- Email: [email protected]
Nostoc sphaeroides Kützing, an excellent candidate producer for CELSS
Available online at www.sciencedirect.com
Advances in Space Research 48 (2011) 1565–1571 www.elsevier.com/locate/asr
Nostoc sphaeroides Ku¨tzing, an excellent candidate producer for CELSS Zongjie Hao a,b, Dunhai Li a, Yanhui Li a, Zhicong Wang a,b, Yuan Xiao a, Gaohong Wang a,⇑, Yongding Liu a,⇑, Chunxiang Hu a, Qifang Liu a a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Received 29 October 2010; received in revised form 16 June 2011; accepted 30 June 2011 Available online 13 July 2011
Abstract Some phytoplankton can be regarded as possible candidates in the establishment of Controlled Ecological Life Support System (CELSS) for some intrinsic characteristics, the first characteristic is that they should grow rapidly, secondly, they should be able to endure some stress factors and develop some corresponding adaptive strategies; also it is very important that they could provide food rich in nutritious protein and vitamins for the crew; the last but not the least is they can also fulfill the other main functions of CELSS, including supplying oxygen, removing carbon dioxide and recycling the metabolic waste. According to these characteristics, Nostoc sphaeroides, a potential healthy food in China, was selected as the potential producer in CELSS. It was found that the oxygen average evolution rate of this algae is about 150 lmol O2 mg 1 h 1, and the size of them are ranged from 2 to 20 mm. Also it can be cultured with high population density, which indicated that the potential productivity of Nostoc sphaeroides is higher than other algae in limited volume. We measured the nutrient contents of the cyanobacterium and concluded it was a good food for the crew. Based on above advantages, Nostoc sphaeroides was assumed to a suitable phytoplankton for the establishment of Controlled Ecological Life Support System. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food in future space missions. Crown copyright Ó 2011 Published by Elsevier Ltd. on behalf of COSPAR. All rights reserved. Keywords: CELSS; Producer; Nostoc sphaeroides Ku¨tzing; Food; Space flight
1. Introduction Establishment of Controlled Ecological Life Support System (CELSS) is a very important aspect for the research of space biology, because it serves people in long-term space flight and to the settle of environmental problem in some extreme habitats on the ground. CELSS is designed to resolve problems in space missions about oxygen, water and food supplying, carbon dioxide removing, as well as making daily life waste reusable (Olson et al., 1988; Ai
⇑ Corresponding authors at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P R China. Tel.: +86 27 68780035; fax: +86 27 68780123. E-mail addresses: [email protected] (Z. Hao), [email protected] (D. Li), [email protected] (G. Wang), [email protected] (Y. Liu), qifang_199 @163.com (Q. Liu).
et al., 2007). At the same time, it also deep our knowledge into the origin of life on earth and performance principles of the biosphere. Most CELSS are based on edible higher land plants because they are able to produce food for humans and to regenerate atmosphere and water. These researches related were carried out in USA, Russia, Japan, Europe and China (Kawasaki et al., 1998; Salisbury et al., 2003). But one aspect of CELSS based on higher land plants is that it will produce more or less voluminous amount of inconsumable parts. Another aspect is CELSS based on higher plants needs high weight, which is not economical for the highpriced space launching. At the same time, the system needs bigger space for operation but the space is always limited in spacecraft or space-lab. In addition, higher plant needs light with definite direction, so it will need to use other equipment to change light direction or addition light. The shortest life cycle of edible higher plants is more than
0273-1177/$36.00 Crown copyright Ó 2011 Published by Elsevier Ltd. on behalf of COSPAR. All rights reserved. doi:10.1016/j.asr.2011.06.035
1566
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
60 days, which is too long for the need of the crew, and it is not feasible to be controlled (Oguchi et al., 1987; Liu et al., 1997; Konig et al., 2001). Based on these above reasons, it is necessary to develop efficient system to resolve these issues. From previous researches, we think that aquaculture, such as microalgae bioreactor, is a highly promising way to resolve these problems (Blu¨m et al., 2000; Wang et al., 2008). Most microalgae do not differentiate to that inconsumable part in their life cycle, so almost all biomass is edible if a good strain is selected. Microalgae also have high cell division rates, thus a very high algal biomass could be gained within a short time. Microalgae need relatively small space and simple medium for growth, so they are very suitable for the space culture (Dixon et al., 1999). Microalgae bioreactor is a very efficient system to produce large amounts of protein in short time (Wang et al., 2006). The microalgae need CO2 and light energy for photosynthesis, which can be coupled with oxygen production. The produced oxygen was exactly aquatic animals such as fish, snail, lobster even space crew need (Gonzales and Brown, 2006). At the same time, the respirations of the crew or aquatic animals can provide CO2 for the photosynthesis of microalgae (Galston, 1992). Another promising future of this system is that it can be integrated into “intensive aquaculture” which can produce large amounts of protein in a minimal volume. In our experiment, the strain N. sphaeroides has the potential to be candidate for microalgae bioreactor, it was also named Ge-Xian-Mi which is related to a renowned alchemist, and physician in China called Ge Hong (284–364, AD) (Zhao, 1765). Our lab has developed techniques for the large-scale culture of the strain N. sphaeroides. This species is very easy to culture and harvest since the usual colony size is between 5–20 mm, and is regarded as a potential food candidate par excellence for CELSS (Wang et al., 2004, 2007). Traditionally, Nostoc sp. has been good resources for food. It can be prepared as salad, sweet or salty soups or braised or fried food (Supplementary material) (Gao, 1998; Qiu et al., 2002; Rasmussen et al., 2009) and medicine (Tang et al., 2007; Park et al., 2008) in China for a long time. We also tested Spirulina maxim and Spirulina platensis in mass culture experiment, and compared their biomass production ability and oxygen evolution rate with those of N. sphaeroides, Oguchi found Tilapia worked well with the spirulina sp. in 32 days operation (Oguchi et al., 1987). In this study, we have analyzed the nutrient contents of the algae N. sphaeroides and evaluated whether it is a suitable candidate food and producer for future CELSS. 2. Materials and methods 2.1. Cells culture The microalgae N. sphaeroides, a traditional Chinese freshwater food item, was provided by FACHB collection
(Freshwater Algae Culture Collection of Institute of Hydrobiology, The Chinese Academy of Sciences, Huang et al., 1998). N. sphaeroides is filamentous cyanobacteria, and hormogonium is the main way to the reproduction, and hormogonium differentiation can be induced by chemical pollutants, lipid metabolism and other pathological changes, longer filaments mixed with excreted extracelluar polysaccharide come to spherical macrocolonies (Li, 2000; Li et al., 2001). The juvenile colonies are spherical and form gelatinous masses, the sheath of N. sphaeroides plays an important role in protecting cells from environmental stresses (Garcia, 1994). For the lab bench culture, N. sphaeroides was cultured in aqueous BG110 medium (fixed nitrogen-free BG-11, Rippka et al., 1979), and was totally exchanged for fresh medium every 10 days, temperature was maintained at 25 ± 1 °C, pH was controlled at 7.5 by addition of carbon dioxide. Fluorescent lamps providing cold white light with photosynthetic photon flux intensity (PPFD) about 40 lmol photons m 2 s 1 and bubbled with sterile air. There were clear spherical shapes developed from filamentous state in 15 days, and it usually took 30 days for the colonies develop into U 0.1–0.2 mm, then they were collected by 100 mesh screen filter, and immersed in 75% ethanol for 20 s after rinsed by distilled water, then they were inoculated into the airlift bioreactor, the working volume was 300 L, lamp providing cold white fluorescent light was set in the middle of the bioreactor, other environmental control were similar to the lab bench culture. Colony sizes more than U 7 mm is considered appropriate to harvest as products, other standard includes the hardness, color and shape of the cyanobacterium.
2.2. Biomass and oxygen production assay Biomass production of the cyanobacterium colony cultures was measured with wet or dry weight. Growth rate of N. sphaeroides in exponential phage have been compared between colony size of U 1–2 mm and U 3–4 mm. Photosynthetic oxygen evolution rate of N. sphaeroides filamentous in exponential growth phase and colony organisms at U 2–4 mm were both measured in Clark-type oxygen electrode (HansatechÒ, UK) using the method described by Melis et al. (1998). An aliquot of 2 mL culture was loaded in oxygen electrode chamber. Samples were illuminated with increasing light intensity, which was provided by a quartz-iodine source, and filtered by Neutral Density Filters (HansatechÒ, UK). The rates of oxygen evolution under each of these conditions were recorded continuously for 5 min. The results were plotted to show the light saturation curves of photosynthesis on per chlorophyll a. Culture solutions Chl a was measured by filtering a variable volume of sample (5–10 mL) onto a Whatman GF/F glass fiber filter (25 mm), followed by grinding into filaments, Chl a was then extracted in 95% (v/v) ethanol for
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
1567
dures were as follows: take crushing sample 50–100 mg into hydrolysis tube, add 10 mL 6 mol L 1 HCl, degassing seal, hydrolysis for 22 h under 110 °C temperature, analysis begun after removing the hydrochloride. Elution conditions were 60% of solution A (100% Acetonitrile solution) and 40% of solution B (0.05 M NaAC-H3PO4) over 20 min at a flow rate of 1 mL min 1. Column temperature was maintained at 37 °C and the injection volume was 40 lL. Fatty acid concentrations were determined using gas chromatography (Agilent-6890N, USA) according to the standard of GB/T 17377-2008. Chromatography condition is: column DB-23, length is 30 m, constant pressure is 20.3 psi, temperature (°C): injection was 230 °C, detection was 280 °C, the oven temperature was 180 °C, hold 5 min, 3 °C min 1 increased to 230 °C. Minerals were quantified using an inductively coupled plasma spectrophotometer (Thermo Jarell Ash, Franklin, MA, USA) using methods described by Riche and Brown (1996).
24 h and determined according to Lichtenthaler and Wellburn (1983). The maximum photochemical efficiency of PS II was denoted as yield. It was measured with a Plant Efficiency Analyzer (Hansatech, UK) according to Strasser et al. (1995). All samples were dark-adapted for 15 min prior to measurements. 2.3. Nutrient content assay Collected colony cultures (U 2–5 mm, 60 days after inoculation) of N. sphaeroides were washed in distilled water and then oven dried at 80 °C for 12 h, this would be repeated for at least three times to get constant weight and then stored in desiccators until analyzed. Total dry weight of N. sphaeroides was 100 g used in all the nutrient analysis, triplicate have done in each nutrient analysis treatments, and showed no significant difference. Substance finally acquired is brown and black color, finely ground, plate like with crossed lines. Crude protein, crude fat, polysaccharide and ash were measured according to the approved method of the Chinese Standard Agency for food. Algae protein content was based on the results of crude nitrogen by the method after Kjeldahl (Hiller et al., 1948), 5 g dried samples were used in the protein analysis. Crude fat was estimated by soxhlet extraction (Sahasrabudhe and Smallbone, 1983). Ashes were measured by 2–3 g dried samples, put into crucible, burned in Muffle furnace under 550 °C ± 25 °C for 5 h, the errors were within 0.5 mg were guaranteed after repeated burn. The amino acid compositions of N. sphaeroides were determined by amino acid analyzer (Hitachi-L8800) according to the standard of GB/T5009.124-2003. Proce-
Fig. 1. Colony size of N. sphaeroides shown by a ruler. Colony sizes of U 30 mm would take 100–150 days after hormogonium inoculation.
Dry weight of N. sphaeroides (g mL-1)
0.060
1-2mm group 3-4mm group 0.045
0.030
0.015
0
3
6
9
12
15
18
21
24
Time (days) Fig. 2. Growth curve of N. sphaeroides, compared between colony size at U 1–2 mm and U 3–4 mm. It was cultured in aqueous BG110 medium, the illumination of fluorescent light is at a PPFD of 40 lmol photons m 2 s 1 with 12 h:12 h day/light cycle and maintained in 25 °C throughout the test.
1568
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
3. Results 3.1. Biomass and oxygen production rates of N. sphaeroides Colony size of N. sphaeroides in diameter is 20 mm is shown in Fig. 1. The biomass production rate and oxygen evolution rate are the key parameters of producer for CELSS. It was found that biomass production rate of N. sphaeroides is about 10–15 g (dry weight) m 2 day 1 (300 L) in mass culture. Growth rate of N. sphaeroides have been compared between colony size of U 1–2 mm and U 3– 4 mm, the former one is 0.0014 g ml 1 day 1, the latter one is 0.0005 g ml 1 day 1 in exponential phase. According to Fig. 2, U 1–2 mm growth rate is 4 times of that U 3– 4 mm colony size. It was found that with the increasing cyanobacterium colony size, the growth rate is declining, so it is inferred that the optimal biomass production occurs when in smaller size. Table 1 shows the oxygen evolution rate is about 150 lmol O2 mg 1 Chl a h 1, which means that 300 L cyanobacteria culture can produce 5000 L oxygen per day, while Sprulira sp. oxygen production is 110–140 lmol O2 mg 1 Chl a h 1 under 40 lmol photons m 2 s 1.
3.2. Nutrient value of N. sphaeroides The proximate nutrient composition of N. sphaeroides is shown Table 2, which indicated the protein content accounted for 30.45% of biomass. Amino acid concentrations are shown in Table 3, which indicated that there are high total amino acids in our indoor cultured sample. The highest concentration amino acid was Asp, then Glu and Thr. These results are consisted to the protein contents in the sample. The mineral concentration of N. sphaeroides are indicated in Table 4, which showed that there were high contents of Selenium, Calcium and Iron in indoor cultured sample while there are not determined Potassium in the sample. Fatty acid concentrations are shown in Table 5, which indicated that the highest concentration fatty acid was Palmitic acid, then Para Amino Benzoic Acid and Linoleic acid. There were also some unsaturated fatty acids in
Table 1 Primary production of N. sphaeroides, cultured in aqueous BG110 medium, the illumination of fluorescent light is at a PPFD of 40 lmol photons m 2 s 1 with 12 h:12 h day/light cycle and maintained in 25 °C throughout the test. The colony size is U 2–5 mm, and the hormogonium is 8 days after inoculation.
Oxygen evolution rate (lmol O2 mg 1 Chl a h 1) Respiration rate (lmol O2 mg 1 Chl a h 1) Yield Biomass production max (g m
2
day 1)
Colony
Hormogonium
125–160
158.2 ± 2.3
4.6 ± 1.0
30.5 ± 1.7
0.538 ± 0.002 250 ± 10.5
0.436 ± 0.005 10–15 (300 L)
Table 2 Main nutrient composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. N. sphaeroides Protein (%) Crude fat (%) Ash Polysaccharide (%) Carbohydrate
30.45 3.1 6.8 59 65.8
Table 3 Amino acid composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Amino acid (lg g 1) Asp Ser Gly Cys Met Leu Phe His Pro Total
3.48 1.34 1.76 0.15 0.13 1.78 1.43 0.21 0.77 23.43
Amino acid (lg g 1) Thr Glu Ala Val Ile Tyr Lys Arg
2.33 2.77 1.87 1.65 1.29 0.28 0.83 1.36
Table 4 Mineral composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Mineral (mg kg 1)
N. sphaeroides
Calcium Iron Potassium Magnesium Zinc Selenium
13.14 1.37 0 0.56 0.34 5.76
the sample, which indicated the sample is healthy as food of the crew. 4. Discussion In principle, the designed CELSS can perform supplying of oxygen, water and food, removing of carbon dioxide, as well as making daily life waste reusable. Most CELSS 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
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571 Table 5 Fatty acid composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Fatty acid (mg kg 1) Dodecanoic acid Palmitic acid Heptadecoic acid Stearic acid Linoleic acid Arachidonic acid
Fatty acid (mg kg 1)
1.2
Myristic acid
9.4
841.2
Para Amino Benzoic Acid Heptadecenoic acid
489.8
11.7 13.4 395.8 3.3
Oleic acid Linolenic Acid Eicosapentaenoic Acid
7.0 253.1 163.2 9
system, plant’s long life cycle and special light requirement limited the application of higher plant bioreactor in space exploration. From previous researches we assume that aquatic organisms, such as phytoplankton, is a highly promising way to resolve these problems because of their high contents of protein and vitamin, shortest generation time, high oxygen production and relative easiness to grow and resistance to changes in environmental conditions, lower production costs. Another promising feature of this system is that it can be integrated into “intensive aquaculture” which can produce large amounts of protein in a minimal volume. In our experiments, the strain N. sphaeroides is one of traditionally healthy foods in China and our lab has developed some techniques for its large-scale culture, with which many kinds of delicious dishes were also developed in restaurant. In this research, it was found that the polysaccharide content was 59% in our sample which is easily digested for human being. Protein content needed by humans is between 23% and 25%, it accounted 30.45% of N. sphaeroides biomass which is closer to human needs, while Sprulira sp. is more than 50%. In addition, there are below 10% ash and fat which have low waste ratio than many plants food. The results of mineral concentration of N. sphaeroides were also showed that there were high content of Selenium, Calcium and Iron, which are essential elements for human body growth. Reported vitamin concentration results also indicated that there were high concentrations of total Vitamin B1, B2, C and E in the sample (Liu, 2000), which is helpful to life normal metabolism. In order to determine the quality of the amino acid and fatty acid in the sample, we carried out experiment to analyze the content of these components in more detail, which showed that amino acid and fatty acid of the sample were of high quality and the ratio to different component is close to the recommended level for human. These results showed that the culture sample of N. sphaeroides is excellent candidate food. The processed food, take salad and soup for example, the nutrient level did not change, as to the fried cyanobac-
1569
terium, if it cooked appropriately, there was no nutrient loss or even more nutritional mixed with other material, including vitamins and protein. Surly it can be used as the sole food considering of its nutrient level, and it is easily digested by humans because of its thin cell wall and rich in polysaccharide. However, we propose it as a supplementary food in the CELSS because of its taste and human unique diet psychology. It can be eaten with rice, corn and wheat. Plus, N. sphaeroides mixed with animal protein in fried and soup diets can serve as a complete and well-balanced source of amino acids to meet human requirements. For producer in CELSS, the most important characteristic is high productivity rate. The results of our experiment showed that under the same PPFD about 40 lmol photons m 2 s 1, the biomass production rate of N. sphaeroides is about 10–15 g (dry weight) m 2 day 1(300 L) in mass culture, which is higher than that of Sprulira sp. (7– 8 g (dry weight) m 2 day 1 (300 L)). Another important characteristic of producer is high oxygen evolution rate. In our experiment, it was found that the oxygen evolution rate of N. sphaeroides is about 150 lmol O2 mg 1 Chl a h 1, which means that 30 L culture can produce 500 L oxygen per day to provide for an adult (about 440– 860 L person 1 day 1) respiration. Sprulira sp. oxygen evolution rate is between 110 and 140 lmol O2 mg 1 Chl a h 1 with the PPFD of 40 lmol photons m 2 s 1 (Vonshak et al., 1996; Torzillo et al., 1998; Jimenez et al., 2003). Compared with Spirulina sp. production ability and oxygen evolution rate, N. sphaeroides can support more human consumption. Spirulina sp. has an optimal growth temperature of around 35 °C and no net growth occurs below 18 °C, which is a high cost in the biomass production. N. sphaeroides colony size at U 2–5 mm is considered suitable for the CELSS construction. Firstly, when in smaller colony size, the growth rate of cyanobacterium is higher. Secondly, the optimal oxygen production rate is in filamentous state, it can produce oxygen without a polysaccharide sheath. However, the colony did not show significantly lower oxygen evolution rate. The third consideration is optimal nutrient level for humans is bigger colony size, because with increasing colony sizes, carbohydrate is increasing. Human diet usually need relatively higher contents of carbohydrate, and it also showed U 2– 5 mm size is closer to that 50% content (Deng, 2006; Ma, 2010). In order to be used in future CELSS, appropriate bioreactor with the cyanobacterium should be developed. Usually, the colony size of the N. sphaeroides is about U 2– 30 mm, it is easy to be harvested and processed for food, but it also needs different bioreactors for culturing. We have designed new bioreactor on ground application (unpublished data) with high velocity stir and air exchange rate since colony growth affect air and nutrient exchange rate in reactor, and shear force has a less effluence on colony than on cell culture. Another problem is the lamps we used in experiments are fluorescent lamps, which will produce a lot of thermal energy and be a big burden for space
1570
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
experiment energy supply. Light emitting diode (LED) and microwave lights were thought to be very good alternatives. Besides, air exchange in lipid culture under space microgravity environment is another problem for bioreactor; one membrane technique was developed to remove the dissolved oxygen immediately, which can separate gas from liquid in weightlessness condition easily (Ai et al., 2007). Considering the mass culture collection, filtration and low speed centrifugation (50–100 r min 1) which needs relatively lower energy consumption are enough for N. sphaeroides colonies to be harvested (Olaizola, 2003). In fact, we had developed one model system after considering these problems, but we need to improve the reactor system further for the future space experiment. 5. Conclusion We have isolated one strain of cyanobacteria, N. sphaeroides, which is one of traditional healthy food in China. The possibility of using it as candidate food and producer for future CELSS was evaluated. The productivity rate and the oxygen evolution rate were high, which indicated the cyanobacterium have advantages in biomass production and oxygen regeneration, and it can grow into colony with size between U 2 and 30 mm which is easy to be harvested and prepared for food process. The results for nutrient content of the cyanobacterium also showed it was a high quality and suitable food for the crew. The cyanobacterium can be suitable to be cultured with high density, and we plan to develop appropriate bioreactor with the cyanobacterium for supplying oxygen and food to the crew in future. Acknowledgements The work was supported by an Academy-Locality cooperation project from Chinese Academy of Sciences (No ZNWH-2011-004), the Project of Chinese Manned Spaceflight, Natural Science Foundation of China (No 30970688), and Natural Science Foundation of China (No 31000061). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.asr.2011. 06.035. References Ai, W.D., Guo, S.S., Qin, L.F., et al. Development of a ground based microalgae photobioreactor experimental facility for using in space. Space Medicine & Medical Engineering 20 (3), 165–169, 2007. Blu¨m, V., Andriske, M., Paris, F., et al. The C.E.B.A.S.-minimodule: behavior of an artificial aquatic ecological system during spaceflight. Advances in Space Research 26, 253–262, 2000. Deng, Z.Y. Studies on mass culture of Nostoc sphaeroides and N. commune (Cyanophyta) and the structural and physiological characteristics of N.
sphaeroides. Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2006. Dixon, M.A., Grodzinski, B., Cote, R., et al. Sealed environment chamber for canopy light interception and hydrocarbon analyses. Advances in Space Research 24, 277–281, 1999. Galston, A.W. Photosynthesis as a basis for life support on earth and in space: photosynthesis and transpiration in enclosed spaces. Bioscience 42, 490–493, 1992. Gao, K.S. Chinese studies on the edible blue-green algae, Nostoc flagelliforme: a review. Journal of Applied Phycology 10, 37–49, 1998. Garcia, P.F. A model for internal self-shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreens. Limnology and Oceanography 39, 1704–1717, 1994. Gonzales, J., Brown, P. Nile tilapia Oreochromis niloticus as a food source in advanced life support systems: initial considerations. Advances in Space Research 38, 1132–1137, 2006. Hiller, A., Plazin, J., Van Slyke, D.D. A study of conditions for KJELDAHL determination of nitrogen in proteins-description of methods with mercury as catalyst, and titrimetric and gasometrical measurements of the ammonia formed. Journal of Biological Chemistry 176 (3), 1401–1420, 1948. Huang, Z.B., Liu, Y.D., Paulsen, B., et al. Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures. Journal of Phycology 34, 962–968, 1998. Jimenez, C., Cossio, B.R., Niell, F.X. Relationship between physicochemical variables and productivity in open ponds for the production of Spirulina: a predictive model of algal yield. Aquaculture 221 (1–4), 331–345, 2003. Kawasaki, Y., Koike, J., Ijiri, K., et al. Microorganisms and plant of Autonomous Biological Systems (ABS) samples. Biological Sciences in Space 12, 373–376, 1998. Konig, B., Dunne, M., Slenzka, K. Earth life support for aquatic organisms, system and technical aspects. Advances in Space Research 27, 1523–1528, 2001. Li, D.H. Studies on morphogenesis, development and physiology of N. sphaeroides Ku¨tzing (cyanobacterium). Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2000. Li, D.H., Liu, Y.D., Song, L.R. Hormogonia mass differentiation from N. sphaeroides Ku¨tz. (cyanobacterium) and the comparison of structural characteristics between hormogonia and vegetative filaments. Phycological Research 49, 81–87, 2001. Lichtenthaler, H.K., Wellburn, A.R. Determinations of total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochemical Society Transactions (London) 63, 591–592, 1983. Liu, J.L. Study on nutrient composition of Nostoc sphaeroides Ku¨tz. Chinese Traditional and Herbal Drugs 31 (17), 862–864 (In Chinese), 2000. Liu, Y.D., Zhang, Y.M., Chen, H.F., et al. Study on the structure and function of a hydrobiological closed ecosystem in space. Chinese Journal of Space Science 17 (Suppl.), 73–77, 1997. Ma, R. Studies on the industrial culture of Nostoc sphaeroides and Nostoc Commune. Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2010. Melis, A., Neidhardt, J., Benemann, J.R. Dunaliella salina (Chlorophyta) with small antenna sizes exhibit higher photosynthetic productivities and photon efficiencies than normally pigmented cells. Journal of Applied Phycology 10, 515–525, 1998. Oguchi, M., Otsubo, K., Nitta, K., et al. Food production and gas exchange system using blue-green alga (Spirulina) for CELSS. Advances in Space Research 7 (4), 7–10, 1987. Olaizola, M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomolecular Engineering 20 (4-6), 459–466, 2003. Olson, R.L., Oleson, M.W., Slavin, T.J. CELSS for advanced manned mission. HortScience 23, 275–286, 1988.
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571 Park, Y.K., Rasmussen, H.E., Ehlers, S.J., et al. Repression of proinflammatory gene expression by lipid extract of Nostoc commune var sphaeroides Ku¨tzing, a blue-green alga, via inhibition of nuclear factorkappa B in RAW 264.7 macrophages. Nutrition Research 28 (2), 83– 91, 2008. Qiu, B.S., Liu, J.Y., Liu, Z.L., et al. Distribution and ecology of the edible cyanobacterium Ge-Xian-Mi (Nostoc) in rice fields of Hefeng County in China. Journal of Applied Phycology 14, 423–429, 2002. Rasmussen, H.E., Blobaum, K.R., Jesch, E.D., et al. Hypocholesterolemic effect of Nostoc commune var. sphaeroides Ku¨tzing, an edible bluegreen alga. European Journal of Nutrition 48 (7), 387–394, 2009. Riche, M., Brown, P.B. Availability of phosphorus from feedstuffs fed to rainbow trout. Oncorhynchus mykiss. Aquaculture 142, 269–282, 1996. Rippka, R., Deruelles, J., Waterbury, J.B., et al. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111, 1–61, 1979. Sahasrabudhe, M.R., Smallbone, B.W. Comparative-evaluation of solvent-extraction methods for the determination of neutral and polar lipids in beef. Journal of the American Oil Chemisty’s Society 60 (4), 801–805, 1983. Salisbury, F.B., Campbell, W.F., Carman, J.G., et al. Plant growth during the greenhouse II experiment on the Mir orbital station. Advances in Space Research 31 (1), 221–227, 2003. Strasser, R.J., Srivastava, A., Govindjee Polyphasic chlorophyll a fluorescence transients in plants and cyanobacteria. Photochemistry and Photobiology 61, 32–42, 1995.
1571
Tang, J., Hu, Z.Y., Chen, X.W. Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from N. sphaeroides. American Journal of Chinese Medicine 35 (5), 887–896, 2007. Torzillo, G., Bernardini, P., Masojidek, J. On-line monitoring of chlorophyll fluorescence to assess the extent of photoinhibition of photosynthesis induced by high oxygen concentration and low temperature and its effect on the productivity of outdoor cultures of Spirulina platensis (Cyanobacteria). Journal of Phycology 34 (3), 504– 510, 1998. Vonshak, A., Torzillo, G., Accolla, P., et al. Light and oxygen stress in Spirulina platensis (cyanobacteria) grown outdoors in tubular reactors. Physiologia Plantarum 97 (1), 175–179, 1996. Wang, G.H., Chen, H.F., Li, G.B., et al. Population growth and physiological characteristics of microalgae in a miniaturized bioreactor during space flight. Acta Astronautica 58, 264–269, 2006. Wang, G.H., Hu, C.X., Li, D.H., et al. The response of antioxidant systems in N. sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants. Advances in Space Research 39 (6), 1034–1042, 2007. Wang, G.H., Li, G.B., Li, D.H., et al. Real-time studies on microalgae under microgravity. Acta Astronautica 56 (2), 131–137, 2004. Wang, G.H., Liu, Y.D., Li, G.B., et al. A simple closed aquatic ecosystem (CAES) for space. Advances in Space Research 41 (5), 684–690, 2008. Zhao, X.M. The Supplement to Compendium of Materia Medica. Reprinted in 1983 by People’s medical Publishing House. People’s medical Publishing House, Beijing, People’s Republic of China, 1765.
Advances in Space Research 48 (2011) 1565–1571 www.elsevier.com/locate/asr
Nostoc sphaeroides Ku¨tzing, an excellent candidate producer for CELSS Zongjie Hao a,b, Dunhai Li a, Yanhui Li a, Zhicong Wang a,b, Yuan Xiao a, Gaohong Wang a,⇑, Yongding Liu a,⇑, Chunxiang Hu a, Qifang Liu a a
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Received 29 October 2010; received in revised form 16 June 2011; accepted 30 June 2011 Available online 13 July 2011
Abstract Some phytoplankton can be regarded as possible candidates in the establishment of Controlled Ecological Life Support System (CELSS) for some intrinsic characteristics, the first characteristic is that they should grow rapidly, secondly, they should be able to endure some stress factors and develop some corresponding adaptive strategies; also it is very important that they could provide food rich in nutritious protein and vitamins for the crew; the last but not the least is they can also fulfill the other main functions of CELSS, including supplying oxygen, removing carbon dioxide and recycling the metabolic waste. According to these characteristics, Nostoc sphaeroides, a potential healthy food in China, was selected as the potential producer in CELSS. It was found that the oxygen average evolution rate of this algae is about 150 lmol O2 mg 1 h 1, and the size of them are ranged from 2 to 20 mm. Also it can be cultured with high population density, which indicated that the potential productivity of Nostoc sphaeroides is higher than other algae in limited volume. We measured the nutrient contents of the cyanobacterium and concluded it was a good food for the crew. Based on above advantages, Nostoc sphaeroides was assumed to a suitable phytoplankton for the establishment of Controlled Ecological Life Support System. We plan to develop suitable bioreactor with the cyanobacterium for supplying oxygen and food in future space missions. Crown copyright Ó 2011 Published by Elsevier Ltd. on behalf of COSPAR. All rights reserved. Keywords: CELSS; Producer; Nostoc sphaeroides Ku¨tzing; Food; Space flight
1. Introduction Establishment of Controlled Ecological Life Support System (CELSS) is a very important aspect for the research of space biology, because it serves people in long-term space flight and to the settle of environmental problem in some extreme habitats on the ground. CELSS is designed to resolve problems in space missions about oxygen, water and food supplying, carbon dioxide removing, as well as making daily life waste reusable (Olson et al., 1988; Ai
⇑ Corresponding authors at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P R China. Tel.: +86 27 68780035; fax: +86 27 68780123. E-mail addresses: [email protected] (Z. Hao), [email protected] (D. Li), [email protected] (G. Wang), [email protected] (Y. Liu), qifang_199 @163.com (Q. Liu).
et al., 2007). At the same time, it also deep our knowledge into the origin of life on earth and performance principles of the biosphere. Most CELSS are based on edible higher land plants because they are able to produce food for humans and to regenerate atmosphere and water. These researches related were carried out in USA, Russia, Japan, Europe and China (Kawasaki et al., 1998; Salisbury et al., 2003). But one aspect of CELSS based on higher land plants is that it will produce more or less voluminous amount of inconsumable parts. Another aspect is CELSS based on higher plants needs high weight, which is not economical for the highpriced space launching. At the same time, the system needs bigger space for operation but the space is always limited in spacecraft or space-lab. In addition, higher plant needs light with definite direction, so it will need to use other equipment to change light direction or addition light. The shortest life cycle of edible higher plants is more than
0273-1177/$36.00 Crown copyright Ó 2011 Published by Elsevier Ltd. on behalf of COSPAR. All rights reserved. doi:10.1016/j.asr.2011.06.035
1566
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
60 days, which is too long for the need of the crew, and it is not feasible to be controlled (Oguchi et al., 1987; Liu et al., 1997; Konig et al., 2001). Based on these above reasons, it is necessary to develop efficient system to resolve these issues. From previous researches, we think that aquaculture, such as microalgae bioreactor, is a highly promising way to resolve these problems (Blu¨m et al., 2000; Wang et al., 2008). Most microalgae do not differentiate to that inconsumable part in their life cycle, so almost all biomass is edible if a good strain is selected. Microalgae also have high cell division rates, thus a very high algal biomass could be gained within a short time. Microalgae need relatively small space and simple medium for growth, so they are very suitable for the space culture (Dixon et al., 1999). Microalgae bioreactor is a very efficient system to produce large amounts of protein in short time (Wang et al., 2006). The microalgae need CO2 and light energy for photosynthesis, which can be coupled with oxygen production. The produced oxygen was exactly aquatic animals such as fish, snail, lobster even space crew need (Gonzales and Brown, 2006). At the same time, the respirations of the crew or aquatic animals can provide CO2 for the photosynthesis of microalgae (Galston, 1992). Another promising future of this system is that it can be integrated into “intensive aquaculture” which can produce large amounts of protein in a minimal volume. In our experiment, the strain N. sphaeroides has the potential to be candidate for microalgae bioreactor, it was also named Ge-Xian-Mi which is related to a renowned alchemist, and physician in China called Ge Hong (284–364, AD) (Zhao, 1765). Our lab has developed techniques for the large-scale culture of the strain N. sphaeroides. This species is very easy to culture and harvest since the usual colony size is between 5–20 mm, and is regarded as a potential food candidate par excellence for CELSS (Wang et al., 2004, 2007). Traditionally, Nostoc sp. has been good resources for food. It can be prepared as salad, sweet or salty soups or braised or fried food (Supplementary material) (Gao, 1998; Qiu et al., 2002; Rasmussen et al., 2009) and medicine (Tang et al., 2007; Park et al., 2008) in China for a long time. We also tested Spirulina maxim and Spirulina platensis in mass culture experiment, and compared their biomass production ability and oxygen evolution rate with those of N. sphaeroides, Oguchi found Tilapia worked well with the spirulina sp. in 32 days operation (Oguchi et al., 1987). In this study, we have analyzed the nutrient contents of the algae N. sphaeroides and evaluated whether it is a suitable candidate food and producer for future CELSS. 2. Materials and methods 2.1. Cells culture The microalgae N. sphaeroides, a traditional Chinese freshwater food item, was provided by FACHB collection
(Freshwater Algae Culture Collection of Institute of Hydrobiology, The Chinese Academy of Sciences, Huang et al., 1998). N. sphaeroides is filamentous cyanobacteria, and hormogonium is the main way to the reproduction, and hormogonium differentiation can be induced by chemical pollutants, lipid metabolism and other pathological changes, longer filaments mixed with excreted extracelluar polysaccharide come to spherical macrocolonies (Li, 2000; Li et al., 2001). The juvenile colonies are spherical and form gelatinous masses, the sheath of N. sphaeroides plays an important role in protecting cells from environmental stresses (Garcia, 1994). For the lab bench culture, N. sphaeroides was cultured in aqueous BG110 medium (fixed nitrogen-free BG-11, Rippka et al., 1979), and was totally exchanged for fresh medium every 10 days, temperature was maintained at 25 ± 1 °C, pH was controlled at 7.5 by addition of carbon dioxide. Fluorescent lamps providing cold white light with photosynthetic photon flux intensity (PPFD) about 40 lmol photons m 2 s 1 and bubbled with sterile air. There were clear spherical shapes developed from filamentous state in 15 days, and it usually took 30 days for the colonies develop into U 0.1–0.2 mm, then they were collected by 100 mesh screen filter, and immersed in 75% ethanol for 20 s after rinsed by distilled water, then they were inoculated into the airlift bioreactor, the working volume was 300 L, lamp providing cold white fluorescent light was set in the middle of the bioreactor, other environmental control were similar to the lab bench culture. Colony sizes more than U 7 mm is considered appropriate to harvest as products, other standard includes the hardness, color and shape of the cyanobacterium.
2.2. Biomass and oxygen production assay Biomass production of the cyanobacterium colony cultures was measured with wet or dry weight. Growth rate of N. sphaeroides in exponential phage have been compared between colony size of U 1–2 mm and U 3–4 mm. Photosynthetic oxygen evolution rate of N. sphaeroides filamentous in exponential growth phase and colony organisms at U 2–4 mm were both measured in Clark-type oxygen electrode (HansatechÒ, UK) using the method described by Melis et al. (1998). An aliquot of 2 mL culture was loaded in oxygen electrode chamber. Samples were illuminated with increasing light intensity, which was provided by a quartz-iodine source, and filtered by Neutral Density Filters (HansatechÒ, UK). The rates of oxygen evolution under each of these conditions were recorded continuously for 5 min. The results were plotted to show the light saturation curves of photosynthesis on per chlorophyll a. Culture solutions Chl a was measured by filtering a variable volume of sample (5–10 mL) onto a Whatman GF/F glass fiber filter (25 mm), followed by grinding into filaments, Chl a was then extracted in 95% (v/v) ethanol for
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
1567
dures were as follows: take crushing sample 50–100 mg into hydrolysis tube, add 10 mL 6 mol L 1 HCl, degassing seal, hydrolysis for 22 h under 110 °C temperature, analysis begun after removing the hydrochloride. Elution conditions were 60% of solution A (100% Acetonitrile solution) and 40% of solution B (0.05 M NaAC-H3PO4) over 20 min at a flow rate of 1 mL min 1. Column temperature was maintained at 37 °C and the injection volume was 40 lL. Fatty acid concentrations were determined using gas chromatography (Agilent-6890N, USA) according to the standard of GB/T 17377-2008. Chromatography condition is: column DB-23, length is 30 m, constant pressure is 20.3 psi, temperature (°C): injection was 230 °C, detection was 280 °C, the oven temperature was 180 °C, hold 5 min, 3 °C min 1 increased to 230 °C. Minerals were quantified using an inductively coupled plasma spectrophotometer (Thermo Jarell Ash, Franklin, MA, USA) using methods described by Riche and Brown (1996).
24 h and determined according to Lichtenthaler and Wellburn (1983). The maximum photochemical efficiency of PS II was denoted as yield. It was measured with a Plant Efficiency Analyzer (Hansatech, UK) according to Strasser et al. (1995). All samples were dark-adapted for 15 min prior to measurements. 2.3. Nutrient content assay Collected colony cultures (U 2–5 mm, 60 days after inoculation) of N. sphaeroides were washed in distilled water and then oven dried at 80 °C for 12 h, this would be repeated for at least three times to get constant weight and then stored in desiccators until analyzed. Total dry weight of N. sphaeroides was 100 g used in all the nutrient analysis, triplicate have done in each nutrient analysis treatments, and showed no significant difference. Substance finally acquired is brown and black color, finely ground, plate like with crossed lines. Crude protein, crude fat, polysaccharide and ash were measured according to the approved method of the Chinese Standard Agency for food. Algae protein content was based on the results of crude nitrogen by the method after Kjeldahl (Hiller et al., 1948), 5 g dried samples were used in the protein analysis. Crude fat was estimated by soxhlet extraction (Sahasrabudhe and Smallbone, 1983). Ashes were measured by 2–3 g dried samples, put into crucible, burned in Muffle furnace under 550 °C ± 25 °C for 5 h, the errors were within 0.5 mg were guaranteed after repeated burn. The amino acid compositions of N. sphaeroides were determined by amino acid analyzer (Hitachi-L8800) according to the standard of GB/T5009.124-2003. Proce-
Fig. 1. Colony size of N. sphaeroides shown by a ruler. Colony sizes of U 30 mm would take 100–150 days after hormogonium inoculation.
Dry weight of N. sphaeroides (g mL-1)
0.060
1-2mm group 3-4mm group 0.045
0.030
0.015
0
3
6
9
12
15
18
21
24
Time (days) Fig. 2. Growth curve of N. sphaeroides, compared between colony size at U 1–2 mm and U 3–4 mm. It was cultured in aqueous BG110 medium, the illumination of fluorescent light is at a PPFD of 40 lmol photons m 2 s 1 with 12 h:12 h day/light cycle and maintained in 25 °C throughout the test.
1568
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
3. Results 3.1. Biomass and oxygen production rates of N. sphaeroides Colony size of N. sphaeroides in diameter is 20 mm is shown in Fig. 1. The biomass production rate and oxygen evolution rate are the key parameters of producer for CELSS. It was found that biomass production rate of N. sphaeroides is about 10–15 g (dry weight) m 2 day 1 (300 L) in mass culture. Growth rate of N. sphaeroides have been compared between colony size of U 1–2 mm and U 3– 4 mm, the former one is 0.0014 g ml 1 day 1, the latter one is 0.0005 g ml 1 day 1 in exponential phase. According to Fig. 2, U 1–2 mm growth rate is 4 times of that U 3– 4 mm colony size. It was found that with the increasing cyanobacterium colony size, the growth rate is declining, so it is inferred that the optimal biomass production occurs when in smaller size. Table 1 shows the oxygen evolution rate is about 150 lmol O2 mg 1 Chl a h 1, which means that 300 L cyanobacteria culture can produce 5000 L oxygen per day, while Sprulira sp. oxygen production is 110–140 lmol O2 mg 1 Chl a h 1 under 40 lmol photons m 2 s 1.
3.2. Nutrient value of N. sphaeroides The proximate nutrient composition of N. sphaeroides is shown Table 2, which indicated the protein content accounted for 30.45% of biomass. Amino acid concentrations are shown in Table 3, which indicated that there are high total amino acids in our indoor cultured sample. The highest concentration amino acid was Asp, then Glu and Thr. These results are consisted to the protein contents in the sample. The mineral concentration of N. sphaeroides are indicated in Table 4, which showed that there were high contents of Selenium, Calcium and Iron in indoor cultured sample while there are not determined Potassium in the sample. Fatty acid concentrations are shown in Table 5, which indicated that the highest concentration fatty acid was Palmitic acid, then Para Amino Benzoic Acid and Linoleic acid. There were also some unsaturated fatty acids in
Table 1 Primary production of N. sphaeroides, cultured in aqueous BG110 medium, the illumination of fluorescent light is at a PPFD of 40 lmol photons m 2 s 1 with 12 h:12 h day/light cycle and maintained in 25 °C throughout the test. The colony size is U 2–5 mm, and the hormogonium is 8 days after inoculation.
Oxygen evolution rate (lmol O2 mg 1 Chl a h 1) Respiration rate (lmol O2 mg 1 Chl a h 1) Yield Biomass production max (g m
2
day 1)
Colony
Hormogonium
125–160
158.2 ± 2.3
4.6 ± 1.0
30.5 ± 1.7
0.538 ± 0.002 250 ± 10.5
0.436 ± 0.005 10–15 (300 L)
Table 2 Main nutrient composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. N. sphaeroides Protein (%) Crude fat (%) Ash Polysaccharide (%) Carbohydrate
30.45 3.1 6.8 59 65.8
Table 3 Amino acid composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Amino acid (lg g 1) Asp Ser Gly Cys Met Leu Phe His Pro Total
3.48 1.34 1.76 0.15 0.13 1.78 1.43 0.21 0.77 23.43
Amino acid (lg g 1) Thr Glu Ala Val Ile Tyr Lys Arg
2.33 2.77 1.87 1.65 1.29 0.28 0.83 1.36
Table 4 Mineral composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Mineral (mg kg 1)
N. sphaeroides
Calcium Iron Potassium Magnesium Zinc Selenium
13.14 1.37 0 0.56 0.34 5.76
the sample, which indicated the sample is healthy as food of the crew. 4. Discussion In principle, the designed CELSS can perform supplying of oxygen, water and food, removing of carbon dioxide, as well as making daily life waste reusable. Most CELSS 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
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571 Table 5 Fatty acid composite of N. sphaeroides, collected cyanobacterium colony size is U 2–5 mm, washed in distilled water and then oven dried at 80 °C for 12 h, and then stored in desiccators until analyzed. N. sphaeroides colonies were cultured in aqueous BG110 medium, the culture temperature is 25 °C, with a PPFD of 40 lmol photons m 2 s 1. Fatty acid (mg kg 1) Dodecanoic acid Palmitic acid Heptadecoic acid Stearic acid Linoleic acid Arachidonic acid
Fatty acid (mg kg 1)
1.2
Myristic acid
9.4
841.2
Para Amino Benzoic Acid Heptadecenoic acid
489.8
11.7 13.4 395.8 3.3
Oleic acid Linolenic Acid Eicosapentaenoic Acid
7.0 253.1 163.2 9
system, plant’s long life cycle and special light requirement limited the application of higher plant bioreactor in space exploration. From previous researches we assume that aquatic organisms, such as phytoplankton, is a highly promising way to resolve these problems because of their high contents of protein and vitamin, shortest generation time, high oxygen production and relative easiness to grow and resistance to changes in environmental conditions, lower production costs. Another promising feature of this system is that it can be integrated into “intensive aquaculture” which can produce large amounts of protein in a minimal volume. In our experiments, the strain N. sphaeroides is one of traditionally healthy foods in China and our lab has developed some techniques for its large-scale culture, with which many kinds of delicious dishes were also developed in restaurant. In this research, it was found that the polysaccharide content was 59% in our sample which is easily digested for human being. Protein content needed by humans is between 23% and 25%, it accounted 30.45% of N. sphaeroides biomass which is closer to human needs, while Sprulira sp. is more than 50%. In addition, there are below 10% ash and fat which have low waste ratio than many plants food. The results of mineral concentration of N. sphaeroides were also showed that there were high content of Selenium, Calcium and Iron, which are essential elements for human body growth. Reported vitamin concentration results also indicated that there were high concentrations of total Vitamin B1, B2, C and E in the sample (Liu, 2000), which is helpful to life normal metabolism. In order to determine the quality of the amino acid and fatty acid in the sample, we carried out experiment to analyze the content of these components in more detail, which showed that amino acid and fatty acid of the sample were of high quality and the ratio to different component is close to the recommended level for human. These results showed that the culture sample of N. sphaeroides is excellent candidate food. The processed food, take salad and soup for example, the nutrient level did not change, as to the fried cyanobac-
1569
terium, if it cooked appropriately, there was no nutrient loss or even more nutritional mixed with other material, including vitamins and protein. Surly it can be used as the sole food considering of its nutrient level, and it is easily digested by humans because of its thin cell wall and rich in polysaccharide. However, we propose it as a supplementary food in the CELSS because of its taste and human unique diet psychology. It can be eaten with rice, corn and wheat. Plus, N. sphaeroides mixed with animal protein in fried and soup diets can serve as a complete and well-balanced source of amino acids to meet human requirements. For producer in CELSS, the most important characteristic is high productivity rate. The results of our experiment showed that under the same PPFD about 40 lmol photons m 2 s 1, the biomass production rate of N. sphaeroides is about 10–15 g (dry weight) m 2 day 1(300 L) in mass culture, which is higher than that of Sprulira sp. (7– 8 g (dry weight) m 2 day 1 (300 L)). Another important characteristic of producer is high oxygen evolution rate. In our experiment, it was found that the oxygen evolution rate of N. sphaeroides is about 150 lmol O2 mg 1 Chl a h 1, which means that 30 L culture can produce 500 L oxygen per day to provide for an adult (about 440– 860 L person 1 day 1) respiration. Sprulira sp. oxygen evolution rate is between 110 and 140 lmol O2 mg 1 Chl a h 1 with the PPFD of 40 lmol photons m 2 s 1 (Vonshak et al., 1996; Torzillo et al., 1998; Jimenez et al., 2003). Compared with Spirulina sp. production ability and oxygen evolution rate, N. sphaeroides can support more human consumption. Spirulina sp. has an optimal growth temperature of around 35 °C and no net growth occurs below 18 °C, which is a high cost in the biomass production. N. sphaeroides colony size at U 2–5 mm is considered suitable for the CELSS construction. Firstly, when in smaller colony size, the growth rate of cyanobacterium is higher. Secondly, the optimal oxygen production rate is in filamentous state, it can produce oxygen without a polysaccharide sheath. However, the colony did not show significantly lower oxygen evolution rate. The third consideration is optimal nutrient level for humans is bigger colony size, because with increasing colony sizes, carbohydrate is increasing. Human diet usually need relatively higher contents of carbohydrate, and it also showed U 2– 5 mm size is closer to that 50% content (Deng, 2006; Ma, 2010). In order to be used in future CELSS, appropriate bioreactor with the cyanobacterium should be developed. Usually, the colony size of the N. sphaeroides is about U 2– 30 mm, it is easy to be harvested and processed for food, but it also needs different bioreactors for culturing. We have designed new bioreactor on ground application (unpublished data) with high velocity stir and air exchange rate since colony growth affect air and nutrient exchange rate in reactor, and shear force has a less effluence on colony than on cell culture. Another problem is the lamps we used in experiments are fluorescent lamps, which will produce a lot of thermal energy and be a big burden for space
1570
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571
experiment energy supply. Light emitting diode (LED) and microwave lights were thought to be very good alternatives. Besides, air exchange in lipid culture under space microgravity environment is another problem for bioreactor; one membrane technique was developed to remove the dissolved oxygen immediately, which can separate gas from liquid in weightlessness condition easily (Ai et al., 2007). Considering the mass culture collection, filtration and low speed centrifugation (50–100 r min 1) which needs relatively lower energy consumption are enough for N. sphaeroides colonies to be harvested (Olaizola, 2003). In fact, we had developed one model system after considering these problems, but we need to improve the reactor system further for the future space experiment. 5. Conclusion We have isolated one strain of cyanobacteria, N. sphaeroides, which is one of traditional healthy food in China. The possibility of using it as candidate food and producer for future CELSS was evaluated. The productivity rate and the oxygen evolution rate were high, which indicated the cyanobacterium have advantages in biomass production and oxygen regeneration, and it can grow into colony with size between U 2 and 30 mm which is easy to be harvested and prepared for food process. The results for nutrient content of the cyanobacterium also showed it was a high quality and suitable food for the crew. The cyanobacterium can be suitable to be cultured with high density, and we plan to develop appropriate bioreactor with the cyanobacterium for supplying oxygen and food to the crew in future. Acknowledgements The work was supported by an Academy-Locality cooperation project from Chinese Academy of Sciences (No ZNWH-2011-004), the Project of Chinese Manned Spaceflight, Natural Science Foundation of China (No 30970688), and Natural Science Foundation of China (No 31000061). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.asr.2011. 06.035. References Ai, W.D., Guo, S.S., Qin, L.F., et al. Development of a ground based microalgae photobioreactor experimental facility for using in space. Space Medicine & Medical Engineering 20 (3), 165–169, 2007. Blu¨m, V., Andriske, M., Paris, F., et al. The C.E.B.A.S.-minimodule: behavior of an artificial aquatic ecological system during spaceflight. Advances in Space Research 26, 253–262, 2000. Deng, Z.Y. Studies on mass culture of Nostoc sphaeroides and N. commune (Cyanophyta) and the structural and physiological characteristics of N.
sphaeroides. Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2006. Dixon, M.A., Grodzinski, B., Cote, R., et al. Sealed environment chamber for canopy light interception and hydrocarbon analyses. Advances in Space Research 24, 277–281, 1999. Galston, A.W. Photosynthesis as a basis for life support on earth and in space: photosynthesis and transpiration in enclosed spaces. Bioscience 42, 490–493, 1992. Gao, K.S. Chinese studies on the edible blue-green algae, Nostoc flagelliforme: a review. Journal of Applied Phycology 10, 37–49, 1998. Garcia, P.F. A model for internal self-shading in planktonic organisms and its implications for the usefulness of ultraviolet sunscreens. Limnology and Oceanography 39, 1704–1717, 1994. Gonzales, J., Brown, P. Nile tilapia Oreochromis niloticus as a food source in advanced life support systems: initial considerations. Advances in Space Research 38, 1132–1137, 2006. Hiller, A., Plazin, J., Van Slyke, D.D. A study of conditions for KJELDAHL determination of nitrogen in proteins-description of methods with mercury as catalyst, and titrimetric and gasometrical measurements of the ammonia formed. Journal of Biological Chemistry 176 (3), 1401–1420, 1948. Huang, Z.B., Liu, Y.D., Paulsen, B., et al. Studies on polysaccharides from three edible species of Nostoc (Cyanobacteria) with different colony morphologies: comparison of monosaccharide compositions and viscosities of polysaccharides from field colonies and suspension cultures. Journal of Phycology 34, 962–968, 1998. Jimenez, C., Cossio, B.R., Niell, F.X. Relationship between physicochemical variables and productivity in open ponds for the production of Spirulina: a predictive model of algal yield. Aquaculture 221 (1–4), 331–345, 2003. Kawasaki, Y., Koike, J., Ijiri, K., et al. Microorganisms and plant of Autonomous Biological Systems (ABS) samples. Biological Sciences in Space 12, 373–376, 1998. Konig, B., Dunne, M., Slenzka, K. Earth life support for aquatic organisms, system and technical aspects. Advances in Space Research 27, 1523–1528, 2001. Li, D.H. Studies on morphogenesis, development and physiology of N. sphaeroides Ku¨tzing (cyanobacterium). Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2000. Li, D.H., Liu, Y.D., Song, L.R. Hormogonia mass differentiation from N. sphaeroides Ku¨tz. (cyanobacterium) and the comparison of structural characteristics between hormogonia and vegetative filaments. Phycological Research 49, 81–87, 2001. Lichtenthaler, H.K., Wellburn, A.R. Determinations of total carotenoids and chlorophyll a and b of leaf extracts in different solvents. Biochemical Society Transactions (London) 63, 591–592, 1983. Liu, J.L. Study on nutrient composition of Nostoc sphaeroides Ku¨tz. Chinese Traditional and Herbal Drugs 31 (17), 862–864 (In Chinese), 2000. Liu, Y.D., Zhang, Y.M., Chen, H.F., et al. Study on the structure and function of a hydrobiological closed ecosystem in space. Chinese Journal of Space Science 17 (Suppl.), 73–77, 1997. Ma, R. Studies on the industrial culture of Nostoc sphaeroides and Nostoc Commune. Doctoral Thesis. Institute of Hydrobiology, the Chinese Academy of Sciences, Wu Han, 2010. Melis, A., Neidhardt, J., Benemann, J.R. Dunaliella salina (Chlorophyta) with small antenna sizes exhibit higher photosynthetic productivities and photon efficiencies than normally pigmented cells. Journal of Applied Phycology 10, 515–525, 1998. Oguchi, M., Otsubo, K., Nitta, K., et al. Food production and gas exchange system using blue-green alga (Spirulina) for CELSS. Advances in Space Research 7 (4), 7–10, 1987. Olaizola, M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomolecular Engineering 20 (4-6), 459–466, 2003. Olson, R.L., Oleson, M.W., Slavin, T.J. CELSS for advanced manned mission. HortScience 23, 275–286, 1988.
Z. Hao et al. / Advances in Space Research 48 (2011) 1565–1571 Park, Y.K., Rasmussen, H.E., Ehlers, S.J., et al. Repression of proinflammatory gene expression by lipid extract of Nostoc commune var sphaeroides Ku¨tzing, a blue-green alga, via inhibition of nuclear factorkappa B in RAW 264.7 macrophages. Nutrition Research 28 (2), 83– 91, 2008. Qiu, B.S., Liu, J.Y., Liu, Z.L., et al. Distribution and ecology of the edible cyanobacterium Ge-Xian-Mi (Nostoc) in rice fields of Hefeng County in China. Journal of Applied Phycology 14, 423–429, 2002. Rasmussen, H.E., Blobaum, K.R., Jesch, E.D., et al. Hypocholesterolemic effect of Nostoc commune var. sphaeroides Ku¨tzing, an edible bluegreen alga. European Journal of Nutrition 48 (7), 387–394, 2009. Riche, M., Brown, P.B. Availability of phosphorus from feedstuffs fed to rainbow trout. Oncorhynchus mykiss. Aquaculture 142, 269–282, 1996. Rippka, R., Deruelles, J., Waterbury, J.B., et al. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology 111, 1–61, 1979. Sahasrabudhe, M.R., Smallbone, B.W. Comparative-evaluation of solvent-extraction methods for the determination of neutral and polar lipids in beef. Journal of the American Oil Chemisty’s Society 60 (4), 801–805, 1983. Salisbury, F.B., Campbell, W.F., Carman, J.G., et al. Plant growth during the greenhouse II experiment on the Mir orbital station. Advances in Space Research 31 (1), 221–227, 2003. Strasser, R.J., Srivastava, A., Govindjee Polyphasic chlorophyll a fluorescence transients in plants and cyanobacteria. Photochemistry and Photobiology 61, 32–42, 1995.
1571
Tang, J., Hu, Z.Y., Chen, X.W. Free radical scavenging and antioxidant enzymes activation of polysaccharide extract from N. sphaeroides. American Journal of Chinese Medicine 35 (5), 887–896, 2007. Torzillo, G., Bernardini, P., Masojidek, J. On-line monitoring of chlorophyll fluorescence to assess the extent of photoinhibition of photosynthesis induced by high oxygen concentration and low temperature and its effect on the productivity of outdoor cultures of Spirulina platensis (Cyanobacteria). Journal of Phycology 34 (3), 504– 510, 1998. Vonshak, A., Torzillo, G., Accolla, P., et al. Light and oxygen stress in Spirulina platensis (cyanobacteria) grown outdoors in tubular reactors. Physiologia Plantarum 97 (1), 175–179, 1996. Wang, G.H., Chen, H.F., Li, G.B., et al. Population growth and physiological characteristics of microalgae in a miniaturized bioreactor during space flight. Acta Astronautica 58, 264–269, 2006. Wang, G.H., Hu, C.X., Li, D.H., et al. The response of antioxidant systems in N. sphaeroides against UV-B radiation and the protective effects of exogenous antioxidants. Advances in Space Research 39 (6), 1034–1042, 2007. Wang, G.H., Li, G.B., Li, D.H., et al. Real-time studies on microalgae under microgravity. Acta Astronautica 56 (2), 131–137, 2004. Wang, G.H., Liu, Y.D., Li, G.B., et al. A simple closed aquatic ecosystem (CAES) for space. Advances in Space Research 41 (5), 684–690, 2008. Zhao, X.M. The Supplement to Compendium of Materia Medica. Reprinted in 1983 by People’s medical Publishing House. People’s medical Publishing House, Beijing, People’s Republic of China, 1765.