Polymer Research in Microgravity - American Chemical Society

Chapter 14

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 31, 2018 | https://pubs.acs.org Publication Date: August 14, 2001 | doi: 10.1021/bk-2001-0793.ch014

Production of Bacterial Polyesters in Simulated Microgravity Radhika Thiruvenkatam 1

1

and Carmen

Scholz

2,*

2

Departments of Chemical Engineering and Chemistry, University of Alabama at Huntsville, John Wright Drive, Huntsville, AL 35899

The synthesis of bacterial polyesters was demonstrated in simulated microgravity, by growing Azotobacter vinelandii UWD in the NASA-Bioreactor. Bacterial growth in simulated microgravity differed significantly from that observed in conventional shake flask experiments. Cells tended to grow in a cluster-like pattern. Bacterial cells started to produce polymer immediately after exposing them to conditions of simulated microgravity, no lag time was observed. Within the first 24 hours of the fermentation, bacterial polyester production was threefold higher in the bioreactor than in conventional shake flasks used as control. In an effort to differentiate between the effects of microgravity and the diffusion based aeration of the bioreactor, a gas supply profile was developed that led to similar amounts of dissolved oxygen in the bioreactor and in shake flask experiments. The bacterial growth behavior in the bioreactor was studied by monitoring the glucose and oxygen consumption and was compared to that in conventional shake flask experiments.

© 2001 American Chemical Society

Downey and Pojman; Polymer Research in Microgravity ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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Introduction Our society is challenged with the need for an increasing number of consumer goods and with the need for their disposal after their usability has been exhausted. New resources for materials production have to be explored and it is imperative to establish alternative disposal and recycling procedures. The aspects of degradability and ability to recycle become even more crucial when a closed system, like a spaceship leaving the Earth or a habitat on a distant planetary body, is considered. Materials production and management of solid waste become an imminent and intertwined problem when the production of materials, their use and eventual disposal are confined to such a limited space. Due to stringent restrictions in mass that a mission can carry from the Earth, materials must be chosen judiciously and, where possible, should be designed for several recycling cycles. The more diversity, with respect to the materials properties, that each use-recycling step allows, the greater the self-sufficiency of the travelling system. In the near future, space exploration will continue to focus on Lower Earth Orbit, i.e. on board the Space Shuttle and the International Space Station. The latter one is a unique platform to test the reliability of a closed travelling system with no immediate and only limited access to supplies from Earth. The success of the International Space Station will, in part, depend on the reliability of the Advanced Life Support System operating in the station. Once a reliable system has been established that guarantees the safety and provision of the crew with food, and manages the recycling of carbon dioxide and waste that is generated in the course of human activity in space, missions will start to focus on deep space exploration. N A S A plans human missions to planetary bodies in our solar system, with the Moon and Mars as immediate destinations after 2010. Even though, missions lasting longer than 600 days, the minimum for a round trip to Mars, are beyond today's technical capabilities, it is imperative to explore now avenues that will result in the realization of these daring plans. Some of the most important issues are the protection of the crew from cosmic radiation, the regeneration of air, water and food, and a management of solid waste, that guarantees optimum resource recovery and a safe environment in the vehicle or habitat. It will be mandatory that medium and long-term space missions produce their food on board, crops have been already identified that will be suited for vehicular activities, the near future goal, and planetary activities in the more distant future. One way to combine the management of generated waste and the recovery of resources from solid waste, is the utilization of biological systems. They are unique for this task since they are characterized by a set of properties, which distinguish them from any other "production" facility. They regenerate, are energy and size efficient and they can adapt to changing environments. In



205 addition, careful interference with biological pathways results in the formation of tailored products designed for specific properties and uses. Special emphasis is placed upon microorganisms, which are extremely versatile with respect to their carbon sources and adaptability to changing environments. Microorganisms are known to degrade organic material ranging from cellulose, to crude oil and toxic waste. Additionally, with the tools of recombinant D N A techniques in place, the capabilities of microorganisms can be expanded beyond their natural capacity and tailored to almost any task. Microorganisms not only degrade organic material, they are also capable of producing useful materials, and they are already exploited for this purpose; e.g. yeasts secrete biologically active glycolipids in comparatively large quantities (1,2), other microorganisms produce surfactants that are useful in the degreasing of machinery (3,4). Bacteria, in particular E. coli, have been studied extensively and the application of recombinant D N A techniques led to their exploitation for the synthesis of biomedically relevant proteins (5,(5). Other bacterial strains, as elaborated below, produce structural materials, biopolyesters, in significant amounts. Based on their versatility, ability to regenerate, ease of handling and most importantly the fact that they thrive in an aqueous environment, thus, omitting the need for harsh chemicals or extreme synthesis conditions, microorganisms can be an ideal candidate for waste management and materials production onboard a spaceship. Ideally, waste will be transformed into a new and valueadded material that can undergo several steps of recycling once its usability is exhausted. A s indicated above, microorganisms could be useful for several different types of application onboard a spaceship. The present study demonstrated the feasibility of producing biopolyester by microorganisms in simulated microgravity. The biological system under investigation in this study was Azotobacter vinelandii UWD, a bacterial strain known to produce ρο1ν(βhydroxybutyrate), P H B , a biopolyester of the family of ρο1ν(βhydroxyalkanoates), PHAs. Microorganisms have been studied in microgravity before, in particular on the Spacelab missions. A l l of these studies focused however, on microorganisms associated with the human body or pathogens. Most of these studies found a different growth behavior in microgravity as compared to bacterial growth on Earth. Space flight experiments have shown that fermentation experiments performed in microgravity were characterized by shortened lag times and increased final cell densities (7-13). The mechanisms leading to enhanced proliferation are not fully understood yet. It is assumed that the quiescent conditions in microgravity might cause fewer disruptions of the cell-cell interactions leading to genetic exchange. Even though the mechanism is not yet understood, the influence of increased radiation in low orbit can be ruled out as the determining factor. Fermentation experiments that were performed in the Shuttle using a reference centrifuge to simulate l g gravity yielded a bacterial


206 growth behavior that was comparable to the growth behavior observed on Earth (14-17).


Biopolyesters produced by bacteria A wide variety of microorganisms produces PHAs for the purpose of carbon and energy storage (18,19). The most common biopolyester and the one produced by the majority of biopolyester-producing microorganisms is PHB, see Figure 1. P H B is produced by aerobic as well as anaerobic microorganisms. Polymerase enzymes become activated by environmental changes such as the shortage of one or more nutrients. Under experimental conditions, nitrogen and/or oxygen limitation are employed to initiate polymerase activity. Bacterial polyesters are unique with respect to some of their properties, they are isotactic, biodegradable and biocompatible. Moreover, they are highly UV-resistant and thermoplastic. A l l chiral Carbon atoms in the polymer backbone are in perfect R-configuration, see Figure 1. C H

3

fnu \ ( p

H c r

H

r\

2 ) x / °

^CH

2

x=0, 1: short chain PHA e.g. χ = 0: Pofy(P-hydroxubutyrate) (PHB)

χ > 2 : long-chain PHA

Figure 1: Structure of bacterial polyesters Perfectly isotactic polymers are not readily synthesizable by conventional synthetic routes; this is a characteristic that can be only achieved by enzyme catalysis. The isotacticity of bacterial polyesters is attributed to the enzymatic synthesis at the active site of the polymerase enzyme. Among others, isotacticity is essential for biodegradability (20), and biocompatibility (21). The isotacticity is also largely responsible for the physical properties of the material. The melting point of the polymer is about 170 °C. Due to its high crystallinity (> 80%), the polymer crystallizes readily in helix conformation, and the presence of large spherulitic crystals that form upon cooling or solvent evaporation, makes P H B a rather brittle and stiff material. The incorporation of hydroxyvalerate repeat units into P H B leads to the formation of a biocopolyester: poly(P-hydroxybutyrate-co-valerate) (PHBV) with properties reminiscent of polypropylene. The incorporation of H V units influences the materials properties, e.g. melting point and the enthalpy of fusion go through a minimum at 35 mole% H V units. PHAs can be easily extracted and used to fabricate useful materials and objects. P H B and P H B V have been tested for a variety of applications, some of which are listed in Table 1.



207 Table 1 : Commercial application of PHB and PHBV US Patent/Reference BioApplication polyester Baptist, 3,072,538 (1963) P H B and Packaging materials Baptist, 3,107,172 (1963) PHBV (bottles, containers) Webb, A . 4,900,299 (1990) Holmes, P.A. 4,620,999 (1986) Films, laminates Noda, I. 5,536,564 (1996) Personal hygiene articles Noda, I. 5,489,470(1996) Shiotani, T. 5,292,860 (1994) Fibers for nonwoven fabrics Steel, M X . 4,603,070 (1986) Kauffman, T. 5,169,889 (1992) Hot-melt adhesive Seholz, C. (22) Biomedical applications The current draw-back to the use of PHAs on Earth is their cost of production. Even though it is an environmentally benign process, currently it is cheaper to produce thermoplastic materials with similar mechanical properties from petroleum. On a long-term space mission however, there is no access to raw materials from Earth, and in addition, an accumulation of organic waste occurs. These waste products can not be discarded since it is imperative to conserve and transform the energy inherent in the waste. Moreover, any process performed onboard a spacecraft needs to comply with safety regulations and should be as non-intrusive as possible. Since fermentation processes are performed at ambient temperatures and do not require the use of harsh chemicals, they are an ideal approach to the production of the structural and other materials, potentially from waste products.

Simulation of microgravity using the NASA- Bioreactor Data and information that are available today on the bacterial production of biopolyesters were gathered so far on Earth, i.e.lg gravity. To test the bacterial capability of producing biopolyesters on a space station or onboard a traveling spacecraft, that is, in microgravity, it was necessary to mimic some aspects of microgravity in ground-based experiments. The NASA-Bioreactor, see Figure 2, was originally designed for the investigation of unrestricted, threedimensional mammalian cell development and for the protection of mammalian cells from extensive shear forces during take-off and landing of the Space Shuttle. The cylindrical device, 95 mm in diameter and 8 mm in height, is engineered to accommodate the unrestricted development of a three-dimensional cytoarchitecture. Cells and cell aggregates rotate at the same speed as the vessel itself, thus constant randomization of the gravity vector subjects the cells to a continuous state of simulated free fall. The rotation of the bioreactor prevents


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settling of the cells, and at the same time maintains a quiescent environment that can not be accomplished by conventional stirring or experiments in shake flasks. Damaging turbulence is minimized in the bioreactor, and cells do not collide with vessel walls or any other damaging objects, such as stirrer blades. Destructive shear forces are minimized because this system has no impellers, bubbles, or agitators. A l l transport processes are diffusion based. The system is aerated with moistened air, oxygen, or a mixture thereof through membrane guaranteeing bubble free gas supply.

Figure 2: Cell mass accumulating in the N A S A Bioreactor

Experimental Strain information: The bulk of this study was performed using the bacterial strain Azotobacter vinelandii UWD ( A T C C 53799). Stock cultures of A. vinelandii UWD were maintained at a growth medium ( A T C C medium 1771) supplemented with 20 % (v/v) glycerol at -80°C. For polymer production cells were grown under nitrogen limiting conditions. One m L of the stock culture was aseptically inoculated into the minimum medium composed of K H P 0 0.2g/L, K H P 0 0.8g/L, C a S 0 χ 2 H 0 0.1g/L, M g S 0 x 7 H 0 0.2g/L, C H C O O N H l . l g / L , F e S 0 χ 7 H 0 0.005g/L, N a M o 0 χ 2 H 0 0.00025g/L, Ferric Ammonium Citrate 0.06g/L, Yeast Extract 0.5 % wt/vol, Glucose 3% wt/vol. A.vinelandii was grown in the NASA-Bioreactor and for control experiments in conventional shake flask experiments. Typically, 250 m L medium was prepared, medium and glucose were autoclaved separately and combined after they cooled to room temperature. After the medium was inoculated with 1 m L of a rapidly thawed stock culture, 55mL was transferred aseptically into the bioreactor through the fill port. The bioreactor was tilted during the transfer and filled completely to obtain an air bubble-free medium. The remaining culture served as control experiments and was grown in a dented 500 m L Erlenmeyer flask. The bioreactor was placed in an incubator at 30°C 2

2

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and rotated at speeds increasing from 8 to 31 rpm as the cell mass grew. The bioreactor was aerated with hydrated air or hydrated oxygen. It was imperative to moisten the gas prior to introduction to prevent the evaporation of the medium that would lead to the formation of disturbing air-bubbles in the bioreactor. Shake flask experiments were performed in a New Brunswick Shaker Incubator at 200 rpm and 30°C.

Analysis: Bacterial cell growth in the control experiments was monitored conventionally by determining the optical density at λ = 540 nm using a Spectronic 20+ spectrophotometer. Since this assay is not applicable for the N A S A Bioreactor, bacterial cell growth had to be monitored via the dissolved oxygen using an Orion 850 D O meter or by monitoring the glucose consumption. The percentage saturation of oxygen in the medium was read with the probe that was calibrated with distilled water (101.7%). Dissolved oxygen in the bioreactor was measured by transferring the medium into a 100 m L beaker. A new fermentation had to be conducted after each measurement, as the medium was rendered non-sterile. The glucose content in the medium at different time points was determined by the use of a glucose oxidase assay (Trinder, Sigma Diagnostics). Samples of l m L were collected from the shake flask and bioreactor using sterile disposable syringes. Cells were filtered off and the supernatant was used with the assay. A l l assays were performed in duplicate and results recorded using a Spectronic 20+ spectrophotometer. Dry cell mass (biomass) was determined gravimetrically. The cells were harvested by centrifugation (8000 rpm, 20 min), lyophilized and refluxed with methylene chloride or chloroform for 24 hours. After filtering the cells, the solvent was concentrated in a roto-evaporator and the polymer precipitated into cold methanol, dried and weighed. Structure determination was accomplished by *H N M R using a Bruker 400 M H z instrument. Chloroform-d was used as solvent and solutions were adjusted to 0.5 % (w/v). Chemical shifts were recorded in parts per million (ppm) downfield from 0.00 ppm using tetramethylsilane as internal reference. 6

Results and Discussion Poly(P-hydroxybutyrate), a biodegradable polyester has been synthesized for the first time under simulated microgravity conditions by fermentation of the bacterial strains Azotobacter vinelandii UWD and Alcaligenes latus. Bacterial polymer production in the bioreactor differs from that in conventional shake flask experiments in the following aspects:


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> > > >

Altered cell growth behavior N o or negligible lag time Immediate polymer production Restricted polymer formation at extended growth times

The term bioreactor is used from here on to indicate experimental conditions that simulate microgravity, that is, constant state of free fall due to randomization of the gravity vector, allowing unrestricted, three-dimensional cell growth. Other aspects of true microgravity, as for instance exposure to elevated levels of radiation, were not realized. Bacterial growth in the bioreactor differs significantly from conventional shake flask experiments. Cells tended to grow in clusters, several small clusters became first visible after about 3 hours. Over a time period of 21 hours these clusters agglomerated into one cluster that was suspended in the center of the Bioreactor. B y gradually adjusting the rotation speed of the bioreactor from initially 8 rpm to 31.5 rpm after 18 hours, the cell clusters were maintained in the center of the bioreactor, preventing them from colliding with the reactor wall. Figure 2 shows the progress of such a cluster formation.

Figure 3: Comparison of polymer production by A . vinelandii UWD grown on minimum medium in shakeflasksand in the NASA Bioreactor (Reproduced with permission from Polymer Preprints, 2000,41(4), 1064 .)

A l l experiments in the bioreactor were compared to conventional shake flask experiments. Whereas the bacterial growth in the shake flask can be easily monitored by the optical density which is directly depended on the number of cells per volume unit, the monitoring of the cell density in the bioreactor was not possible, due to the cluster-like growth pattern, see Figure 2. Other analysis techniques had to be explored to reliably monitor the cell growth in the


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bioreactor. Two analysis techniques were determined to be suited for the evaluation of bacterial activity: the consumption of glucose and the oxygen profile. The aeration in the bioreactor differs significantly from that in the shake flask. Aeration in shake flasks was accomplished by gas-exchange at the fermentation-broth-air surface area that was generated by the vigorous shaking motion. The bioreactor was aerated by a pump that delivered the moistened gas air bubble-free via a membrane to the reactor. A l l transport processes in the bioreactor were based on diffusion.

Grovtfh curve and polymer production of A vinelandii 2.4

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22 2 - 1.8 - 1.6 - 1.4

"B>

- 1.2 Φ Ε 1 >» -0.8

ο α.

-0.6 -0.4 - 0.2 0 10

15

2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 Time, hours

Figure 4: Growth of A. vinelandii grown on minimum medium and polymer yield Bacterial fermentation in the Bioreactor led to a higher polymer production in the initial phase of the fermentation as compared to that achieved in shakeflasks, see Figure 3. After 24 hrs of fermentation, the polymer yield in the Bioreactor was about threefold of that obtained in shake flask experiments. A t 24 hours 85 mg/L polymer were produced in the Bioreactor , whereas in the same time only 30 mg/L polymer were produced in the shake flasks. After 48 hours of fermentation however, the polymer production in the shake flask clearly superceded that in the bioreactor, 2.2 g/L in the shake flask as compared to 0.24 g/L in the bioreactor. The polymer production in the shake flask is so low in the first 24 hours since the cells are in their lag phase as it is clearly shown in the growth curve of A. vinelandii, see Figure 4. A. vinelandii exhibits a lag phase of about 20 hours with the polymer production starting shortly after the cells went into their exponential growth phase. Exhibiting a lag phase is a


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typical behavior for bacterial cells grown in conventional experiments.

fermentation

In the initial stage of the fermentation, up to 24 hours, polymer production is more efficient in the bioreactor. Nutrients were transformed directly into the storage polymer, without extensive cell proliferation and the respective build-up of biomass, see Figure 5. A s the fermentation proceeds, polymer production in the shake flask is eventually higher than that in the bioreactor. It is believed that the cluster-like growth pattern of the cells in simulated microgravity is the main reason for the eventual decrease in polymer production. This cluster-like growth pattern resulted in reduced mass transfer to and from the cell clusters. As the cells metabolized, they secreted waste products, mainly acetate, which accumulated in the center and immediate vicinity of the clusters. Since all mass transport processes were solely diffusion based, transport of the waste away from the cells and transport of nutrients to the cells was comparatively slow, probably going to the extent that the cells in the center of the clusters were eventually completely depleted of oxygen and nutrients. The build-up of metabolites in conjunction with reduced nutrient supply led to the observed lower polymer incorporation at a later stage of the fermentation in the Bioreactor. Most importantly, the lag phase described above for bacterial fermentation in conventional shake flask experiments was not observed when the cells were grown in simulated microgravity. A s discussed above, the cells started immediately to produce polymer.

Figure 5: Comparison of biomass production by A. vinelandii UWD grown on minimum medium in shake flash and in the NASA Bioreactor (Reproduced with permission from Polymer Preprints, 2000, 41(4), 1064 .)


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In an effort to differentiate between the influence of simulated microgravity and differences in the oxygen supply to the cells, the oxygen profile in the bioreactor was adapted to match that of the shake flask as closely as possible. The established oxygen supply profile is given in Table 2. Since the initial phase of the bacterial growth is the one that exhibited significant differences compared to conventional shake flask experiments, the first 6 hours of the fermentation were monitored closely. Table 2: Oxygen supply profile for the operation of the Bioreactor Oxygen supply [L/rnin] Time Period [hr] 0- 1 1.36 χ HT* 1-2 2.40 χ 10* > 3.4 χ 10-' ' 2-6 Oxygen was supplied in the excess of 3.4 χ 10" L/min, a more accurate determination was not possible in the course of this experiment a

a )

Bacterial activity was monitored by recording the glucose consumption and oxygen profile, since the optical density could not be used to monitor the bacterial growth behavior due to the cluster like growth pattern. These experiments aimed at confirming or dismissing the hypothesis that bacterial growth is influenced by simulated microgravity. Hence, all fermentation parameters were kept identical to those in shake flask experiments. Figure 6 shows the consumption of glucose in the bioreactor and in the shake flask within the first four hours of the fermentation. Glucose was consumed in the bioreactor at a greater rate than in the shake flask experiments, indicating a higher bioactivity, that is, no lag phase was observed. The same tendency was observed by monitoring the dissolved oxygen for the first 6 hours of the fermentation, again oxygen was consumed at a much higher rate in the bioreactor than in the shake flasks, Figure 6. It was rather difficult to match the oxygen profile in the Bioreactor to that of the shake flask since no direct measurement of the dissolved oxygen was possible during a fermentation experiment. Supplying the Bioreactor with air resulted in a continuous decrease in the oxygen level (data not shown). Supplying the Bioreactor with the oxygen supply profile given in Table 2, led to the oxygen balance shown in Figure 6, matching the conditions in the shake flask up to 2 hours. Then, the oxygen level decreased due to the bioactivity of the cells. Independent of the amount of oxygen delivered to the fermentation broth in the Bioreactor, this distinct, steep decrease in oxygen levels was observed within one to three hours of inoculation and could not be counteracted by increasing the oxygen supply. This result indicated that biological activity started in the Bioreactor within one hour of inoculation, whereas in the shake flask experiments constant oxygen level



214 between 85 and 90 % were maintained. Both, oxygen and glucose consumption indicated an elevated bioactivity of the bacterial cells in simulated microgravity. These results confirmed that the microorganisms did not undergo the conventionally observed, extended lag phase, when grown in microgravity. As shown above by the consumption of oxygen and glucose, bacterial cells grown in microgravity showed only a negligible lag phase, bacterial growth and polymer production started immediately after inoculation. In summary, the bacterial growth phenomena observed in the bioreactor can be attributed to the effect that microgravity has on the cells and not to an altered gas balance. Glucose consumption and dissolved oxygen in Shake flask and Bioreactor

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Figure 6: Glucose consumption and Dissolved Oxygen profile in the Bioreactor and in conventional shake flask experiments (SF) In an effort to demonstrate that the effects of microgravity on bacterial polymer production are of general nature, initial experiments were conducted in the bioreactor using another bacterial strain, Alcaligenes latus ( A T C C 29713). The growth and polymer production data confirmed the results obtained with A. vinelandii, that is, shortened lag time and immediate polymer production. The amount of polymer produced was higher than in the respective control shake flask experiments. Further investigations will aim at studying the growth behavior of this strain in simulated microgravity and under the condition of an adjusted oxygen profile.


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Future work Future research will concentrate on quantifying the microbial polymer production in simulated microgravity applying the established oxygen-profile. Initial results indicated that the polymer production in the bioreactor increased significantly when oxygen is supplied to the bioreactor. After three hours of fermentation, 110 mg/L biopolymer were obtained. This represented a threefold increase in polymer production in the bioreactor aerated with oxygen as compared to experiments when the bioreator was aerated with air only. Further detailing is necessary to reconfirm these preliminary data.

Acknowledgement We thank Dr. Richard Ashby from U S D A , Eastern Regional Devision for providing the A. vinelandii UWD bacterial strain. Financial support by N A S A EPSCoR grant NCC5-391 and U A H Minigrant #3-87585 is greatly acknowledged.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

Isoda, H.; Kitamoto, D. Shinmoto, H.; Matsumara, M.; Nakahara, T. Biosci.Biotech.Biochem. 1997, 61(4), 609 Scholz, C. Mehta, S.; Nicolosi, R.; Bisht, K.; Guilmanov. V. Gross, R.A. Polymer Preprints 1998, 39 (2), 168 Gutnick, D.L. Rosenberg, E. United States Patent 4,276,094 Gutnick, D.L. Nestaas, E.; Rosenberg, E.; Sar, N. United States Patent 4,883,757 Shine, J.; Dalgarno, L. Nature 1975, 254, 34 Goeddel, D.V. in: Gene Expression Technology. Methods in Enzymology 185, New York Academic Press, 1990 Klaus, D.; Simske, S.; Todd, P.; Stodieck, L. Microbiology 1997, 143(2) 449 Mattoni, R.H.T. Bioscience 1968, 18, 602 Moatti, N.; Lapchine, L.; Gasset, G.; Richoilley, G.; Templier, J.; Tixador, R. Naturwissenschaften 1986, 73, 413 Mennigmann, H.D.; Lange, M. Naturwissenschaften 1986, 73, 415 Ciferri, O.; Tiboni, O.; DiPasquale, G.; Orlandoni, A.M.; Marchesi, M.L. .; Naturwissenschaften 1986, 73, 418 Mattoni, R.H.; Ebersold, W.T.; Eiserling, F.A.; Keller, E.C.; Romig, W.R.; The experiments of Biosatellite 2 (ed. J. Saunders) N A S A SP 1971, 104, 304 Planel, H. Tixador, R.; Nefedov, I.G.; Gretchko, G.; Richoilley, G. Advances in Space Science 1981, 1, 95



216 14. Mennigmann, H.D.; Heise, M.; Fifth European Symposium on Life Science and Space Research, Arcachon, France, Conference Proceedings 1983, 83 15. Mennigmann, H.D. BioEngineering 1988, 4, 20 16. Mennigmann, H.D.; Lange, M. in: B I O R A C K on Spacelab D1 E S A SP-1091 (ed. N. Longdon, V. David) ESA Publ ESTEC, Noordwijk, 1987, 374 17. Gmünder, F.K.; Cogoli, A. Appl. Microgravity Techn. 1988, 1, 115 18. Doi, Y. Microbial Polyesters VCH, New York 1990 19. Steinbüchel, A. in: Biomaterials: Novel Materials from Biological Sources (ed. D. Byrom) Macmillan Publishers Ltd. London 1991, 123 20. Huisman, G.W.; Wonink, E.; Meima, R.; Terpstra, P.; Witholt, B. J.Biol.Chem. 1991, 266(4), 2191 21. 21 Marois, Z.; Zhang, Z.; Vert, M.;Deng, X.; Lenz, R.W.; Guidon, R. J.Biomater. Sci. Polym. Ed. 1999, 10(4), 483 22. Scholz, C. in: Polymers from Renewable Resources Biopolyesters and Biocatalysis (ed. C.Scholz and R.A. Gross) A C S series 764, 2000, 328


Polymer Research in Microgravity - American Chemical Society

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