i g u r e 7 .

I f t h e r e i s any e f f e c t of v e r t i c a l i n c u b a t i o n upon o r i e n t a t i o n of c e l l d i v i s i o n , i t i s probably s m a l l and d i f f i c u l t t o reproduce. Cultured Human C e l l s i n Weightlessness The above c o n c l u s i o n s concerning a l a c k of obvious e f f e c t of t h e g r a v i t y v e c t o r on t h e o r i e n t a t i o n of mammalian c e l l d i v i s i o n i s borne o u t i n t h e s t u d i e s of Montgomery ( 3 6 ) , i n which c u l t u r e d human WI-38 f i b r o b l a s t s were grown d u r i n g t h e 59-day m i s s i o n of Skylab. The p o p u l a t i o n doubling time i n f l i g h t , 22.3 t 3 . 1 h r d i d n o t d i f f e r s i g n i f i c a n t l y from The speed of c e l l m i g r a t i o n on t h e c u l t u r e v e s s e l s u r f a c e was t h a t a t l g , 20.4 t 4.8. t h e same, and no u l t r a s t r u c t u r a l o r k a r y o t y p i c d i f f e r e n c e s could be observed. C e l l s t h a t had rounded f o r m i t o s i s d i d n o t even r e q u i r e t h e g r a v i t a t i o n a l f o r c e t o r e a t t a c h t o t h e s u r f a c e upon which t h e y were growing. Experiments i n t h e l a b o r a t o r y and i n space i n d i c a t e t h a t t h e c e l l d i v i s i o n p r o c e s s i n c u l t u r e d mammalian c e l l s i s r a t h e r s e n s i t i v e t o t h e i n f l u e n c e of g r a v i t y . DISCUSSION Some of t h e s e concepts l e a d t o i n t e r e s t i n g q u e s t i o n s concerning t h e r o l e of g r a v i t y i n o r g a n i c o r chemical e v o l u t i o n . For example, one might a s k would t h e i d e a l shape of an organism i n t h e absence of g r a v i t y always be a s p h e r e ? I n o t h e r words, would an organism e v o l v i n g i n space b e s p h e r i c a l r a t h e r t h a n s h a p e l y a s organisms evolved on e a r t h i n t h e p r e s e n c e of g r a v i t y ? A t t h e s u b c e l l u l a r o r o r g a n e l l e l e v e l even more s e r i o u s q u e s t i o n s p e r s i s t : Do p a r t i c l e s t h a t sediment i n p l a n t cytoplasm r e a l l y behave a s g e o t r o p i c s e n s o r s ? I f t h e y do, how do t h e y inform t h e c e l l what t o do? Does g r a v i t a t i o n a l stress l e a d t o a n i n t r a c e l l u l a r c o n t r a c t i l e response? Many of t h e s e c o n s i d e r a t i o n s overlook t h e e x i s t e n c e of i n t e r n a l c e l l u l a r membranes which, i n e u k a r y o t i c c e l l s , e x i s t i n g r e a t abundance. Perhaps t h e s e d i m e n t a t i o n of p a r t i c l e s i n c e l l s h a s been c o n s i d e r e d too s i m p l i c i s t a l l y and one needs t o i n c l u d e c o n s i d e r a t i o n s of such phenomena a s t h e Dorn e f f e c t i n which an e l e c t r i c f i e l d r e s u l t s when a p a r t i c l e sediments, Such f i e l d s can b e a s g r e a t a s 20 m i l l i v o l t s . Also, d r o p l e t s e d i m e n t a t i o n should probably be given more s e r i o u s c o n s i d e r a t i o n a s i t i s a phenomenon r e l a t e d t o l a r g e r hydrodynamic u n i t s whose d e n s i t y depends on p a r t i c l e concentration. Other q u e s t i o n s of b i o l o g i c a l i n t e r e s t i n c l u d e , Why a r e p l a n t tumors not g e o t r o p i c ? Do p l a n t tumor c e l l s d i s r e g a r d g r a v i t y ? Is something missing i n t h e i r d i f f e r e n t i a t e d s t r u c t u r e ? Also, s i m p l e p l a n t s such a s t h e mold, Phycomyces, respond t o g r a v i t y w i t h o u t p o s s e s s i n g any apparent sedimenting cytoplasmic p a r t i c l e s . Research on e a r t h and i n s p a c e h e s n o t y e t l e d t o c o n c r e t e evidence t h a t sedimenting i n t r a c e l l u l a r p a r t i c l e s p l a y a r o l e i n determining t h e r e l a t i o n s h i p between c e l l u l a r a c t i v i t i e s and t h e g r a v i t y v e c t o r . ACKNOWLEDGMENTS The c e l l c u l t u r e o r i e n t a t i o n experiments were c a r r i e d o u t under t h e combined s u p p o r t of t h e U.S. Atomic Energy Commission and t h e N a t i o n a l A e r o n a u t i c s and Space Administrat i o n w i t h t h e t e c h n i c a l a s s i s t a n c e of Mrs. J e a n Luce a t t h e U n i v e r s i t y of C a l i f o r n i a . The e l e c t r o n microscopy was performed by Helge Dalen under p a r t i a l s u p p o r t of U.S. Atomic The f l u o r e s c e n c e microscopy was performed by M r . Energy Commission Grant AT(30-1)-3834. Alex L. M i l l e r and M r . A l l a n F. Worton under p a r t i a l s u p p o r t of U.S. N a t i o n a l Cancer H e l p f u l d i s c u s s i o n s have been provided by D r s . J. W, I n s t i t u t e C o n t r a c t NOl-CB-43984. Tremor, C, A . Tobias, and E. C. P o l l a r d .

REFERENCES ( 1 ) 0. S c h u l t z e , Die k u n s t l i c h e Erzeugung von Doppelbildungen b e i F r o s c h l a r v e n m i t B i l f e abnormer G r a v i t a t i o n s w i r k u n g . Arch, Entw.-mech. (1894) 269-305. ( 2 ) G. b i o l o g y experiments i n reduced g r a v i t y , in on in (3) F. Saunders, Ed., The Experiments of B i o s a t e l l i t e II,NASA SP-204 (1971) ( 4 ) J. W. Tremor and K. Souza, Development of t h e gravity-compensated f r o g egg. Am. Zool. 2 (1969) 1118 ( 5 ) R. Young, P. Deal, K. Souza, and 0 . W h i t f i e l d , A l t e r e d g r a v i t a t i o n a l f i e l d e f f e c t s on t h e f e r t i l i z e d f r o g egg. Exp. C e l l Res. 59 (1970) 267-271. ( 6 ) R. S. Young and J. W. Tremor, W e i g h t l e s s n e s s and t h e d e v e l o p i n g f r o g egg. L i f e S c i e n c e s and Space Research V I , North-Holland, Amsterdam (1968) 87-93. ( 7 ) R. S. Young and J. W. Tremor, The e f f e c t of w e i g h t l e s s n e s s on t h e d i v i d i n g egg of Rana p i p i e n s . BioScience 18 (1968) 609-615. (8) B. S. Young, J. W. t r e m o r , R. Willoughby, R. L. C o r b e t t , K. A. Souza, and P. D. S e b e s t a , The e f f e c t of w e i g h t l e s s n e s s on t h e d i v i d i n g eggs of Rana p i p i e n s . i n The Experiments of B i o s a t e l l i t e 11, J. F. Saunders, Ed., NASA SP-204 (1971) 251-271. ( 9 ) S. W. Gray and B. F. Edwards, The e f f e c t of w e i g h t l e s s n e s s on t h e growth and o r i e n t a t i o n of r o o t s and s h o o t s of monocotyledonous s e e d l i n g s . i n The Experiments of B i o s a t e l l i t e 11, J . F. S a u n d e r s , Ed., NASA SP-204 (1971) 1 2 3 - 1 6 6 7 (10) B. F. Edwards, W e i g h t l e s s n e s s experiments on B i o s a t e l l i t e 11, L i f e S c i e n c e s and Space Research 7, W. V i s h n i a c and F. G. F a v o r i t e , Eds., North-Holland, Amsterdam (1969) 84-92. (11) L. J. Audus, The mechanism of p e r c e p t i o n of g r a v i t y by p l a n t s . Symp. Soc. Exp. B i o l . 16 (1962) 197-226. (12) L. J. Audus, Geotropism and t h e modified s i n e r u l e ; a n i n t e r p r e t a t i o n based on t h e amyloplast s t a t o l i t h theory. Physiol. Plant. 1 7 (1964) 737 (13) B. G. Pickard and K. V. Thimann, G e o t r o p i c r e s p o n s e of wheat c o l e o p t i l e s i n a b s e n c e of a m y l o p l a s t s t a r c h . J . Gen. P h y s i o l . 49 (1966) 1065-1086. (14) J. Shen-Miller and C. M i l l e r , I n t r a c e l l u l a r d i s t r i b u t i o n of mitochondria a f t e r g e o t r o p i c s t i m u l a t i o n of t h e o a t c o l e o p t i l e . P l a n t P h y s i o l . 50 (1972) 51-54. i n Plant (15) J . Shen-Miller, P a r t i c i p a t i o n of t h e Golgi a p p a r a t u s i n geotropism. Growth Substances 1970, D. J. C a r r , Ed., S p r i n g e r - V e r l a g , B e r l i n (1972) 7 3 8 - 7 4 4 , ( 1 6 ) J. Shen-Miller, The Golgi a p p a r a t u s and geotropism. i n Hormonal R e g u l a t i o n i n P l a n t Growth and Development, H. Kaldewey and Y . Vardar, Eds., V e r l a g Chemie, Weinheim (1972) 365-376. (17) J . Shen-Miller and C. M i l l e r , D i s t r i b u t i o n and a c t i v a t i o n of t h e Golgi a p p a r a t u s i n geotropism. P l a n t P h y s i o l . 49 (1972) 634-639. (18) J . Shen-Miller and R. R. Hinchman, G r a v i t y s e n s i n g i n p l a n t s : A c r i t i q u e of t h e s t a t o l i t h t h e o r y . BioScience 24 (1974) 643-651. ( 1 9 ) E. C. P o l l a r d , T h e o r e t i c a l c o n s i d e r a t i o n s on l i v i n g systems i n t h e absence of mechanical s t r e s s . J. T h e o r e t . B i o l . 8 (1965) 113-123. ( 2 0 ) R. P e r r y , A. H e l l , and M. E r r e r a , The r o l e of t h e n u c l e o l u s i n r i b o n u c l e i c a c i d and p r o t e i n s y n t h e s i s 1. I n c o r p o r a t i o n of c y t i d i n e i n t o normal and n u c l e o l a r i n a c t i v a t e d HeLa c e l l s . Biochim. Biophys. Acta 49 (1961) 47-57. (21) I. Deak, F u r t h e r experiments on t h e r o l e of t h e n u c l e o l u s i n t h e t r a n s f e r of RNA from nucleus t o cytoplasm. J . C e l l S c i . 13 (1973) 395-401. (22) D. Brown and I. Dawid, S p e c i f i c gene a m p l i f i c a t i o n i n o o c y t e s . S c i e n c e 160 (1968) 272-280. (23) J. Fawcett, The C e l l : An A t l a s of F i n e S t r u c t u r e , W. B. S a u n d e r s , P h i l a d e l p h i a (1966). (24) R. C. Weisenberg, G. G. B o r i s y , and E. W. T a y l o r , The c o l c h i c i n e - b i n d i n g p r o t e i n of mammalian b r a i n and i t s r e l a t i o n t o m i c r o t u b u l e s . Biochemistry 1 (1968) 4466-4479. (25) R. P o l l a c k , M. Osborne, and K. Weber, P a t t e r n s of o r g a n i z a t i o n of a c t i n and myosin i n normal and transformed c u l t u r e d c e l l s . Proc. Nat. Acad. S c i . U. S . A. 72 (1975) 994-998.

7

(26) N. S . McNutt, L , A . Cubp, and P. H. Black, C o n t a c t - i n h i b i t e d r e v e r t a n t c e l l l i n e s from SV-40 transformed c e l l s . I V . Microfilament d i s t r i b u t i o n and c e l l shape i n untransformed, t r a n s f o r m e d , and r e v e r t a n t Balb/C 3T3 c e l l s . J , C e l l B i o l . 56 (1973) 412-428. (27) S. B. C a r t e r , E f f e c t s of c y t o c h a l a s i n s on mammalian c e l l s . Nature (1967) 261-264. (28) D. M, P r e s c o t t , B, Myerson, and J . Wallace, E n u c l e a t i o n of mammalian c e l l s w i t h c y t o c h a l a s i n B. Exp. C e l l Res. 2 (1972) 480-485. (29) H. Dalen and T. J . Nevalainen, D i r e c t epoxy embedding f o r v e r t i c a l s e c t i o n i n g of c e l l s grown a s a monolayer on M i l l i p o r e f i l t e r . S t a i n Technol, 43 (1968) 217-220. (30) A . L. M i l l e r , A. F. Horton, and R. L. McCarl, P r e p a r a t i o n of f l u o r e s c e n t c o n t r a c t i l e p r o t e i n s and e v a l u a t i o n of t h e i r a p p l i c a t i o n t o c y t o l o g y by f l u o r e s c e n t l i g h t and e l e c t r o n microscopy. J. C e l l B i o l . 67 (1975) 282a. (31) E. L. Schneider and N. Salzman, I s o l a t i o n and zonal f r a c t i o n a t i o n of metaphase chromosomes from human d i p l o i d c e l l s . S c i e n c e 167 (1970) 1141-1143. (32) H. J. Burki, T . J . Regimbal, J r . , and H, C. Mel, Zonal f r a c t i o n a t i o n of mammalian metaphase chromosomes and d e t e r m i n a t i o n of t h e i r DNA c o n t e n t . P r e p a r a t i v e Biochem. 2 (1973) 157-182. (33) A. D. K e i t h and W. S n i p e s , V i s c o s i t y of c e l l u l a r protoplasm. S c i e n c e 183 (1974) 666-668. (34) P. Todd, D e f e c t i v e mammalian c e l l s i s o l a t e d from x - i r r a d i a t e d c u l t u r e s . Mutation Res. 5 (1968) 173-183. (35) G . W. Barendsen, T. L. J . Beusker, A. J. Vergroesen, and L. Budke, E f f e c t s of d i f f e r e n t i o n i z i n g r a d i a t i o n s on human c e l l s i n t i s s u e c u l t u r e , 11. B i o l o g i c a l experiments. R a d i a t i o n Res. 13 (1960) 841-849. (36) P . O'B. Montgomery, Montgomery, P . O I B . J r . j J.E. Cook, R.C. Reynolds, J.S. Paul,

L, Hayflick, D. Stock, W.W. Schulz, S. Kimzey, R. G. T h i r o l f , T. Rogers, D. Campbell, and J . Morrell , 1974. "The Response of Single Human Cell s t o Zero G r a ~tiy " In. The Proceedings of t h e Skylab Life Sciences Synyposuim, NASA document TM X-58154, pp 467-491 (37) Moskvitin, E.V. and E.N. Vaulina. Experiment with a physiologically a c t i v e Chlorel.1a Culture on "Soyuz-9' spaceship. Space Biology and Aerospace Medicine 9(3) : 7-10 (1975).

.

TABLE I. HYDRODYNAMIC VALUES FOR A METAPHASE CHROMOSOME ( S E E F I G U R E 3) USED FOR A P P L I CHROMOSOMES HAVE BEEN EXAMINED HYDRODYNAMICALLY I N I S O U T I O N CAT ION T O EQUATION (3) ( 3 1 , 3 2 ) , AND CYTOPLASMIC V I S C O S I T Y HAS BEEN S T U D I E D BY PARAMAGNETIC RESONANCE (33).

.

V = 2nr2k = 2 5

X

10-l2 cm 3

g = 9 8 0 cm/sec 2 p-p,

=

1.35-1.04

= 0.31 g/cm 3

3J3V/4n= 2 . 1 x cm q = 5 + 2 dyn-sec/cm 2

TABLE 11.

ANGULAR INTERVALS ( S E E FIGURE 4 ) USED T O CLASSIFY ORIENTATION OF MITOTIC CELLS AND COLONIES ON HORIZONTAL AND VERTICAL CULTURE FLASKS.

Figure 1.- Electron micrographs of v e r t i c a l s e c t i o n s of c u l t u r e d human l i v e r c e l l s grown on h o r i z o n t a l Millipore f i l t e r s . The l o c a t i o n of n u c l e o l i i s v a r i a b l e , and (~icro~raph courtesy s of Helge Dalen t h e n u c l e i tend t o b e broader a t t h e base. ( r e f . 291.)

Figure 2.- Fluorescence micrographs of c u l t u r e d human embryonic lung c e l l s f i x e d i n acetone, e x t r a c t e d w i t h g l y c e r o l , and "stained" with f l u o r e s c e n t antibody a g a i n s t heavy meromyosin t o show presence of myosin ( l e f t ) and "stained" with heavy meromyosin i n a d d i t i o n t o t h e same f l u o r e s c e n t antibody t o show presence of a c t i n i n filaments ( r i g h t ) . ( ~ i c r o g r a p h scourtesy of Alex L. M i l l e r ( r e f . 3 0 ) . )

Figure 3.- Assumed properties of a metaphase chromosome suspended in cytoplasm. (See table I.)

DIVIDING CELL rCULTURE

/

Figure 4.- Illustration of analysis of orientation of mitosis in horizontal and vertical cell culture flasks. The diatram defines the mitosis orientation angle 0 .

, I

1.21.0-

0.8

-

0.4 0.2 -

--sll)

V/H RAT I0 0.6

M3-I MITOSES

0.00 1 2 3 4 5 6 ANGLE 8 Figure 5.- Histogram showing the ratios of mitoses in vertical to those in horizontal culture flasks at each interval of the mitosis orientation angle defined in figure 4 and in table 11.

9,

V/H RATIO

ANGLE 8 Figure 6.- Histogram showing the ratios of M3-1 cell colonies in vertical to those in horizontal culture flasks oriented with their long axes in each interval of the colony orientation angle 0 , defined in figure 4 and in table 11.

1.0 0.8

V/H 0.6 RATIO 0.4 0.2 ''1

2

3 4 5 ANGLE 8

6

Figure 7.- Histogram showing the ratios of T-1 (two-cell) colonies in vertical to those in horizontal culture flasks having their plane of division in each interval of the division plane angle 0 , defined in figure 4 and in table 11.

BIOPROCESSINC: PROSPECTS FOR SPACE ELECTROPHORESIS Milan Bier Veterans Administration Hospital and University of Arizona Tucson, Az. 85723

ABSTRACT The basic principles of electrophoresis will be reviewed in light of its past contributions to biology and medicine. Preliminary experiments aboard of Apollo 14 and 16, Skylab and the recent Apollo-Soyuz Mission have confirmed the feasibility and advantages of a possible space electrophoresis facility. This has to be viewed primarily a s a unique national research resource, which may eventually yield significant benefits f o r the advancement of biomedical knowledge and i t s technological utilization. Primary objectives of the facility should be the increase of resolution and throughput for critical electrophoretic fractionation of living cells and biologically active macromolecules.

INTRODUCTION The technology of bioprocessing comprises two distinct categories of activity; on the one hand, there is need 10 grow and propagate various organisms through bacterial, mold o r tissue culture and much work has been dedicated t o optimize techniques and isolate strains possessing maximal activity. On the other hand, specific products have to be isolated from the biomass, often a difficult problem in view of the complexity of composition of living matter, and the similarity of the species to be separated. As a result, separation processes have acquired a unique importance in biotechnology, reflecting their importance in basic biochemistry. Both of these aspects of bioprocessing may benefit from space research, and the last two speakers have discussed specialized aspects of space effects on growth and reproduction. In a later presentation, Dr. Barlow will show a n example of where cell activity may have been enriched by separation in space. My present paper will focus on possible contribution of space electrophor e s i s , a separation process which has been identified a s being most likely to benefit from the nearzero gravity environment prevailing in orbiting spacecraft. The possible contribution of NASA's space capabilities t o bioprocessing should be evaluated not only in t e r m s of the economic importance of this industry, but primarily in t e r m s of its sccial impact, through advances i n medicine and its contributions to the quality of our lives.

PREmING PAGE BLANK NOT PPL

E LECTROPHOR ESlS

Electrophoresis (1) i s defined a s the transport of electrically charged species under the influence of a direct current electrical field. Most materials in aqueous solution or suspension acquire an electrical charge due to ionization of their functional groups, ion absorption, or other more complex phenomena, and a r e therefore attracted by electrodes of opposite polarity. The charged species may be simple ions, complex macromolecules or even particles, such a s living cells, emulsion droplets, clay, etc. Their migration velocity in unit electsical field i s referred to as their electrophoretic mobility, and is a complex function not only of their electrical charge, but also of their molecular size, shape and hydration, a s well a s the dielectric characteristics of the solvent. As a result, electrophoresis is capable of providing a high degree of characterization of individual ionized species, which is most important for macromolecular systems and living cells, where structural parameters a r e difficult to determine. Based on this uniqueness of information provided by electrophoresis, a number of applications have been developed. To categorize them in their broadest outlines, these a r e a s follows: (a) Identification and characterization of an ionized species. (b) Determination of the quantitative composition of a complex mixture. (c) Actual isolation of components of a mixture, separation being achieved on the basis of differences in transport rates.

Originally, electrophoresis was carried out in free solutions but it was soon recognized that problems arise due to convective disturbances in the bulk of fluid. We can categorize several major causes of these disturbances: (a) The solute to be separated, if present in significant concentratibn, adds to the density of the supporting electrolyte. This difference in density between solution and pure solvent causes gravity-caused convective flow, unless means a r e found to prevent it. (b) In some instances the particles may be sufficiently large to sediment noticeably. While there a r e techniques which utilize differential sedimentation to accomplish meaningful separations, within the context of electrophoresis such sedimentation is usually undesirable, especially a s it i s superimposed on the convective flow of the suspension a s a whole, described above. (c) The passage of electric current causes heating of the solution. As the vessels a r e externally cooled, a radial temperature gradient arises, again causing gravity-conditioned convection. (d) The electric charge exhibited by the vessel walls within which electrophoresis is carried out causes an electroosmotic streaming of the fluid. This disturbance is independent of gravity and is a consequence of the electrical properties of the system a s a whole. In order to eliminate some o r all of the above problems, a variety of techniques has been evolved, and a systematic classification i s next to impossible. Thus, the techniques a r e differentiated according to their primary purpose (preparative or analytical), the mode of operation

(batchwise o r continuous flow), shape of vessel (cylindrical, flat, annular, etc.), and other operational parameters. In the context of this paper, the most important classification is based on the anticonvective means employed to circumvent the effects of gravity-. Three basic approaches were taken: (a) migration can be carried out in gels, where all convective flow is prevented, (b) fine porous structures of packed granules, or the interstitial spaces of filters and various specially developed membranes a r e also effective in preventing gross fluid motion without interfering in molecular transport, and (c) a density gradient can be artificially created within the liquid by using a nonmigrating solute such a s sucrose, of sufEicient steepness to overcome the density gradients caused by the electrophoretic process. To these three approaches, we now must add the radically new concept to avoid gravity altogether, by using orbiting spacecraft. The soundness of this approach has been confirmed in pilot experiments conducted aboard Apollo 14 and 16 (2), Skylab (3), and the recent Apollo-Soyuz Mission (4). A s defined above, electrophoresis is a separation process occurring within the bulk of the liquid phase (and not at the electrodes) and i s based on the differences in electrical transport rates. Electrophoresis alone, however, does not provide for the ultimate separation of various molecular species of proteins present, a s their mobilities may be overlapping. Highest resolution i s obtained if a second separation parameter is employed by introducing an element of discontinuity into the liquid phase. Two methods a r e most often used, In high density gel electrophoresis an element of molecular sieving i s superimposed on the electrical separation process by progressively increasing the density of the supporting gel matrix. In isoelectric focusing a continuous pH gradient is established and the proteins become immobilized at the pH corresponding to their characteristic isoelectric point (mobility of proteins i s pH dependent the narrow pH zone of zero mobility is the isoelectric point). The separation obtainable by isoelectric focusing is comparable to that in high density gels, and both a r e much superior in resolution to plain electrophoresis.

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ELECTROPHORESIS OF LIVING CELLS Because of its nondestructive nature, electrophoresis is one of the few separative methods applicable to living cells. Nevertheless, in comparison to proteins, cell electrophoresis is only in its infancy. Most of the techniques and instruments developed for protein electrophoresis a r e not applicable, and cell electrophoresis has remained the province of a few highly specialized laboratories. One of the great merits of the NASA program i s that it has focused the attention of a number of scientists here and abroad on this long neglected field. At present, cell separation is the main objective of NASA's space electrophoresis facility. Basic knowledge in this field is sorely needed. While there is a multitude of analytical electrophoretic methods applicable to proteins, until quite recently there was only one method suitable for cell electrophoresis. This technique involves direct visual microscopic measurement of electrophoretic migration velocity of individual cells, and has remained essentially unchanged for over 50 years (5,6). It is an inberently slow and unreliable method, burdensome and tedious

for the observer. A s a result, while there is adequate information on some normal cell populations, such a s red blood cells and lymphocytes, there a r e a l m ~ s no t reliable data on changes of cell properties in most clinical o r pathologic conditions, This situation is intolerable, since the present state of the a s would readily permit computer assisted automation of the microscopic method, resulting in rapid accumulation of important basic data on cell mobilities in health and disease. It i s hoped that through NASA sponsorship, such an instrument will soon become available. Other alternatives to more rapid accumulation of data involve the measurement of the Doppler Effect caused by migrating particles under laser illumination (7). Both of these two types of instruments a r e operable in presence of gravity, but at zero gravity their scope of application would be extended to larger cells, characterized by rapid sedimentation in a normal gravity field. Moreover, such instruments will be essential for the space facility, to provide real time information on the quality of separation achieved in space in the preparative instruments. Similar considerations prevail in preparative electrophoresis. Several techniques have been developed, including thin film free-flow electrophoresis (8), stable-flow electrophoresis (9), electromagnetophoresis (lo), and rotationally stabilized instruments (11, 12), but most have remained almost exclusively in the hands of their original developers. This is largely due t o their complexity and thepaucity of basic analytical data, which a r e indispensable in pinpointing the most important areas of preparative application, Moreover, the throughput of the instruments is limited, and their resolution less than optimal. It was previously emphasized that highest resolution of proteins is obtained only when a second discriminating parameter is superimposed on electrophoresis, a s in high density gel electrophoresis. The same situation may prevail with cells, and we a r e presently developing a system where electrophoretic separation is followed by an in-line discrimination according to cell size. Other secondary discrimination factors may be usable, such a s presence of fluorescent markers. Such systems bear the promise of much higher resolution than that obtainable by electrophoresis only.

SPACE ELECTROPHORESIS Gravity is not an unmitigated enemy of electrophoresis, and, to the contrary, in numerous techniques it is utilized t o great advantage. The determining factor is the objective one seeks and one has to consider in different light separation of proteins and that of living cells. With proteins, there is a profusion of excellent methods for analytical o r micropreparative work, i.e. fractionation and separation of products on a small, laboratory scale operation, and no foreseeable advantage is to be gained from a zero gravity facility. The situation is different when scaling up of these techniques to larger volumes is attempted. In this realm, ground-based electrophoresis has failed completely and all attempts to scale up high resolution micropreparative procedures have been unsuccessful. The zero gravity facility may provide the hoped-for breakthrough by allowing the use of novel instruments specifically designed for the weightless environment. A s an example, in our laboratory we are currently engaged in collaborative efforts t o purify two trace components of human serum, Somatomedin, a growth promoting polypeptide, and Phagocytosis Recognition Factor, a potential antitumor agent. The technique utilized is isotachophoresis, a relatively new variant amongthe many electrophoretic techniques (13), and we have designed a novel type of instrument (14) particularly suited for space use. A diagram of this instrument is shown in Fig. 1. It is constructed from a parallel array of feeder spacers and knife edge separators, assuring laminarity of liquid flow., Time does not per-

Fig. 1. Schematic presentation of a miniaturized flow electrophoresis apparatus designed for space electrophoresis. The parallel array of spacers and knife edge separators provide for laminar flow within the cell. The apparatus is envisioned for rapid flow-through with minimal migration distance, and may be particularly suited for isotachophoresis

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mit to go into the rationale why this apparatus promises a higher throughput in space operation than that of other similar continuous flow instruments available on ground. There a r e obviously a great number of other proteins which may benefit from space processing, including clotting factors, enzymes, and other protein hormones.

If the prospects of space electrophoresis were to be realized for proteins, there would be an immediate widespread usage for the products. Nevertheless, the primary emphasis of the current NASA program i s centered on separations of living cells. The reason for it i s that most techniques to circumvent gravity effects developed for protein electrophoresis are not applicable to cells. Moreover, cell electrophoresis i s only in its infancy, and the development of better techniques may result in significant advancement of our knowledge of cell biology. This is the most opportune time for such a development, a s it is only in recent years that cell biology has come into i t s own, with the recognition that apparently similar cells may have a variety of distinct functions. This i s particularly true of lymphocytes, the mediators of immune reactions, which a r e of direct and immediate importance in such diverse areas of medicine a s allergies, autoimmune diseases, leukemia and other forms of lymphocyte neoplasias, resistance to cancer, etc. Thus, there is a widespread current interest in cell separations by any and all means. Reliance on electrophoresis i s based on the a s yet fragmentary but significant evidence that functional, pathologic, genetic and environmental factors affect the electrophoretic behavior of lymphocytes (15). The recent Apollo-Soyuz Mission provided an opportunity to test two prototypes of instruments suitable for zero gravity operation. The NASA prepared flight module was similar t o those previously flown in Apollo 14 and 16 (2), though more ambitious in its aims. The essential part of the instrument was the electrophoresis column, reproduced schematically in Fig. 2. The two electrode compartments were detachable from the main body of the column, permitting a total of eight different columns to be tested. The columns were preloaded with sterile buffer, and the samples to be electrophoresed were frozen in liquid nitrogen till immediately before use by the astronauts. The samples cbrmined kidney cells, lymphocytes and fresh and fixed lymphocytes. Photographs could be taken during the run to record the migration of the cells, while at the end of the run, the

Fig. 2. Schematic diagram of the column assembly for the static electrophoresis experiment carried out a s part of the ApolloSoyuz Mission. The center part of the column i s detachable from the electrode compartments on both ends.

Fig. 3. Schematic drawing of the continuous flow electrophoresis apparatus used in the Apollo-Soyuz Mission. The cell itself is in the center. Accessories provide for frozen sample storage, sample insertion, and buffer circulation.

columns were frozen in situ, and returned to earth in liquid nitrogen. The kidney cells were subsequently grown in tissue culture, demonstrating the viability of the cells s o recovered and we will hear more about it later on. Time does not permit to discuss in greater details the other experiments, o r the apparatus, A second apparatus was also included in this Mission. It was designed by Hannig of the Max Planck Institute for Biochemistry in Munich, Germany, and was constructed by the Messerschmitt-Bolkow-Blohm consortium, and financed by the German Government. The diagram of this apparatus is presented in Fig. 3, and it can be readily seen that it i s far more complex than the first apparatus. It is an automated and miniaturized version of the well known continuous flow instrument of Hannig (8), and contained three samples: a mixture of human and rabbit erythrocytes, a mixture of B and T lymphocytes, and a suspension of bone marrow cells. Their migration was followed photometrically, and no recovery of fractions was intended.

Both of above instruments were space adaptations of ground based equipment, and represent prototypes of two basic concepts in electrophoresis: stationary fluid versus continuous flow operations. Their designs were kept within the narrow constraints inherent in experiments aboard manned rockets. The availability of the Shuttle will probably eliminate most of these constraints, and there have been already several proposals within the NASA program of instruments more specifically designed for the space application.

CONCLUSIONS

The near-zero gravity environment of orbiting spacecraft may present some unique advantages for a variety of processes, by abolishing the major source of convection in fluids. As the ground-based development of electrophoresis was heavily influenced by the need to circumvent the effects of gravity, this prvcess should be a prime candidate for space operation. NeverTheless, while a space facility for electrophoresis may overcome the limitations imposed by gravity, it will not necessarily overcome a11 problems inherent in electrophoresis. These a r e , mainly, electroosmosis and the dissipation of the heat generated by the electric field. The NASA program has already led to excellent coatings t o prevent electroosmosis, while the need for heat dissipation will continue to impose limits on the actual size of equipment. It is also not excluded that, once the dominant force of gravity is eliminated, disturbances in fluid stability may originate from weaker forces, such a s surface tension. There is a s yet no consensus onthe best apparatus for space electrophoresis. Reflecting the diversity of ground-based electrophoresis instruments, it is likely that more than one instrument will be needed for the space facility a s well. It is important t o consider the nature of the possible advantages t o be derived from space electrophoresis: these a r e only a matter of degree. In all separation processes, one has t o consider the factors of resolution and throughput, there being usually a trade-off between the two. For cell separation i n space, both may be of importance, while f o r proteins, only a quantum increase of throughput would constitute a definitive advantage. To achieve either o r both of these advantages, optimization of the design of the space instruments is essential. Less than the best designed instruments may well jeopardize the whole program, particularly when one considers that its operators in space may not have the skills of principal investigators o r highly skilled technicians. The interdisciplinary expertise available t o NASA offers a unique opportunity not only to optimize the space but also ground-based equipment. The space facility for electrophoresis has to be considered primarily a s a unique r e s e a r c h tool for the advancement of our knowledge of cell biology, though its potential technological applications should not be overlooked. In view of the cost of space experimentation, greatest c a r e should be given t o the selection of candidate materials for space processing. To accomplish this, it would be desirable if the NASA program were to be integrated o r correlated with the work of other agencies o r organizations having a more primary interest in the health field. The process of selection should encompass a thorough evaluation of the material by ground-based electrophoresis and the consideration of alternatives. A s we a r e dealing with a rapidly advancing area of science, maximum,flexibility in planning is essential for both, instrument design and their application.

ACKNOWLEDGEMENT: This research was supported in part by NASA Contract NAS8-29566.

REFERENCES

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I. M. Bier, ed., "Electrophoresis Theory, Methods and Applications," Vol, l and Vol, 11, Academic Press, New Voxk, I959 and 1967,

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2 , R S, Snyder et al, F r e e fluid particle electrophoresis on Apollo 16. Separ, Purif 2:259-282, 1973,

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.

.

. Methods

3. M. Bier, J 0. N Hinckley and A , J K, Smolka: Potential Use of Isotachophoresis in Space, Protides Biological Fluids, XXll Colloquim (H Peeters, ed .), pp 673-678, Pergamon Press, New York, N. Y. 1975

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.

4. Final reports in preparation.

5. H. A. Abramson, L oS. Moyer and M. H. Gorin: "Electrophoresis of Proteins, " Hafner Publishing Company, New York, 1964. 6. C. C. Brinton and M. A. Lauffer in "Electrophoresis, " M, Bier, ed,, Vol. 1, pp 427-492, Academic Press, New York, 1959, 7. Under commercial development by Pen Kem, Inc., Croton-On-Hudson, New York,

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. .

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8. K Hannig in "Methods in Microbiology, " J R Norris and D. W Robbins, eds Press, New York, 1971.

., Academic

9. C. Mel, Science 132:1255-56, 1960. 10. A. Kolin, J. Chromatog. 17:532-537, 1965. 11. S. Hjerten, "Free Zone Electrophoresis, " Almquist and Wiksells Boktryckeri AB, Uppsala, 1967. 12. A. J. K. Smolka and M. Bier: Isotachophoresis of Living Cells (Abstract), FASEB, 1975, U.S. Patent Specifications submitted t o NASA.

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13, A. Kopwillem, W. G. Merriman, R M. Cuddeback, A .J .KOSmolka and M. Bier: .Serum Protein Fractionation by Isotachophoresis Using Amino Acid Spacers, J. Chromatography (in press).

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14. M. Bier, A .J K. Smolka and A. Kopwillem: Preparative Electrophoresis at Zero Gravity. J Colloid and Interface Science (in press).

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15. M. Bier: Potential Contribution of the Space Program t o the Advancement of Electrophoresis and i t s Biomedical Applications, i n "Future Space Programs 1975", Committee on Science and Technology, U. S, House of Representatives, Vol. 2, p, 4, U,S. Government Printing Office NO. 052-070-02891-2, 1975.

E l e c t r o p h o r e t i c S e p a r a t i o n of Human Kidney C e l l s a t Zero Gravity

Grant H. Barlow*, S. LaVera Lazer*, Annemarie Rueter* and Robert A1 1en** *Experimental Biology D i v i s i o n , Abbott L a b o r a t o r i e s , North Chicago, I1 1 . and **Biotechnology Branch, NASA, George C. Marshal 1 Space F l i g h t C e n t e r , Alabama

Work supported by C o n t r a c t from NASA, No. NAS-8-30591

Acute thromhoen~bolicv a s c u l a r d i s e a s e remains the g r e a t e s t s i n g l e cause of n i o r t a l i t y i n the middle and e l d e r age population of the U . S . I n r e c e n t y e a r s the use of f i b r i n o l y t i c therapy has shown t h e f e a s i bi 1 i ty in vivo i n such d i s e a s e s as pulmonary emboli and deep vein of c l o t l y s i s thrombosis.

The UPET ( 1 ) and USPET ( 2 ) mu1 t i c e n t e r e d c l i n i c a l t r i a l s

have demonstrated t h e s a f e t y of such therapy. The presence i n u r i n e of a substance capable of e f f e c t i n g t h e t r a n s f o r m a t i on of plasmi nogen t o pl asmi n, t h e agent necessary t o bring about f i b r i n o l y s i s was f i r s t described by Williams ( 3 ) i n 1951 and i n t h e following y e a r by Astrup and S t e r n d o r f f ( 4 ) .

Sobel ( 5 ) et a1 assigned

t h e name urokinase (UK) t o t h i s a c t i v a t o r i n 1952. I t was apparent from t h e s t a r t t h a t t h e l o g i s t i c s of u r i n e supply and t h e c o s t of production made t h e u r i n e d e r i v a t i v e of the enzylne impracticable.

For t h e 4 m i l l i o n u n i t dose used i n t h e c l i n i c a l t r i a l s

a t l e a s t 1500 l i t e r s of u r i n e were required and t h e c o s t was p r o h i b i t i v e . These f a c t s motivated us t o search f o r another source of t h i s f i b r i n o l y t i c a g e n t ; and s i n c e t h i s agent i s p r o t e i n i n n a t u r e , i t i s not d e s i r a b l e f o r imniunogenic r e a s o n s , t o use o t h e r animal sources a s i d e from man. In 1959, B a r n e t t and Baron ( 6 ) demonstrated t h e production of a plasmi nogen a c t i v a t o r from KB c e l l s , derived from a human epidermoid carcinoma and primary monkey kidney c e l l s .

In a l a t e r paper ( 7 ) t h e

same a u t h o r s showed t h a t many continuous primary c e l l c u l t u r e s would produce e i t h e r o r both plas~iiinogen and protease a c t i v a t o r s .

P a i n t e r and

Charles ( 8 ) deliionstrated t h e accun~ulationof a f i b r i n o l y t i c agent i n c u l t u r e s of primary monkey kidney c e l l s and i n an e s t a b l i s h e d l i n e of canine renal c e l l s .

This agent was shown t o be an a c t i v a t o r of plasminogen

with p r o p e r t i e s s i m i l a r t o those of urokinase.

F i n a l l y , Bernik and Kwaan

( 9 , 10) demonstrated ( a ) f i b r i n o l y t i c a c t i v i t y i n c u l t u r e s of human

kidneys, ( b ) t h a t t h i s a c t i v i t y was imniunoloyical ly i n d i s t i n g u i s h a b l e from u r i n a r y urokinase and ( c ) t h a t t h i s f i b r i n o l y t i c agent was produced t o the g r e a t e s t degree i n c u l t u r e s of human renal c e l l s froin a 26 week o l d f e t u s .

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32

These f i n d i n g s and the development by Weiss and S c h l e i c h e r

( 1 1 , 1 2 ) of c e l l equipment known a s t h e Mass Tissue Culture Propagator (MTCP) which allows f o r the c u l t u r i n g of c e l l s on a l a r g e s c a l e prompted

us t o i n i t i a t e a program t o produce urokinase from human embryo kidney cells.

The b a s i c methodology has been described (13) and t h e r e s e a r c h

has continued f o r improvement of t h e process. The observation by Bernik and Kwaan ( 1 0 ) using a f i b r i n s l i d e technique t h a t only about 5% of t h e c e l l s produced a c t i v a t o r led t o t h e design of t h e experiments described here.

A method was sought t o i s o l a t e

t h e producing c e l l s t h a t could be used e v e n t u a l l y on a l a r g e s c a l e . One p o s s i b l e way i s e l e c t r o p h o r e t i c a l l y .

However, a drawback i n t h e e l e c t r o p h o r e s i s

o f c e l l s i s t h e l o s s of r e s o l v i n g power due t o t h e sedimentation of t h e c e l l s i n t h e media.

An e l e c t r o p h o r e t i c s e p a r a t i o n a t zero g r a v i t y

should obviously negate t h i s drawback.

Thus, the experiments described

here were performed on t h e Apolly-Soyuz space mission.

Methods Electrophoresis including instrurncntation, electrophoretic conditions ang g e l s l i c i n g techniques wi 11 be d e s c r i b e d elsewhere by NASA ( 1 4 ) . C e l l V i a b i l i t y was performed u s i n g a 0.4% s t o c k s o l u t i o n o f e r y t h r o s i n e B made i n phosphate b u f f e r , pH 7 ( 1 5 ) . Urokinase A c t i v i t y determined u s i n g a m o d i f i c a t i o n o f t h e f i b r i n p l a t e t e c h n i q u e as d e s c r i b e d by Braknlan ( 1 6 ) . HGCF A c t i v i t y determined u s i n g human bone marrow c e l l s i n a m o d i f i c a t i o n o f t h e method d e s c r i b e d by S t a n l e y and M e t c a l f ( 1 7 ) . E l e c t r o p h o r e t i c Mobi 1 it y determined by t h e method d e s c r i b e d by Seaman ( 1 9 ) . Results The f r o z e n f r a c t i o n s were thawed r a p i d l y a t 3 7 ' ~ , c e n t r i f u g e d and resuspended i n growth media.

The f r a c t i o n s were weighed and t a r e d t o

d e t e r m i n e t h e w e i g h t o f each f r a c t i o n . determined.

The pH on each f r a c t i o n was a l s o

These r e s u l t s a r e shown i n Table I. An a l i q u o t was taken

f o r v i a b l e c e l l c o u n t and based on t h i s i n f o r m a t i o n t h e c e l l s were cultured.

The d i s t r i b u t i o n o f v i a b l e c e l l s i s shown i n F i g u r e 1.

As

can be seen about 4 subpopulations o f c e l l s can be i d e n t i f i e d . A f t e r 28 days o n l y f r a c t i o n s between 11 and 19 had reached confluency. The o t h e r f r a c t i o n s were removed f r o m t h e c u l t u r e p l a t e s and t e s t e d f o r u r o k i n a s e a c t i v i t y by f i b r i n p l a t e method and showed no f i b r i n o l y t i c activity.

The sequence o f events w i t h t h e c o n f l u e n t p l a t e s a r e shown

i n Table 2 and o f t h e s u b c u l t u r e i n Tables 3 and 4 .

Those p l a t e s t h a t

went t o p r o d u c t i o n were p u t on p r o d u c t i o n media and t e s t e d f o r u r o k i n a s e a c t i v i t y a t v a r i o u s times.

The c e l l s t h a t were s u b c u l t u r e d were

removed from t h e d i s h e s w i t h EDTA and then r e c u l t u r e d .

Table 5 shows the r e s u l t s of t h e urokinase production obtained with t h e primary and subrul t u r e 1 c e l l s a f t e r 35 days on production. There i s an obvious enrichment of urokinase a c t i v i t y i n Fraction 15 when i t i s recorded a s u n i t s of a c t i v i t y per 100 c e l l s . An optimization i s a l s o seen i n several o t h e r f r a c t i o n s .

Control experiment with t h e same c e l l s

a t ground base c o n d i t i o n s gave t h e value 0.28/100 c e l l s . The s u b c u l t u r e 2 c e l l s d i d not produce urokinase when placed on production media. P a r t of t h e c e l l s from s u b c u l t u r e 2 were analyzed f o r m o b i l i t y d i s t r i b u t i o n and t h e r e s u l t s on t h r e e such f r a c t i o n s i s shown i n Figures 2 , 3 and 4.

The m a t e r i a l from SC-1 was a l s o t e s t e d f o r the presence of Human Granulocyte Conditioning Factor and t h e r e s u l t s a r e shown i n Table 6. Di scussion The r e s u l t s show t h a t c e l l s can be s e p a r a t e d under s t e r i l e c o n d i t i o n s and r e t u r n e d from o r b i t i n such a manner t h a t they r e t a i n e d t h e i r a b i l i t y t o grow in cul t u r e . The e l e c t r o p h o r e s i s i n space showed good s e p a r a t i o n of t h e kidney c e l l s i n t o subpopulations.

The r e s u l t s i n d i c a t e d t h a t t h e r e were a t

l e a s t 3 and maybe 4 subpopulations.

This r e s u l t i s i n agreement with

some r e s u l t s obtained using t h e e n d l e s s be1 t e i e c t r o p h o r e s i s . Even though each f r a c t i o n showed v i a b l e c e l l s by the s t a i n technique and they a l l a t t a c h e d t o the g l a s s s u r f a c e only the few f r a c t i o r i s bctwceri 11 and 20 grew.

The reason f o r t h i s i s not known. The only p o s s i b l e

e x p l a n a t i o n i s t h a t t h e non growers were more s e n s i t i v e t o unfavorable c o n d i t i o n s and t h e r e f o r e could not recover from t h e shock t o grow i n culture.

The d a t a i n d i c a t e s an enrichment of producing c e l l s i n t h e a r e a c e n t e r i n g around F r a c t i o n 15.

The r e s u j t s can be i n t e r p r e t e d t o show an

incomplete r e s o l u t i o n between a producing c e l l population and a non producing p o p u l a t i o n .

Considering t h e f a c t t h a t t h e c o n d i t i o n s of t h e

experiment were n o t optimized f o r kidney c e l l s but g e n e r a l i z e d f o r t h r e e d i f f e r e n t separations, t h i s r e s u l t i s not surprising.

This i s probably

why t h e c e l l s f a i l e d t o produce a t s u b c u l t u r e 2 when normally under optimi zed growth condi t i o n s they produce t o s u b c u l t u r e 7 . I t would appear t h a t t h e a r e a f o r maxiniunl production of Hunian Granulocyte Conditioning F a c t o r does not c o i n c i d e with t h a t f o r urokinase. This i s a very i n t e r e s t i n g f i n d i n g and i n d i c a t e s t h a t t h e two products a r e most l i k e l y n o t produced by t h e same c e l l . The a n a l y t i c a l m o b i l i t y d a t a a t t h e s u b c u l t u r e 2 d a t a shows each f r a c t i o n a t t h i s s t a g e t o have a r a t h e r broad d i s t r i b u t i o n .

This i s

r a t h e r disappoi nti'ng i n t h a t one would a n t i c i p a t e a r a t h e r s h a r p d i s t r i b u t i o n based on t h e narrow f r a c t i o n one s t a r t s w i t h .

This i s probably explained

by t h e f a c t t h a t t h e s t a r t i n g f r a c t i o n i s heterogeneous and t h e c e l l attachment and growth p a t t e r n i s a random e v e n t which can broaden a t each r e c u l t u r e l e v e l .

More a n a l y s e s should help prove t h i s p o i n t .

U P E T T r i a l s , C i r c u l a t i o n XLVII S u p p l . 11, ( 1 9 7 3 ) .

29,1606,

USPET T r i a l s , J . Am. Med. Ass.

(1974).

W i l l i a m s , J . W . , Baldwin, R . L . , S a u n d e r s , \#I. M . and S q u i r e , P.G., 9 . Am. Chem. Soc.

74,1542,

(1952).

A s t r u p , T. and S t e r n d o r f f . , P r o c . Soc. E x p t l . B i o l . Med.

81,

675, ( 1 9 5 2 ) . S o b e l , G . W . , Mohler, S . R . , J o n e s , N . W., G u e s t , M. M . , Arner. J . P h y s i o l .

171,1968

Dowdy, A . B . C . , and (1952).

B a r n e t t , E . V . and Baron, S . , P r o c . Soc. E x p t l . B i o l . Med.

102,

308, ( 1 9 5 9 ) . Baron, S. and B a r n e t t , E . V . ,

Proc. Soc. E x p t l . B i o l . Med.

103,

527, ( 1 9 6 0 ) . P a i n t e r , R . H . and C h a r l e s , A . F . , Amer. J . P h y s i o l .

202,

1125, ( 1 9 6 2 ) . B e r n i k , M. B. and Kwaan, H. C . , J . Lab. C l i n . Med.

70,650,

( 19 6 7 ) . B e r n i k , M. B. and Kwaan, H . C . , J . C l i n . I n v e s t .

48,

1740, ( 1 9 6 9 ) .

Weiss, R . E. and S c h l e i c h e r , J . B . , Biotech. and Bioeng.

10,

601, ( 1 9 6 8 ) . S c h l e i c h e r , J . B. and Weiss, R . E . , Biotech. and Bioeng.

10,

617, ( 1 9 6 8 ) . Barlow, G . H,, R u e t e r , A.,

T r i b b y , I . E . , P r o t e a s e s and B i o l o g i c a l

C o n t r o l , Vol. 2 , Cold S p r i n g s Harbor Laboratory (1975). NASA:

To ve p u b l i s h e d .

15.

P h i l l i p s , H . J . , In T i s s u e C u l t u r e , Methods and A p p l i c a t i o n s . Ed. Kruse and P a t t e r s o n . pg. 406, Academic P r e s s ( 1 9 7 3 ) .

16.

Brakman, K. , F i b r i n o l y s i s :

A Standardized F i b r i n P l a t e Method

and A F i b r i n o l y t i c Assay of Plasminogen.

Scheltema and Holkema

(1967). 17.

S t a n l e y , E . R . and Metcalf, D., J . Lab. C l i n . Med.

75,657,

(1972).

18.

Seaman, G . V. F . , In t h e Red Blood Cell Vol 11, pg. 1135. Acadenii c P r e s s (1 975)

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TABLE 2 SEQUENCE OF EVENTS PRIMARY CULTURE (35 mrn). P e t r i D i s h e s

FRACTION

COriDITION

DISPOSITION

1 dish

Conf 1 uen t

To p r o d u c t i o n

13 1 d i s h

Conf 1 uen t

To p r o d u c t i o n

14 1 dish

Confluent

To s u b c u l t u r e ( 2 )

15 1 d i s h

Conf 1 u e n t

To p r o d u c t i o n

16 1 dish

Conf 1 u e n t

To s u b c u l t u r e

17 2 d i s h e s

Confluent

To p r o d u c t i o n ( I ) , t o s u b c u l t u r e ( 1 1

19 1 dish

Confluent

To s u b c u l t u r e

11

B Control

1 dish

Confluent

To p r o d u c t i o n

D Control

1 dish

Confluent

To p r o d u c t i o n

TABLE 3 SEQUENCE OF EVENTS SUBCULTURE 1

CONDITION

DISPOSITION

Confluent

To s u b c u l t u r e - 2 5 crn2 f l a s k

Confl uent

To production

505 Conf 1uen t

To production

50% Conf 1uent

Recul t u r e - n o growth

Conf 1uent

To s u b c u l t u r e 2-25 c a 2 f l a s k

Conf 1uen t

To production

Conf 1uen t

To s u b c u l t u r e 2-25 c s 2 f l a s k

Conf 1u e n t

To production

TABLE 4 SEQUENCE OF EVENTS SUBCULTURE 2 Fraction

Condition

Disposition

14-2

Conf 1uen t

To Production

14-2

Confluent

To M o b i l i t y Detn.

17-2

Conf 1uent

To Production

17-2

Conf 1uen t

To M o b i l i t y Detn.

19-2

Confluent

To Production

19-2

Conf 1uen t

To M o b i l i t y Detn.

RESULTS

P R I M A R Y CULTURE FRACTION

UI< ASSAY

unitsjdish 11

13 15 17 B CONTROL D CONTROL

VIABLE CELLS XI o5

45 535 240 225

.07 .696 .I2 .744

61 81

,068 .288 SUBCULTURE 1

85 1 24 205 359

.60 .I32 .9 .222

TABLE 6 RESULTS

Human Granulocyte Conditioning F a c t o r Subculture 1

Fraction

HGCF lColoni es formed)*

14-1 16-1 17-1 19-1 *Colonies formed c o r r e c t e d f o r c o n t r o l p l a t e

ELECTROPHORESIS FOR BIOLOGICAL PRODUCTION Louis R. McCreight General Electric Company Space Sciences Laboratory Abstract P r e p a r a t i v e electrophoresis m a y provide a unique method for meeting ever m o r e stringent purity requirements. Prolonged near z e r o gravity i n space may p e r m i t t h e operation of preparative electrophoresis equipment with 100 t i m e s g r e a t e r throughput than i s currently available. Some experiments with Influenza Virus Antigen, Erythropoietin and Antihemophaliac F a c t o r , along with p r o c e s s and economic p r o j e c tions, will b e briefly reviewed. Lntroduction The idea of preparing biologicals of improved purity and specificity in space h a s both g r e a t technical and economic basis. It could become a multi-billion dollar business and be a n important u s e for the STS. There i s however a g r e a t deal of r e s e a r c h and development to p e r f o r m first. This paper reviews s o m e of the e a r l y work; f r o m the initiation of the idea through s o m e flight demonstrations and on to the c u r r e n t development of a sounding rocket experimental unit and Some ground based separations work. A brief concluding section then outlines some of the projections f o r possible future preparative electrophoresis i n space. Early History of Electrophoresis in Space Electrophoresis h a s been widely used for s e v e r a l decades, p r i m a r i l y based on the work of Tiselius, for analysis of biological materials. T h e r e a r e now a n estimated 30, 000 r e s e a r c h and analysis personnel who utilize the technique in the U. S. alone and s e v e r a l hundred technical papers a r e based on this work annually. Unlike other analysis and p r o c e s s techniques it h a s not been possible however to s c a l e up the electrophoretic analytical technique to provide a t r u l y preparative s c a l e of o p e r a tion. This i s p r i m a r i l y due to gravity induced convection and sedimentation which can be sufficiently counteracted by such approaches a s the u s e of gels, orientation, cooling, and s m a l l dimensions in the c a s e of analytical devices, but a r e too restrictive t o p e r m i t scaling up to a n economical preparative level. Thus in a "brain storming" type of discussion on space processing ideas among staff m e m b e r s of the Wyeth Laboratories and General Electric Space Sciences Laboratory i n the spring of 1969, preparative scale electrophoresis was suggested among the s e v e r a l ideas a t that meeting. Several possible product examples such a s vaccines, hormones, enzymes, and cells w e r e suggested by Wyeth a s well a s other organizations over the next year or so. About a y e a r l a t e r , specific R&D work was initiated on the idea and almost i m m e d iately an opportunity a r o s e to have a flight demonstration on Apollo 14, Ln about four months, we then designed and developed a s m a l l flight demonstration shown in Figures 1 and 2,

Samples of salmon s p e r m DNA, hemoglobin, and a mixture of r e d and blue dye w e r e chosen to r e p r e s e n t a broad range of molecular weights and to demonstrate electrophoretic mobility under microgravity conditions. Only the r e d and blue dye w e r e expected to be, and were, electrophor etically separated. Although the r e s u l t s w e r e not up to our high expectations, the r e d and blue dyes did s e p a r a t e but the photography did not provide clear pictures. The biologicals w e r e destroyed, apparently by bacteria, during the four month flight and quarantine period, but the engineering aspects of the unit w e r e excellent and w e r e r e u s e d on l a t e r flights. A second flight demonstration was then scheduled on Apollo 16 with again about four months to develop it. Steps w e r e taken to overcome the problems of the Apollo 14 flight, namely: a tripod and lens extension tube s y s t e m was provided to improve the photography (Figure 3 ) and P S L (polystyrene latex) was suggested by the USRA (University Space R e s e a r c h Association) a s a m o r e stable non-biological sample. Along with t h e choice of P S L a s the sample, ground based work using s u c r o s e solution density gradients was suggested and used to indicate ( F i g u r e 4) and define the separation of the 0.2 and 0.8 micron P S L which was flown a s a m i x t u r e in the upper tube and individually in the bottom and middle tubes, respectively, of the apparatus.

An example of the r e s u l t s of the Apollo 16 demonstration i s shown in Figure 5 with a ground based view f o r comparison. A clear indication of the possible improvements in electrophoretic separation performed in space is indicated even though electroosmosis and some bubbles a r e a l s o indicated. The l a t t e r two problems w a r r a n t some brief mention.

-

The bubbles w e r e , we now believe, caused by the permeability of the silicone tubing especially when it i s subjected to a rapid external depressurization a s was the custom on Apollo flights. During the development of the Apollo 16 unit t h e r e had been s o m e indication of s t r e s s corrosion problems with the Lexan used to fabricate the electrophoresis cells. This was p r i m a r i l y due to the u s e of thin wall sections f o r g r e a t e r transparency a s compared to the m o r e m a s s i v e monolithic machined block used for Apollo 14. While the design was indeed demanding of the full capabilities of the Lexan, i t was the best choice of t r a n s p a r e n t plastic and probably not the source of fluid leaks that permitted bubbles to form. The electroosmosis i s t h e r e s u l t of the high zeta potential on the walls of the e l e c t r o phoresis chambers. At that t i m e t h e r e was no low zeta potential coating available which would a d h e r e adequately t o the chamber walls, and ground based t e s t s in s u r c r o s e density gradients indicated no benefit f r o m the u s e of a collodion coating a s compared to leaving the Lexan uncoated. Since applying a coating may have been detrimental to the Lexan f r o m the s t r e s s - c o r r o s i o n standpoint, it was decided to leave it uncoated. The problem then i s one of either not being able to t r a n s l a t e t h e density gradient work to the flight demonstration unit o r in not being able to obtain ground based r e s u l t s f r o m the flight demonstration unit that would show the electroosmosis problem. Happily, the remaining problems with these two flight demonstrations have been o v e r come in the m o r e major experiment MA-011 (which on the other hand had some other difficulties which a r e being a s s e s s e d and reported separately). The MX-0 11 also again m a d e use of the phase s e p a r a t o r s and s m a l l peristaltic pump plus other technologies f r o m +OH-o 14 and 16. Thus it appears that while each flight h a s c o r r e c t e d the deficienc i e s of the previous flight, new problems have a r i s e n by virtue of the changes made in

the demonstration m a t e r i a l s , o r design, o r equipment, a s each unit was being hurriedly developed for a singular flight opportunity. It i s therefore highly satisfying to s e e the sounding rocket and space shuttle flight schedules and the possibility for repeated flights of an experiment until it i s satisfactory fbr a s long a s warranted.

Equipment - With the completion of the sufficiently satisfactory Apollo 14 and 16 flights, our attention was turned toward meeting the original and still d e s i r a b l e goal of developing a truly preparative unit for space experiments. We chose the continuous flow type of unit a s offering the g r e a t e s t e a s e of inserting and removing samples and s a m p l e fractions. While the engineering of such a unit r e q u i r e s ingenuity, it i s not extremely difficult and the basic idea for the electrophoresis cell i s to simply make it thicker than the 0.5 to 1.5 m m commonly used for such units on earth. It was estimated that the c e l l in such a unit for space could be a s much a s 8-10 m m thick and provide an improvement by a factor of about 5 - 10 in resolution or an improvement of about 80- 100 in throughput. This much g r e a t e r performance i s simply due to being able to scale up the thickness without gravity induced convection and to i n c r e a s e the sample concentration without sedimentation problems. This h a s now been demonstrated with a 4 m m thick cell, a t l e a s t partially, on t h e ASTP-h4A-014 experiment by Hannig. A s i m i l a r unit has a l s o been developed i n our laboratory for sounding rocket usage. It is shown in F i g u r e 6 a s currently equipped with a c a m e r a for data acquisition. Other work is now underway on modifications to p e r m i t collecting up to 50 fractions of sample and to detect them by a U. V. scanner system. These a r e being done on a schedule to permit a flight test in late 1976. The cell i s 5 m m thick by 5 c m wide and has a 10 c m long electrode section. It i s supplied with approxiC coolant, and samples by the u s e of a passive refrigerant system. The mately ~ O buffer, possible operating conditions such a s flow r a t e s , v o l t s / c m a c r o s s the cell, etc. a r e v e r y broad and can be adjusted over s e v e r a l decades by a choice of gear ratios on the pumps and by plug-in power supplies, a s well a s by "fine tuning" electrically up to a short t i m e before flight. Math Modeling and Computer Simulation - P r i o r to designing the current sounding rocket unit, a math model was prepared both to aid the design and to predict the performance of thick cell electrophoretic s e p a r a t o r s in space. A s e p a r a t e publication i s in preparation on this work s o it will only be briefly reviewed here. F i g u r e 7 shows t h e factors conside r e d i n t h e m a t h model along with indications of which a r e controllable. As compared to previous efforts to d e s c r i b e the operation of a continuous electrophoresis cell, the p a r a m e t e r s in our model a r e allowed to interact and a r e calculated primarily a s to t h e i r effect on resolution and secondarily, throughput. Some typical r e s u l t s for a r e a l i s t i c although hypothetical separation of four samples (as defined by zeta potential and other conditions in each of t h r e e different thickness flow cells) a r e shown in Figures 8, 9, and 10. Extensive ground based testing in prototype electrophoresis cells built for the previously described sounding rocket, a s well a s with other electrophoresis units, has generally corroborated the calculations for a t l e a s t low levels of power. A plot of the power v e r s u s resident t i m e (as a m e a s u r e of flow r a t e ) and thickness for stable and unstable conditions (hydraulically) i s presented in Figure l I. Unfortunately, gravitationa l convection induced by the joule heating in these thick cells occurs at levels of power (10-15 watts) which a r e an o r d e r of magnitude below the power levels useful for separation.

Therefore, while the calculations and ground based t e s t s a t low levels a r e in good a g r e e ment, the m o r e realistic level t e s t s will have to be accomplished in space under m i c r o gravity conditions,

- Ground based t e s t s for both the development of equipment and for establishing operating conditions f o r biologicals a r e being accomplished in a commercially available unit shown in F i g u r e 12. It i s a Beckman C P E I1 which i s well designed for model studies and to which we a r e making additions and changes for m o r e easily handling biologicals. These include fused silica windows and a U. V. scanner, for example. Studies ranging f r o m single experiments, so f a r , to about 20 experiments, plus numerous calibration r u n s with PSL, have been undertaken with each of various biologicals including: Hepatitis Vaccine Sperm Lymphocytes AHF Erythr opoietin Influenza Virus Antigen The r e s u l t s a r e generally encouraging but not n e c e s s a r i l y easily achieved nor suffi ciently complete. Considerably m o r e effort has to be expended in this a r e a before flight t e s t s since it s e e m s unlikely that a space flight t e s t will accomplish a separation that has not a t l e a s t been shown to be feasible on earth. Examples of some of the separations studied and r e s u l t s obtained a r e shown in Figures 13- 15. Projections for the Future - Contacts in numerous pharmaceutical houses indicate that, while indeed cells of various types and sources a r e a n intriguing problem for separation science, numerous hormones, enzymes, blood and u r i n a r y source m a t e r i a l s , and vaccims need o r would be benefited by a great deal of improvement in purity. The practical limitation on the u s e of electrophoresis to p r e p a r e these products in sufficient purity to be of value i s throughput efficiency. While absolute purity i s required in certain cases, many products a r e only needed in m o r e concentrated f o r m and therefore resolution i s often a subjective p a r a m e t e r which can perhaps be traded off against throughput. An estimate of the throughput in g r a m s l h o u r v e r s u s cell thickness f a r two c a s e s i s shown in Figure 16. The upper right hand a r e a of the figure depicts a high throughput case, i. e. for a c a s e where sufficient resolution i s easily obtainable and the extra capacity of the equipment can be utilized for throughput. The lower portion of the figure depicts a situation w h e r e resolution is to be s t r e s s e d . Ln each c a s e a range of 1-1070 sample concentration i s shown which should be compared with typical ground based practice of using about 0. 1 to 0.570. Ln any case, s o m e 2 to 3 o r d e r s of magnitude improvement in throughput a r e predictable. This i s f a r beyond the degree of improvement for which one might consider simply duplicating the ground based facilities when g r e a t e r throughput i s needed even if resolution w e r e satisfactory. Economic Predictions - T h r e e g e n e r a l a r e a s of potential payoff for this work a r e f o r e seen. F i r s t i s the possibility of the r e s e a r c h and development being beneficial to ground based electrophoresis equipment and techniques. Secondly i s the possibility of preparing m o r e specific s t r a i n s or products i n space which can then be used to culture and produce g r e a t e r quantities of particular products on earth. Thirdly, when the f i r s t two o r other approaches a r e insufficient, products may actually be produced in space. Examples of the f i r s t two approaches already being productive a r e available, Improved electrophoresis equipment and coatings with nearly z e r o zeta potential a r e now available

and a r e examples of the f i r s t a r e a of benefits. Increased yield of Urokinase through t h e improved separation of fetal kidney cells on the Apollo Soyus Test Project flight in the s u m m e r of 1976 i s a n e a r l y indication of a potential benefit in the second a r e a . Pr elimina r y examples of the third a r e a must await further work but may well come f r o m c u r r e n t projects for the sounding rocket and e a r l y shuttle flights. Several products could potentially benefit f r o m t h e s e capabilities and further work i s recommended to establish the n e c e s s a r y protocols and r e f e r e n c e data on which to b a s e flight tests. The human value of m o r e effective biologicals i s of c o u r s e impossible to m e a s u r e , The preparation of p u r e r erythropoietin could f r e e s o m e 15,000 U. S. r e n a l failure patients f r o m repeated blood transfusions. Thus humanitarian and societal motivation in this a r e a i s unusually high, and even g r e a t e r than the basic economic value. Some simple p r o j e c tions for space processing of biologicals can be made based on c e r t a i n assumptions. It i s presumed f i r s t that for efficiency and economy, a s much of the processing a s possible will be done h e r e on Earth. Then, only a reasonably pure concentrate will be taken to space for one m o r e , o r perhaps a few, processing steps. In addition, the l a r g e quantities of water normally used in biological processing a r e presumed to be r e c o v e r a b l e and reusable in space so that this commodity will not need to be completely resupplied f r o m E a r t h for each product. Finally, however, the general r u l e that each biological product should be p r e p a r e d in isolation f r o m other products i n o r d e r to avoid c r o s s contamination i s likely to be necessary. This may necessitate s o m e special schedcling, but should not c r e a t e any insurmountable problems. Vaccines a r e the best defined available product on which to base projections for the future. In the U. S., s o m e 60 million doses of vaccine a r e used annually. If we utilize the World Health Organization's estimates of World population in 1990-2000 a s 5 billion and a s s u m e t h e s a m e r a t e of vaccine applications world-wide a s i s now current in the U. S., we project the need for about 1.5 billion doses of vaccine per year. Using a conservative average number of 100,000 doses per g r a m of active ingredient, we calculate the need f o r 15,000 g r a m s of active ingredients per year. Many currently used and v e r y fine biologic a l products a r e a t best however quite dilute o r i m p u r e (but not n e c e s s a r i l y with harmful impurities). The purity may range f r o m l e s s than 1'70 to about 5070. This i s a s s u m e d to be the starting m a t e r i a l for a space purification operation. Therefore, the weight of starting m a t e r i a l could range f r o m 2 to 100 t i m e s the 15,000 g r a m final product weight derived above. Assuming a conservative average of 50, i t i s expected that some 750 Kg of partially purified vaccines might be used a s the starting materials. In addition, some s e v e r a l hundred kilograms of water would be required. While vaccines generally cost about 20$ per unit to produce, some examples of higher costs for g r e a t e r specificity indicate that $1.00 p e r unit m a y be a n acceptable value. This then indicates a $1. 5 billion dollar activity in vaccines alone, a fraction of which may r e q u i r e space operations. The processing of s o m e other biological products such a s c e l l s and the blood derivatives in space while l e s s specifically calcuable could easily exceed the estimates for vaccines by up to a n o r d e r of magnitude in volume and value.

The support and encouragement for much of this work by NASA through s e v e r a l contracts i s appreciated and acknowledged. In addition, major r o l e s i n various a p s e c t s of this work have been filled by s e v e r a l a s s o c i a t e s especially Dr. R. N. Griffin, R. Je Locker, Dr. J, Giannovario, and F r a n k Cosrni, a s well a s others too numerous to mention. It i s a p l e a s u r e to acknowledge the work of a l l of them.

Figure 1.- Apollo 14 fluid electrophoresis demonstration unit on right with some of the major components at left. These include the three electrophoresis cells machined in a monolithic block of Lexan, with the phase separators below and the peristaltic pump at center. The overall dimensions of the experiment are approximately 4 by 5 by 7 in. plus appurtenances.

Figure 2.- Apollo 14 fluid electrophoresis demonstration unit in opened configuration showing back view of electrophoresis cell in upper portion of box with phase separators and peristaltic pump in middle and fluorescent lamps and potted electronics in lower area.

Figure 3.- Apollo 16 fluid electrophoresis demonstration mockup. Larger window, instruments, and camera-tripod arrangement improve data acquisition.

.-

Figure 4 Electrophoretic separation of 0.2- and 0.8-micrometer polystyrene latex in a sucrose density gradient after 40 minutes during which time the leading band (0.8 micrometer) traveled 8 centimeters and the trailing band 5,6 centimeters using a 0.085 M borate buffe? of pH 8,5.

(a) Flight results.

(b) Ground results. Figure 5.- Results of Apollo 16 fluid electrophoresis demonstration. The flight results clearly show the benefit of reduced gravity on the electrophoretic mobility at 30 V/cm in borate buffer of a mixture of 0.2- and 0.8-micrometer polystyrene latex (FSL) in the upper tube, 0.8-micrometer PSL in the middle tube, and 0.2-micrometer PSL in the lower tube. Equivalent samples in the ground-based results show the detrimental effects of gravity-induced convection and sedimentation.

Figure 6.- Advanced a p p l i c a t i o n s f l i g h t experiment (AAFE) continuous-flow e l e c t r o p h o r e t i c s e p a r a t o r ( r i g h t ) under development f o r use on a sounding rocket. The upper enclosure (middle) with an access door f o r i n s t a l l i n g t h e sample, s e r v i c i n g t h e c w e r a , and s e t t i n g experiment conditions before f l i g h t ; and t h e t e s t and c o n t r o l panel i n c l u d i n g power supply ( l e f t ) a r e a l s o shown.

INTERDEPENDENCE OF CELL VARIABLES VOLUME

VOLTAGE GRADIENT*

7

n

POWER

ELECTRICAL CONDUCTANCE

THERMAL CONDUCTIVIN

TEMPERATURE DISTRIBUTION

Figure 7.- Major f e a t u r e s of a computerized mathematical model of a continuous-flow e l e c t r o p h o r e t i c s e p a r a t o r , ( c o n t r o l l a b l e v a r i a b l e s a r e i n d i c a t e d by a s t e r i s k s . )

CELL > K M X W M TMICXNES% DO7CM SIMPLE DIAM. a(lbU1 SlFLD 4O\'/CY WALL POT'L: 5HV PART. POT'L. 23.29.30. ! CENTERLINE VELLKIIV. RELATIVE MOUNT

Figure 8.- Illustration of calculations for a four-component separation in a 0.5-mm-thick continuous-flow electrophoresis cell. (Note expanded scale ; other assumed conditions as indicated.)

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Figure 9.-

Illustration of a calculated resolution for the same four-component separation as figure 8 in a 1.5-m-thick cell.

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Figure 10.- Illustration of calculated resolution for the same four-component separation as figures 8 and 9 in a 5-mm-thick cell.

ACTIVE AREA 5CM X 1OCM SAMPLE: N E U T R A L DENSITY POLYSTYRENE L A T E X ORIENTATION 20° F R O M VERTICAL

Figure 11.- Experimentally determined regions of stable and unstable operation of the AAFE (fig. 6) electrophoresis cell as a function of power and residence time for a 5-mm-thick cell with other conditions estimated,

154

Figure 12.- Modern laboratory continuous-flow electrophoretic separator.

14

15

16

17

18

19

20

21

22

23

24

25

FRACTION

Figure 13.- Partial concentration of Erythropoietin from protein by electrophoresis.

4

5

6

7

8

9

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11

12

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FRACTION

Figure 14.- Concentration of antihemophiliac factor VIII by continuous-flow electrophoresis.

HEMAGGLUTINATION AND NEURAMIN IDASE ENDOTOXIN

3

I 4

5

6

7

8

9

10

11

12

13

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15

16

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Figure 15.- Separation of influenza virus antigen from endotoxins by continuous-flow electrophoresis.

TO 4.0 A T 10 MM 10 % CQNC

1 % CONC.

0.5

1.5

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10 W IS CONSTANT

CELL THICKNESS, T (MM)

Figure 16.- Calculated throughput for continuous electrophoretic separators as a function of cell thickness and sample concentration for high-resolution (lower curves) and high-throughput examples.

-

N11-11688 SOME QUESTIONS -OF SPACE B IOENG I NEER ING

LASZLO K, N Y I R I FERMENTATION DESIGN, INC, DIVISION OF NEW BRUNSWICK SCIENTIFIC GO,, INC, BETHLEHEM, PA a 18017 j

COLLOQU IUM ON B IOPROCESS ING IN SPACE JOHNSON SPACE CENTER, HOUSTON, TEXAS MARCH 10-12, 1976

1 '

SOME QUEST1ONS OF SPACE B 1OENG 1NEER ING

FERMENTATION DESIGN, INC* DIVISION OF NEW BRUNSWICK SCIENTIFIC CO,, INC,

BETHLEHEM, P A , ,

18017

.

. .for the chief malady of man i s a restless c u r i o s i t y about things which he cannot understand; and i t i s not so bad f o r him to be in e r r o r as to be curious to no purpose. PASCAL (Pensees, Section 1, #18)

ABSTRACT Zero-gravity offers selective effect o n g r o w t h and metabolic activity of unicellular organisms as well as unique opportunities i n purification of organic compounds. These make i t possible to consider the biosynthesis and recovery of certain metabolites economically feasible in space. Design, construction and operation of systems for the above mentioned purposes r e q u i r e s i n t e r d i s c i p l i n a r y actions w i t h i n the scope of a new discipline: space bioengineering. Paper discusses the problems and perspectives o f this d i s c i p l i n e p a r t i c u l a r l y in the application of bioreactor-recovery systems in space to manufacture metabolites of h i g h economic and scientific value. Special attention i s paid to pivotal factors such as various mass transport phenomena, contamination control, automatic control o f optimum environment and synchronization of the operation of the biological (biosynthesis) and the physicochemical (recovery-purification) systems. Although the space bioengineering is i n its early stage of development, i t s role w i l l g r a d u a l l y increase w i t h the f u l l implementation o f the Space Bioprocessing Program.

I n the course o f technical developments, we always witness that the human knowledge o n the p h y s i c a l behavior o f i n o r g a n i c materials precedes the information o n biological systems. T h i s t r a d i t i o n was observed again, most r e c e n t l y , d u r i n g the Apollo a n d S k y l a b e x p e r ments w h i c h demonstrated s i g n i f i c a n t changes in the p h y s i c a l behavior o f f l u i d s and gasses exposed to z e r o - g r a v i t y (1) . On the other hand, c e r t a i n experiments related to the search o f effects of g r a v i t a t i o n a l acceleration, cosmic r a d i a t i o n a n d weightlessness o n l i v i n g systems revealed, among others, the p o s s i b i l i t y o f c u l t u r i n g u n i c e l l u l a r organisms in space. Increased specific g r o w t h r a t e and h i g h e r c e l l d e n s i t y obtained w i t h S. t y p h i m u r i u m (2) as w e l l as h i g h e r frequency of i n d u c t i o n o f bacterioFhage in E . coli K-12 ( 3 ) indicated improved metabolic a c t i v i t i e s p r o b a b l y d u e t o altered physicochemical p r o p e r t i e s o f the l i q u i d s and gases u n d e r near weightless conditions.

-

These r e s u l t s i n i t i a t e d a s t u d y by JORDAN (4) concluding that t h e r e is a p o s s i b i l i t y to enhance the p r o d u c t i v i t y of c e r t a i n o r g a n i c compounds i f the biosynthesis takes place i n space. Most recent experiments w i t h z e r o - g r a v i t y electrophoresis revealed the p o s s i b i l i t y of f i n e separation and ( u l t r a ) p u r i f i c a t i o n of biological materials ( 1 ) . These basic experimental data forecast a potential research a c t i v i t y a n d i n d u s t r i a l application in space environment f o r processing biological materials. A new d i s c i p l i n e seems to shape u p w h i c h may be called as "space b i o e n g i n e e r i n g " . Objective o f t h i s paper i s to analyze some o f the major questions related to t h i s technology. It i s emphasized, however, that the potentials and l i m i t s of t h i s d i s c i p l i n e have not been i d e n t i f i e d y e t . A l l of o u r discussion i s based o n a few experimental data in space and the knowledge accumulated, m o s t l y d u r i n g the last 30 years, i n biochemical engineering. It i s felt, however, that t h i s s t u d y g i v e s some basis w h i c h can be u t i l i z e d by space processing scientists d u r i n g the implementation o f Space Bioprocessing Program.

SPACE BIOENG I NEERING U n i q u e p r o p e r t i e s o f the space environment ( p a r t i c u l a r l y g r a v i t a t i o n a l effects, cosmic r a y s ) i n t e r a c t i v e l y influence the bioprocesses which, henceforward, r e q u i r e s h a r d w a r e a n d technology substantially d i f f e r e n t from the ones employed o n the Earth. Space bioengineering i s concerned w i t h the h a r d w a r e d e s i g n and technologies related to p r o c essing o f o r g a n i c compounds b y means of biosynthesis and r e c o v e r y p u r i f i c a t i o n , a t least one of w h i c h takes place i n space.

As a d i s c i p l i n e ( F i g u r e 1 ) space b i o e n g i n e e r i n g i s closely r e l a t e d @ biochemical ( 5 ) w h i c h g e n e r a l l y deals w i t h the t h e o r y and n of materials a p r a c t i c e of l i q u i d . I t has also a r e l a t i o n s h i p w i t h w h i c h deals w i t h e x t r a t e r r e s t r i a l detection of microorganisms, evaluation of b e h a v i o r o f t e r r e s t r i a l microorganisms i n space and m o n i t o r i n g o f sgacecraft a n d astronaut m i c r o b i a l f l o r a . T h e d i s c i o l i n e has a close r e l a b o n s h i p w i t h the material processing sciences spedially u t i l i z i n g the knowledge o n u n i q u e behavior of l i q u i d s , gases a n d s o l i d s in zero-gravi t y . T a b l e I l i s t s the m a i n areas of c o n c e r n r e l a t e d to a c t i v i t i e s in space b i o e n g i n e e r i n g . A t t h i s time, w e shall address o u r s e l v e s o n l y to few k e y questions closely related to implementation of NASA L i f e Sciences Program in Space, p a r t i c u l a r l y the e a r l y stage of Space S h u t t l e a n d Spacelab experiments. I t i s anticipated, however, that g a i n i n g f u r t h e r p r a c t i c a l information, the scope of discussions w i l l b r o a d e n i n c o r p o r a t i n g s u c h questions as experimental t r i a l o f bioprocessed material in space f o r q u a l i t y c o n t r o l purposes.

PROCESS DESIGN -FOR SPACE EXPERIMENTS A major objective f o r the i n i t i a l stage o f Bioprocessing Program i s the demonstration of usefullness of space b i o s y n t h e s i s a n d biochemical separation techniques. Implementation of biosyntheses a n d r e c o v e r y p u r i f i c a t i o n processes in space, however, faces c o n s t r a i n t s f r o m the v i e w p o i n t of payload, in p a r t i c u l a r r e g a r d i n g the r e q u i r e m e n t of r e l a t i v e l y l a r g e q u a n t i t y of water d u r i n g each step o f the operation. Another important c o n s t r a i n t i s the maintenance of aseptic c o n d i t i o n d u r i n g the c u l t u r e a n d p r o d u c t r e c o v e r y . Usefullness of b i o p r o c e s s i n g c a n b e demonstrated in p r o d u c t i o n o f one o r m o r e o r g a n i c compounds of h i g h s c i e n t i f i c o r medical v a l u e in a q u a n t i t y a p p l i c a b l e f o r a t least experimental purposes. In a n attempt to d e f i n e t h e most p r o m i s i n g materials, T a b l e II l i s t s v a r i o u s o r g a n i c compounds c u r r e n t l y p r o d u c e d b y means of b i o s y n thesis and biochemical r e c o v e r y techniques o n l a b o r a t o r y o r indust r i a l scale. Each process r e p r e s e n t s a t y p e o f metabolic p a t t e r n a n d has a t t r a c t i v e features f r o m experimental p o i n t of v i e w . A c c o r d i n g l y ,

1.

P r o d u c t i o n of c e l l mass (SCP) o r ETOH o n c a r b o h y d r a t e s c a n b e the s u b j e c t of e x p e r i ments o f s h i f t i n g metabolic p a t h w a y in f a v o r o f one p r o d u c t accumulation ( 7 ) ,

2.

B i o s y n t h e s i s o f g l u c o n i c a c i d f r o m glucose i s a classical example of combined a n d staged a c t i v i t y o f v a r i o u s cell-bound, cel I-free enzyme a c t i v i t i e s as we1 l as nonenzymatic c o n v e r s i o n o f a n intermediate i n t o f i n a l p r o d u c t .

Besides, the process i s w e l l k n o w n , therefore, comparative studies can b e easi l y made. 3.

Production o f o x y t e t r a c y c l i n e ( O I C ) has the combined c h a r a c t e r i s t i c s o f the former two processes ( w i t h the exception of nonennymatie catalysis s t e p ) . In addition, the p r o b l e m of contamination i s g r e a t l y r e d u c e d because of the w i d e spectrum of the a n t i b i o t i c a c t i v i t y .

4.

Biosynthesis of v i t a m i n B 1 2 i s a n example of m i x e d c u l t u r e o p e r a t i o n i n c o r p o r a t i n g complex g r o w t h a n d p r o d u c t formation k i n e t i c s .

A s i t i s noted o n T a b l e II w i t h the exception o f the f i r s t process, p r o d u c t r e c o v e r y can b e implemented either by chromatography o r by electrophoresis. O n the other hand, the absolute (scientific, commerical) v a l u e of p r o d u c t s #1 - #4 i s low, whereas the d e s i r e d q u a n t i t y for applicat i o n i s r e l a t i v e l y l a r g e . E v e n in the case of substantial improvement in b i o s y n t h e s i s (assuming f o u r f o l d increase in space) the needs can b e f u l f i l l e d o n l y w i t h m o v i n g of l a r g e q u a n t i t i e s o f w a t e r . Because of these considerations, a n y of the processes has s h o r t r a n g e of a p p l i c a b i l i t y and s c i e n t i f i c v a l u e o n l y in the testing stage o f space biosynthesis a n d r e c o v e r y equipment. P r o d u c t #5 has the advantage o f experimental t r i a l of eucaryote c e l l g r o w t h exposed to space e n v i r o n m e n t as w e l l as p r o d u c t i o n of compounds of s c i e n t i f i c a n d medical significance. In p a r t i c u l a r , p r o d u c t i o n of g r o w t h hormones (GH) , adrenocortical s t e r o i d hormones, t h y r o c a l c i toni n a n d p a r a t h y r o i d hormone may b e l i s t e d h e r e as p r i m e candidates. In addition, electrophoresis i s cons i d e r e d as the b e s t means in separation of the p r o t e i n a n d p o l y peptide compounds f r o m the c u l t u r e medium components ( e . g . from serum) . POSNER, in a s h o r t d i s c u s s i o n (8), d e s c r i b e s the most r e c e n t achievements (notably, d i r e c t r e l a t i o n s h i p between cell mass and GH p r o d u c t i o n , suspension c u l t u r e o f p i t u i t a r y tumor cells, enhancing effect of h y d r o c o r t i s o n e o n GH production, release o f hormones i n t o the e x t r a c e l l u l a r l i q u i d ) . W i t h a potential increase o f cell d e n s i t y to 1012 c e l l s p e r l i t e r f r o m l o 9 cells p e r l i t e r , gonadotropin hormone p r o d u c t i o n can b e substantially augmented (a cautious estimation i s a f o u r f o l d increase in GH p r o d u c t i o n ) . HIMMELFARB and h i s c o w o r k e r s a l r e a d y r e p o r t e d l o 8 cells p e r m l in a perfusion-suspension apparatus (9) . On the basis o f the f i r s t experiments i n space r e l a t i v e to f l u i d a n d gas m i x i n g conditions, i t i s anticipated that the enhanced o x y g e n transfer w i l l i m p r o v e the cell metabolic a c t i v i t y leading to increased cell number, hence l a r g e r hormone p r o d u c t i o n . In case of a c h i e v i n g

10" cells p e r l i t e r , a semicontinuous c u l t u r e can produce about 2 G G H / L / 2 4 h o u r s f o r f u r t h e r r e c o v e r y and p u r i f i c a t i o n . Also

changes i n normal human cells "anchorage dependency" can be anticipated i n ;zero-(;, m a k i n g p r o d u c t i o n of hormones by nonmalignant cells possible.

EQUIPMENT DESIGN GENERAL CONSIDERATIONS

A c c o r d i n g to experiences accumulated in biochemical engineering the f o l l o w i n g m a i n r u l e s m u s t be observed i n the h a r d w a r e design: 1

.

2.

Systems i n t e g r i t y . T h e e n t i r e system consists o f t h r e e major elements: 1) Biosynthesis equipment, 2) Recovery equipment and 3) Process s u p p o r t subsystem. F r o m operation p o i n t of v i e w a l l those elements a r e considered as one unit. T h i s p r i n c i p l e defines the t y p e and number o f m o n i t o r i n g and control elements as w e l l as the mode o f operation o f the system. Systems f l e x i b i l i t y . A t the b e g i n n i n g o f the experiments, p a r t ~ c u l a r l yi n the test staqes a t least t h r e e tyDes o f i-eactions dan b e visualized: 1) Fast, enzymatic conversion o f C O ~ D O UA ~ i ~n t o B ( w h e r e c o m ~ o u n dB i s the subiect o f r e c d v e r y ) , 2) ~ e l a t i v e fast l ~ microbiological proc&s ( d o u b l i n g time = 20-60 minutes, p r o d u c t formation r a t e (dP/dt) = ) 5C/L/HR) and 3) R e l a t i v e l y slow process ( d o u b l i n g time = 10-20 h r s , p r o d u c t f o r m a x r a t e 5G/L/HR).