Real-time studies on microalgae under microgravity

Acta Astronautica 55 (2004) 131 – 137 www.elsevier.com/locate/actaastro

Real-time studies on microalgae under microgravity G.H. Wanga , G.B. Lia , D.H. Lia , Y.D. Liua;∗ , L.R. Songa , G.H. Tongb , X.M. Liub , E.T. Chengb a Institute

b Shanghai

of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, China Institute of Technical Physics, The Chinese Academy of Sciences, Shanghai 200083, China

Received 3 February 2003; received in revised form 19 December 2003; accepted 10 February 2004

Abstract Using remote sensing technique, we investigated real-time Nostoc sphaeroides K9utz (Cyanobacterium) in Closed System under microgravity by SHENZHOU-2 spacecraft in January 2001. The experiments had 1g centrifuges in space for control and ground control group experiments were also carried out in the same equipments and under the same controlled condition. The data about the population growth of Nostoc sp. of experiments and temperature changes of system were got from spacecraft every minute. From the data, we can @nd that population growth of Nostoc sp. in microgravity group was higher than that of other groups in space or on ground, even though both the control 1g group in space and 1g group on ground indicated same increasing characteristics in experiments. The growth rate of 1:4g group (centrifuged group on ground) was also promoted during experiment. The temperature changes of systems are also aAected by gravity and light. Some aspects about those diAerences were discussed. From the discussion of these results during experiment, it can be found that gravity is the major factor to lead to these changes. c 2004 Elsevier Ltd. All rights reserved.  Keywords: Remote-sensing; Real-time: Nostoc sphaeroides K9utz; Microgravity; Population growth

1. Introduction Controlled ecological life support system (CELSS) is an ideal concept based on the earth biosphere to serve people in long-term space Eight and to settle environmental problems in some extreme habitats on ground. It is necessary to select adapted species of organisms from diAerent types so that the system could be operated in rational, precise, eAective and easy-controlled procedure. In principle, the main

∗ Corresponding author. Tel.: +86-27-87884371; fax: +86-27-87875132. E-mail address: [email protected] (Y.D. Liu).

functions of CELSS are in oxygen, water and food supplying, carbon dioxide removing, as well as making daily life waste reusable [1–3]. A lot of aquatic organisms could be regarded as suitable candidates in the establishment of CELSS. In our experiments, the strain Nostoc sphaeroides K9utz is one of the traditional healthy foods in China, which has the potential to be a component of microalgae bioreactor and the food candidate for CELSS [4,5]. At the same time, the process of oxygen evolution and removing CO2 from Nostoc sp. culture are highly eNcient when the better culture condition was provided. This characteristic may contribute to oxygen providing and CO2 removing of CELSS if it was used. The cells of Nostoc sp. also have

c 2004 Elsevier Ltd. All rights reserved. 0094-5765/$ - see front matter
doi:10.1016/j.actaastro.2004.02.005

G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137

Nomenclature Acronyms ABS CELSS

autonomous biological systems closed ecological life support system PC change of concentration C.E.B.A.S. closed equilibrated biological aquatic system FACHB freshwater algae culture collection of institute of hydrobiology LED lighting emitting diode high ratio of protein, so they are a very important and eNcient source of plant protein when they are used in space [6]. For these reasons, we selected Nostoc sp. as the candidate for the experiments. In order to investigate the rule of change of population growth of Nostoc sp. in the closed system under microgravity, we designed the four groups experiments including microgravity group, 1g group in space (centrifuge group in space), 1g group on ground and 1:4g group on ground (centrifuge group on ground as the control group for the centrifuge group in space) to study it further. 2. Materials and methods The species Nostoc sphaeroides K9utz was provided by FACHB collection (Freshwater Algae Culture Collection of Institute of Hydrobiology, The Chinese Academy of Sciences). The basal medium was BG-110 (@xed nitrogen-free BG-11, Rippka et al., 1979) [7]. Four groups (microgravity group, 1g group in space, 1g group on ground and 1:4g group on ground) of Nostoc sp. cultured with the same algae density (85 ml) in four closed chambers (120 ml) which can transmit light. In January 2001, the experiment was carried out in the second Chinese Spacecraft SHENZHOU-2 and on ground at the same time. Special control system was developed to control four diAerent groups under the same culture condition, such as the same light cycle, light intensity, temperature, rotation speed of centrifuges, etc. The experiment lasted 6 days and 15 h. The remote sensing technique was used to study the population growth of microalgae in real time. The equipment on

Concertration of chlorophyll a of Nostoc sp

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3.5 y = -0.002x + 4.0519 R = 0.9592

3 2.5 2 1.5 1 0.5 0 -0.5 0

500

1000

1500

2000

2500

Transmission Light Intensity (Lux)

Fig. 1. The relationship of transmission light intensity (Lux) with the concentration (
g=ml) of chlorophyll a of Nostoc sp.

the spacecraft can get data of our experiments every minute after the spacecraft get on its orbits. The tungsten lamps provide light (2200 Lux, 250–3000 nm) for microalgae growth in a 12-h-dark/12-h-light cycle. When light penetrates the transparent box, the algae cells can absorb some light, the intensity of transmission light will decrease at a rate according to the density of algal cells. In the other side of the box, a light receptor that is photosensitive was designed to measure the transmission light intensity. The light intensity reveals the density of algae indirectly. From the relationship of the density of algae (measured by the concentration of chlorophyll a in Nostoc sp.) and the values of transmission light intensity (Fig. 1), the densities of algae can be calculated from the data of transmission light intensity got from the spacecraft. 3. Results 3.1. The light intensity after penetration can show the population of algae in experiments The density of Nostoc sp. in experiments varied according with the light intensity after the penetration of the culture chamber (Fig. 1). The liner relationship is y = −0:002x + 4:0519 (y represents the concentration of Chlorophyll a of Nostoc sp. and x represents transmission light intensity), R2 value is 0.9592. This means that the concentration of Chlorophyll a of Nostoc sp. and the transmission light intensity n is linear, so the light intensity can be a good parameter for the biomass of Nostoc sp. Our team also applied this method to the experiment of other algae, the experiments were also successful to present other kinds of algae, though every strain has its speci@c linear relationship.

G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137

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Microgravity Group in Space

1 g Group in Space 1000 Transmission Light Intensity (LUX)

Transmission Light Intensity (LUX)

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800 600 400 200

0

0

-200 -1

(A)

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3 Days

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Fig. 2. The population growth of Nostoc sp. in space (
light oA group in space.

1600 1400 1200 1000 800 600 400 200

4

5

6

7

The population growth of Nostoc sp. under microgravity in space was shown in Fig. 2A. The mean values of transmission light intensity decreased every day. The values changed from 1500 to 700 Lux after 7 days of spaceEight. This means that the density of Nostoc sp. (measured by the concentration of chlorophyll a in Nostoc sp.) in this group shifted from 1.052 to 2:652
g=ml. In other words, the density of Nostoc sp. had increased about 150%. The data mean shows that the algae exhibited had high growing rate during the spaceEight. However, there are some surprising data received from the experiment, such as the data on the sixth day of the spaceEight, and the transmission light intensity changed greatly (from 700 to 1100 Lux). The real microgravity may aAect both the growth rate of algae and the quality of measuring. Fig. 2B showed the population of algae of 1g group

7

1 g Group on Ground

600 400 200

(B)

Fig. 3. The population growth of Nostoc sp. on ground (
light oA (1g) on ground.

3.2. The population growth of Nostoc sp. in space

6

800

0

3 Days

5

1000

-200 2

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1200

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light on): (A) microgravity group in space; (B) centrifuged (1g)

1.4 g Group on Ground

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light on): (A) centrifuged (1:4g) group on ground; (B) control

in space (treated by 1g centrifuge). The mean values of transmission light intensity in this group changed from 920 to 710 Lux. This means that the density of Nostoc sp. altered from 2.212 to 2:632
g=ml, or the density of Nostoc sp. had increased only 18% during the spaceEight. Comparing with the data of these two groups, it can be found that the microgravity group had a higher growth rate (150%) during space Eight than 1g group in space (18%). 3.3. The population growth of Nostoc sp. on ground The 1g and 1:4g group (treated by 1g centrifuge on ground) on ground were used as the control groups for the space experiment groups. From Fig. 3A, it can be found that the mean values of transmission light intensity of 1:4g (treated by 1g centrifuge on ground) changed from 1600 to 960 Lux. These data showed that the density of algae changed from 0.852

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G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137 Microgravity Group in Space

1 g Group in Space

37

36

36 Temperature (C˚)

Temperature (C˚)

35 35 34 33 32 31

34 33 32 31

30 30 29

(A)

-1

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1

2

3 4 Days

5

6

7

Fig. 4. The temperature change of groups in space (
light oA in space.

(B)

-1

0

1

2

3 4 Days

6

7

light on): (A) microgravity group in space; (B) centrifuged (1g) group

1 g group on ground

1.4 g Group on ground

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36 Temperature (C˚)

Temperature (C˚)

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Days

Fig. 5. The temperature change of groups on ground (
light oA on ground.

light on): (A) centrifuged (1:4g) group on ground; (B) control (1g)

to 2:132
g=ml, or the density increased 150% during the experiment. During the experiment, the mean value of transmission light intensity of 1g group decreased from 1400 to 1200 Lux. This data exhibited that the density of algae increased from 1.252 to 1:652
g=ml, in other words, the density increased about 32%. Comparing two groups on ground, it can be found that 1:4g group on ground had a higher growth rate during the experiment than the 1g group on ground.

on, the temperature was about 36:7◦ C, otherwise it was 34:20◦ C. In the 1g group in space, the temperature range was in 35 ± 0:65◦ C (Fig. 4B), which was better controlled than in the microgravity group. When the light was on, the temperature increased to 35:72◦ C, but when the light was oA, the temperature decreased to 34:60◦ C.

3.4. The changes in temperature of the groups in space The equipment for temperature control operated well (Fig. 4). The designed temperature control range was 35±3◦ C, but the data from the spacecraft showed the range was 35 ± 1:7◦ C (Fig. 4), which was better than the designed requirement. When the light was

3.5. The changes in temperature of the groups on ground From Fig. 5A, we can @nd that the temperature range of centrifuge group on ground (1:4g group) is 35 ± 0:7◦ C, but of 1g group on ground is 35 ± 1:3◦ C (Fig. 5B), so the centrifuge groups showed better control than 1g group. When the light was on, the temperatures values of 1:4g group was about 35:70◦ C, otherwise the temperature was about 34:60◦ C. In the

G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137

1g group on ground the temperature was about 36:10◦ C when the light was on, but the temperature changed to 34:2◦ C when the light was oA. 4. Discussion In principle, the designed CELSS can perform supply of oxygen, water and food, of removing of carbon dioxide, as well as making daily life waste reusable. Most CELSSes are based on edible higher land plants because they are able to produce food for humans and regenerate atmosphere and water. However, huge weight, enormousness of the system, plant’s long life cycle and special light requirement limited the application of this system in space exploration. From previous researches we assume that aquatic organisms, such as microalgae, is a highly promising way to resolve these problems [2,8–12] because of their high content of protein and vitamin, shortest generation time (18:9 h), high oxygen production (33 mol=m3 day) and relative easiness to grow and resistance to changes in environmental conditions. Another promising feature of this system is that it can be integrated into “intensive aquaculture” which can produce large amounts of animal protein in a minimal volume. In our experiments, the strain Nostoc sp. is one of traditional healthy foods in China. Our lab has developed some techniques for its large-scale culture. Living beings on earth are adapted to natural 1g gravity and are protected by the atmosphere from space radiation. When they stay in space their metabolism change in order to adapt to the new environment [13–15]. These space environmental factors include microgravity, radiation, hypoxia, heat and cold, etc. Those factors always aAect earth life synchronously. In order to understand the eAect of microgravity on the population growth of microalgae in closed system, our experiments were designed with a centrifuge in space to eliminate the eAect of other space factors on algae. From Fig. 2A we can @nd that microgravity aAected the density of Nostoc sp. in the closed system more deeply than 1g gravity (1g centrifuge) in space, even though some of the eAect resulted from experiment errors. The range of densities of algae in microgravity environment (1:60
g=ml) vary about 4 times than that in 1g gravity (0:42
g=ml). Some

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very large data of transmission light intensity (small data of density of algae) resulted from the clone of algae. From these data, we can assume that some of algae lived in the state of spherical clone, but in the 1g gravity, only a few algae lived in the state of clone in the beginning of the Eight, most of them lived in the state of @lament during the Eight. From the mean values of every day data, we can @nd another interesting phenomenon that the algae in microgravity had a higher growth rate than those in 1g gravity. In our previous studies or other experiments in this Eight [15–18], the algae in microgravity always show lower rate of growth than those in 1g gravity. We assume that some of Nostoc sp. in the experiment excited in a state of sentiments, but in other experiments all algae were buoyant. When Nostoc sp. is in 1g gravity, the gravity will force some degree of sentiment in Nostoc sp., which will lead to nutrition exchange in the medium, and if gas exchange is not easy to occur, then it will result in lower rate of growth of algae. But in a microgravity environment, the algae will be at any random location, and the air and nutrition in the chamber will be mixed with algae much better promoting the rate of growth. In the microgravity environment, Nostoc sp. itself may show lower rate of growth in order to cope with microgravity, but the decreasing step back might be compensated by the increase resulting from the complete mixing of gas and medium with algae in microgravity. Comparing the data of the two 1g control groups, we can @nd that the ranges of change of density of both groups (the change of algae density of 1g group on ground is 0:40
g=ml and that of the 1g group in space is 0:42
g=ml) have no diAerences during the Eight. These results show that other space environment factors during our Eight had little or no eAect on the experiments, so microgravity is the major factor that aAects the experiments. These results also show that 1g group in space is a very good control group for the analysis of the data of microgravity group. Comparing the two centrifuge groups, we can @nd that 1:4g group had a higher rate of growth (PC = 1:28
g=ml) than 1g group in space (PC = 0:40
g=ml). We think that 1:4g gravity may promote the exchange of nutrition in the medium than 1g gravity. For the change of temperature in these groups in experiments, we can @nd that light state and gravity

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G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137

can aAect them deeply. When the light was on, all the data of temperature increased (In microgravity group, the temperature increased to 36:70◦ C; In 1g group in space, the temperature increased to 35:65◦ C; the groups on ground also had the same way of change during experiment.). But when the light was oA, the data decreased (In microgravity group, the temperature decreased to 34:20◦ C; In 1g group in space, the temperature decreased to 34:60◦ C; the groups on ground also had the same way of change during experiment.). The lamps we used in experiments are tungsten lamps, which produce a lot of thermal energy that will contribute to the temperature change during the light change state during experiment. At the same time, the heat produced from this source can also increase the burden of the system in controlling temperature, so it is very necessary to develop a lighting system to provide maximum photosynthetic active radiation with minimum power. Light emitting diode (LED) and microwave lights were thought to be very good alternatives [19,20]. In fact, we had developed some equipment lighted by LED, but we need to improve the system further in the future experiment. Besides, for the data of temperature, we can also @nd that 1:4g group on ground had smaller range of temperature change than 1g group on ground, while 1g group in space had a smaller range of temperature change than microgravity group. Since the same lamps were used in all groups, it can be deduced that gravity is the main factor, except the light state, in aAecting the changes in temperature. Gravity can promote the exchange of medium in chamber, so the heat carried by one part of medium can transfer to other parts easily and diAused into the other part of medium or chamber, and so the range of change will be less than that in weightless or weight-losing environment. In order to realize long-time stable operation of CELSS and better understanding of the mechanism of operation of the system, the measuring of key parameters of pH, O2 , CO2 concentration and some monitoring sensors are also needed [20]. Acknowledgements The research was supported by the Chinese Manned Space Flight Project and The Chinese Academy of Sciences (KSCX-SW-322).

References [1] R.L. Olson, M.W. Oleson, T.J. Slavin, CELSS for advanced manned mission, Horticultural Science 23 (1988) 275–286. [2] Y.D. Liu, X.M. Zhang, H.F. Chen, et al., Study on the structure and function of hydrobiological closed eco-system in space, Chinese Journal of Space Sciences 17 (Suppl.) (1997) 73–77 (in Chinese). [3] A.W. Galston, Photosynthesis as a basis for life support on earth and in space: photosynthesis and transpiration in enclosed spaces, Bioscience 42 (1992) 490–493. [4] Z.B. Huang, Y.D. Liu, B. Paulsen, et al., Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with diAerent colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from @eld colonies and suspension cultures, Journal of Phycology 34 (1998) 962–968. [5] D.H. Li, Y.D. Liu, L.R. Song, Hormogonia mass diAerentiation from Nostoc sphaeroides K9utz. (cyanobacterium) and the comparison of structural characteristics between hormogonia and vegetative @laments, Phycological Research 49 (2001) 81–87. [6] Z.H. Hu, Y.D. Liu, Biological responses of algae to space environment, Chinese Journal of Space Sciences 17 (Suppl.) (1997) 14–23 (in Chinese). [7] R. Rippka, J. Deruelles, J.B. Waterbury, M. Herdman, R.Y. Stanier, Generic assignments, strain histories and properties of pure cultures of cyanobacteria, Journal of General Microbiology 111 (1979) 1–61. [8] V. Bluem, F. Paris, Aquatic modules for bioregerative life support systems based on the C.E.B.A.S. biotechnology, Acta Astronautica 48 (2001) 287–297. [9] V. Bluem, M. Andriske, F. Paris, D. Voeste, The C.E.B.A.S.-Minimodule: behaviour of an arti@cial aquatic ecological system during spaceEight, Advances in Space Research 26 (2000) 253–262. [10] V. Bluem, F. Paris, Novel aquatic modules for bioregenerative life-support systems based on the closed equilibrated biological aquatic system (C.E.B.A.S.), Acta Astronautica 50 (2002) 775–785. [11] Y. Kawasaki, J. Koike, K. Ijiri, et al., Microorganisms and plant of Autonomous Biological Systems (ABS) samples, Biology Science and Space 12 (1998) 373–376. [12] Y. Kubo, F. Takasu, R. Shimura, S. Nagaoka, N. Shigesada, Population dynamics of nitrifying bacteria in an aquatic ecosystem, Biology Science and Space 13 (1999) 333–340. [13] Z.L. Hu, Y.D. Liu, L.F. Dai, et al., Space-reEight strains of Anabaena oryza (Blue-green alga) carried by retrievable satellite, Chinese Science Bulletin 41 (1996) 679–683. [14] Z.L. Hu, Y.D. Liu, Cell responses of Dunaliella salina FACHB 435 (Green alga) to microgravitational stimulation by clinorotation, Chinese Science Bulletin 43 (1998) 1737–1741. [15] Y.D. Liu, H.M. Lin, L.F. Dai, et al., The eAect of retrieved satellite space-Eight on Anabeana oryzae and Chlorella

G.H. Wang et al. / Acta Astronautica 55 (2004) 131 – 137 pyrenoidosa, Chinese Science Bulletin 38 (1993) 177–180 (in Chinese). [16] V.A. Kordyum, E.Y. Shepelev, G.I. Meleshko, et al., Biological studies of Chlorella pyrenoidosa (strain LARG-1) cultures grown under space Eight conditions, Life Sciences and Space Research 18 (1980) 199–204. [17] V.N. Sychev, M.A. Levinskikh, O.G. Livanskaia, Study of the growth and development of Chlorella on “Kosmos-1887”, Kosm Biol Aviakosm Med. 23 (1989) 35–39 (in Russian). [18] A.F. Popova, K.M. Sytnik, Peculiarities of ultrastructure of Chlorella cells growing aboard the Bion-10 during 12 days, Advances in Space Research 17 (1996) 99–102.

137

[19] M.A. Dixon, B. Grodzinski, R. Cote, M. Stasiak, Sealed environment chamber for canopy light interception and hydrocarbon analyses, Advances in Space Research 24 (1999) 277–281. [20] B. Konig, M. Dunne, K. Slenzka, Earth life support for Aquatic organisms, system and technical aspects, Advances in Space Research 27 (2001) 1523–1528.