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Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation a
bc
a
a
L. Martínez , M. Otero , A. Morán & A.I. García a
Natural Resources Institute, University of Leon, Avda. Portugal 42, León 24071, Spain
b
Department of Applied Chemistry and Physics, University of León, Campus de Vegazana, León 24071, Spain c
CESAM & Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Accepted author version posted online: 28 May 2013.Published online: 20 Jun 2013.
To cite this article: L. Martínez, M. Otero, A. Morán & A.I. García (2013) Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation, Environmental Technology, 34:24, 3137-3143, DOI: 10.1080/09593330.2013.808238 To link to this article: http://dx.doi.org/10.1080/09593330.2013.808238
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Environmental Technology, 2013 Vol. 34, No. 24, 3137–3143, http://dx.doi.org/10.1080/09593330.2013.808238
Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation L. Martíneza , M. Oterob,c , A. Morána and A.I. Garcíaa∗ a Natural
Resources Institute, University of Leon, Avda. Portugal 42, León 24071, Spain; b Department of Applied Chemistry and Physics, University of León, Campus de Vegazana, León 24071, Spain; c CESAM & Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
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(Received 9 November 2012; final version received 18 May 2013 ) One of the technologies available for coping with problems related to the rise in atmospheric concentrations of carbon dioxide is CO2 biofixation with microalgae or cyanobacteria. The selection of native strains that grow well at the specific site where the technology is to be used will increase the success possibilities of such a technology. Thus, with the aim of finding a suitable local variety for use in a CO2 biofixation system, three recently isolated freshwater strains, Scenedesmus sp., Chlorogonium sp. and Synechocystis sp. were studied. Chlorella sorokiniana was used as a control strain. All the strains were grown under the same culture conditions for seven days of batch culture, and various growth and CO2 biofixation parameters were determined. Synechocystis sp. showed the highest specific growth rate at 1.75 per day (l/d). Results for CO2 biofixation ranged between 0.650 and 0.953 g of carbon dioxide per litre per day (g CO2 /l/d), but differences among native strains were noted, although they were not statistically significant. However, Synechocystis sp. was selected as the most suitable strain for CO2 biofixation, owing to its good capacity to use light in dense cultures, an essential requirement for sustainable commercial systems. Keywords: Synechocystis sp.; cyanobacterium; fresh water microalgae; CO2 biofixation; Chlorella sorokiniana
1. Introduction The global warming is a worldwide issue primarily attributed to the increase of the greenhouse gases (GHGs) in the atmosphere, especially CO2 . The use of biotechnological processes, such as photosynthetic CO2 biofixation, may be viable for reducing emissions of this GHG. However, among photosynthetic organisms, only microalgae and cyanobacteria are capable of direct CO2 reduction of the flue gases of thermo-electric power plants, thanks to their unique characteristics: (1) they are able to assimilate CO2 at higher concentrations than normal in the atmosphere; indeed, their productivity improves considerably in the presence of high CO2 concentrations [1]; (2) they show higher photosynthetic efficiency than crops and trees,[2] (3) they can be grown in controlled systems such as closed photobioreactors and open ponds [3,4]; and (4) they can be cultivated in areas not suitable for agriculture, such as brackish, marine and residual waters, as well as on clayey and saline lands. Moreover, their considerable requirements for nitrogen (N) and phosphorus (P) as nutrients allow these elements to be reduced when wastewaters are used, with the benefit of a reduction in the costs of inorganic nutrients.[3] Existing power plants use air for combustion and generate a flue gas at atmospheric pressure, whose concentration of CO2 is typically less than 15%.[5] The success of ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
applying a CO2 biofixation technology based on microalgae for the reduction of CO2 from these gases depends on the isolation and selection of native freshwater microalgae and cyanobacteria with a strong potential for CO2 biofixation.[6] Native strains are most likely to be successful at the specific site where the technology is to be used. However, the fact of working with native strains implies that the best culture conditions for optimum growth and maximum CO2 biofixation are unknown. Furthermore, these conditions can vary considerably from one strain to another, as they can even belong to different domains. Generally, optimum growth conditions for microalgae and cyanobacteria fall within certain ranges of temperature, pH and concentration of CO2 in air, as emerges from the bibliography published on the subject.[1] According to some studies, the optimum pH for growth tends to be higher than 6 and to fall between 7 and 9.[7–9] With regard to the concentration of CO2 in the air, although some strains reach their optimum growth rates at CO2 concentrations above 5%, including Chlorella sp. RK-1,[10] or Chlorococcum littorale,[11] it has been observed that microalgae and cyanobacteria tend to develop their highest growth rates when there is between 1% and 5% of CO2 in the air.[12] Higher CO2 concentrations negatively affect microalgae growth rates,[12] although microalgae strains capable of tolerating up to 20% CO2 have been cultured under batch
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conditions.[13] Regarding the temperature, the best range for growth is usually from 25◦ C to 35◦ C, but sometimes can be as high as 40◦ C.[3,14–17] Most of the strains investigated for suitability for cultivation under flue gases belonged to the Chlorella genus.[10,16–22] Chlorella are small microalgae (between 2 and 10 μm in size), with cells spherical in shape, and relatively very high maximum growth rates, for instance 0.13 l/h for Chlorella sp. KR-1,[20] or 0.24 l/h for Chlorella sorokiniana.[19] The reported maximum biomass productivity for Chlorella sp. KR-1 was 1.66 g of biomass/l/d (125 ml reactors, 450 μE/m2 /s) [20] and 1.1 g of biomass/l/d for C. sorokiniana (40 l flat reactor, 150 μE/m2 /s).[19] These biomass productivities equate to CO2 biofixation of 2.74 g/l/d and 1.81 g/l/d, respectively, based on 45% C in dry biomass. The aim of the research work presented here was to evaluate the capacity of native strains for their application in a CO2 biofixation system for the flue gases of local power plants, in comparison with C. sorokiniana, which, as it was above reported, is known to have a good CO2 biofixation capacity.
2. Material and methods In order to obtain native microalgae strain, 20 water samples from different origins (sewage treatment plants, manure treatment plants, natural ponds, springs, etc.) were collected and taken to the laboratory where they were cultured in Mann and Myers medium [23] under stirring. Culture under environment temperature, artificial light (200 μE/m2 /s2 ) and a photoperiod of 12/12 h was maintained until microalgae proliferation. If microalgae proliferation did not occur after 15 days under these conditions, the corresponding sample was discarded. With the aim of selecting CO2 resistant strains, inoculums of each of the samples that resulted in microalgae proliferation were cultured by triplicate in the same conditions as above but under a pure CO2 flow (0.5 l/min/l) during five days. In parallel, inoculums of the same samples were cultured by triplicate under an air flow (0.5 l/min/l), this air containing 0.038% of CO2 . Those strains which were grown under the pure CO2 flow were then isolated by the streak plate method. Isolated strains were identified by the Culture collection of Algae and Protozoa, England as Scenedesmus sp., Chlorogonium sp. and Synechocystis sp. These native microalgae and cyanobacteria strains were all cultured under the same culture conditions. Simultaneously, a control strain was cultured under identical conditions. The selected control strain was C. sorokiniana, which was kindly provided by the Chemistry and Materials Science Department of the University of Huelva (Spain). Although the best growth conditions are known for C. sorokiniana, it was cultivated under the same conditions as native strains,
in order to make it feasible to compare the CO2 biofixation results obtained by each strain. After isolation, the control and the native strains were adapted to experimental conditions by sparging the culture medium with 5% CO2 in air under the same temperature and light intensity for three days as an adaptation period. Then, each strain was grown in triplicate in 0.5 l bubble-column photobioreactors installed inside a controlled temperature chamber, as shown in the schematic diagram represented in Figure 1. Photobioreactors were inoculated with biomass suspension taken from the adaptation cultures at their exponential growth phase. The exact volume of suspended biomass was added to ensure a biomass concentration (Cb) of 0.01 g dry weight/l after bringing the volume to 0.5 l with Mann and Myers medium.[23] The temperature in the chamber was adjusted to 25◦ C. As a result, the temperature in the reactors during the period without illumination (night) was 25◦ C and reached 30◦ C ± 1◦ C when the lights were on (day). Homogeneous illumination was provided from the top by a set of six lamps (Phillips TLD 36W, France), using an 8/16 h photoperiod. A mean light intensity (photosynthetically active radiation) of 300 μE/m2 /s was measured on the reactor walls. Sparging air, which came from a small pump installed inside the chamber, was humidified, regulated and filtered through a 0.22 μm filter (Millex® -FG50, Millipore) before being mixed with CO2 . Commercialquality CO2 (N48, 99% purity, Air Liquide, Spain) was semi-automatically controlled, filtered and finally mixed with air for an inlet flow of 5% CO2 in air. The CO2 was not added continuously, but in pulses, with the aim of controlling pH as well as having the necessary carbon source for well-balanced growth. The frequency and duration of CO2 pulses were checked and adjusted daily to minimize pH deviations from the initial pH, which was set at 7. In this way, pH was kept between 7 and 8 along the culture duration. When CO2 was not being injected, an air flow rate of 0.4 l/min/l was sparged through each reactor. The addition of CO2 (at 0.02 l/min/l) did not substantially modify that flow. The above culture conditions were selected on a practical basis, while the pH was set according to the optimum pH data published in the literature.[7–9] Batch cultures were maintained during seven days. Along this time, culture samples (10 ml) were taken daily for 4 h after the lights were switched on, at the same time as pH and temperature were being measured. Changes in the growth of each strain were followed by determining cellular density (N) and Cb. Cb was determined as dry weight by filtering 10 ml of culture through a 0.45 μm Whatman filter (Whatman GF/A, Germany), washed with 20 ml 0.5 N HCl to dissolve precipitated salts. The filter was dried at 105◦ C for 24 h. Cellular density was determined by counting cells using a Thoma counting chamber (Brand GmbH, Germany). The carbon content of each strain was determined in accordance with the D-5373 ASTM method.[24]
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Fluorescent lamps 74 mm
0,5 l-bubble column photobioreactor
260 mm
CO2 controller
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Gas mixing chamber
CO2 flow Electrovalve meter
Gas distributor
Millipore 0.22 µm filter
CO2 cylinder
Air flow meter
Air pump
Figure 1.
Air humidifier
Laboratory-scale experimental setup used for this study.
2.1.
Kinetic parameters defining microbial growth and CO2 biofixation With the aim of comparing growth rates and CO2 biofixation among the strains studied, the following parameters were used. • Specific growth rate (μi , l/h or l/d) for consecutive days of culture (from day i−1 to day i) was calculated as follows [25,26]: μ=
Ln(Ni ) − Ln(Ni−1 ) , ti − ti−1
(1)
where Ni is the cellular density, in cells/ml, at time ti . Time ti was expressed in hours or days, as specified. • Specific growth rate can be also determined as a function of Cb, by replacing cellular density values (N ) with Cb values in Equation (1). • Maximum specific growth rate (μmax , l/h or l/d): was the highest obtained within the seven days of the culture period. • Doubling time (dt , in hours): was the time required for cell division. It can be calculated from specific growth rate for time i (μi , l/h), as described by the following expression [26]: dt,i = Ln(2)/μi .
(2)
• Minimum doubling time (dt,min , in hours) was as determined from μmax (l/h): dt,min =
Ln(2) . μmax
(3)
• Accumulated cellular density (in cells/ml): was the number of cells per millilitre generated during the seven days of the culture period. It was calculated as the difference between cellular density at the end of the culturing period and cellular density at its start. • Accumulated Cb (in g/l): was the quantity of biomass that had been generated by the growth of microalgae or cyanobacteria since the beginning of the culture. It was expressed as grams of biomass per litre of culture. • Daily biomass productivity (Pi , in g/l/d): was the biomass per litre of culture generated during consecutive days: Pi =
(Cbi − Cbi−1 ) , (ti − ti−1 )
(4)
where Cbi , is Cb on day i. Maximum daily productivity (Pmax ) was the highest rate recorded during the seven days of the culture period. • CO2 biofixation (Fi ). This parameter was calculated from biomass productivity and carbon content (%C) for each strain, in accordance with the following
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44 , 12
(5)
where Fi is CO2 biofixation for day i and Pi is biomass productivity on day i, in batch cultures. Maximum CO2 biofixation (Fmax ) was calculated taking Pmax as productivity.
3.
Results and discussion
The selected culture conditions, mostly resembling natural conditions, were to a large extent favourable for the considered aquatic phototrophic micro-organisms here but not optimal for any of them.
Log (cellular densityx107)
(a) 100.0 10.0 1.0 0.1 0.0 0
1
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3
4
5
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7
8
time (d)
(b)
Cb (g/l)
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2.2. Statistical analysis Results in the present study were evaluated by one-way analysis of variance of kinetic parameters. Significant differences between parameters for each strain were determined by the Tukey test, at a level of significance of 95% (p = .05).
Growth curves, representing cellular density (N) vs. time and Cb vs. time may be seen in Figure 2. Each point of the curves represents mean values from three replicates and the error bars, standard deviations between replicates. Cellular density was recorded on a logarithmic scale to ensure correct noting of the exponential growth phase. That phase extends along the first segment of higher slope, all the strains showing an early exponential growth phase, which lasted until the third day of culturing, as can be noted in Figure 2(a). After that, growth rates became slower moving towards the stationary phase. This slowdown must be related with the biomass increase, which makes it difficult for light to penetrate into the culture, owing to the self-shading effect. The absence of lag phase observed in all the growth curves indicates good adaptation to the culture conditions imposed,[28] especially to CO2 injection.[22] This gas was bubbled through at the highest concentration allowing uninhibited growth, as mentioned previously. The results revealed that strains studied needed only three days of prior cultivation with 5% CO2 in the air in order to become adapted to these conditions and not to develop any perceptible lag phase.
Synechocystis
Scenedesmus
C. sorokiniana
Chlorogonium
2.0 1.5 1.0 0.5 0.0
0
1
2
3
4
5
6
7
8
time (d) Synechocystis sp.
Scenedesmus sp.
C. sorokiniana
Chlorogonium sp.
Figure 2. Growth curves for the four strains studied. These are shown in terms of cell density (a) and Cb (b). Each data point indicates the mean and standard deviation of three replicates.
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Table 1. Maximum specific growth rates (μmax ), minimum doubling times (dt,min ), maximum productivity (Pmax ), maximum CO2 biofixation (Fmax ) and carbon content for each strain studied. Strain Synechocystis sp. Scenedesmus sp. C. sorokiniana Chlorogonium sp.
μmax (l/d)
Day of culturing
dt,min (h)
Pmax (g biomass/l/d)
Day of culturing
Fmax (g CO2 /l/d)
Carbon (% db)
1.75 a 1.44 ab 1.25 ab 1.08 b
2 2 1 3
9.5 a 11.5 ab 13.3 ab 15.4 b
0.475 ± 0.101 a 0.403 ± 0.075 a 0.364 ± 0.050 a 0.346 ± 0.005 a
7 2 6 6
0.953 ± 0.100 a 0.768 ± 0.139 a 0.670 ± 0.093 a 0.650 ± 0.055 a
49.8 50.7 50.2 49.9
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Note: Different letters for the same parameters indicate statistically different values (p < .05) while equal letters indicate not statistically significant values (p > .05).
Data for cell density indicate that the cyanobacterium Synechocystis sp. presented the highest values over the entire growth curve, while the microalgae Scenedesmus sp. had the lowest (Figure 2). However, this is not true when representing the concentration of biomass vs. time (Figure 2(b)). This apparent inconsistency finds its explanation in the cell size for each strain. Microscopic observations showed that Synechocystis sp. cells are around 4–6 μm in size. However, an adult Scenedesmus sp. cell can be three or even four times bigger than an adult cyanobacterium cell. C. sorokiniana and Chlorogonium sp. have sizes intermediate between Synechocystis sp. and Scenedesmus sp. Thus, despite cell size differences, the biomass produced along time was similar for the four strains (Figure 2). The maximum specific growth rates and the corresponding minimum doubling times are given in Table 1 for each strain. Different letters refer to statistical differences (p < .05) shown by the Tukey test. The maximum specific growth rate for Synechocystis sp., 1.75 l/d, is statistically different and higher than those achieved by the microalgae. Chlorogonium sp. showed the lowest specific growth rate, while there were no statistical differences between Scenedesmus sp. and C. sorokiniana. However, despite the significant differences, all strains showed the same order growth rate under the culture conditions imposed. Regarding the minimum time for cell division, i.e. the doubling time, it varied from 9.5 h for Synechocystis sp. to 15.4 h for Chlorogonium sp. Synechocystis sp. was the strain that showed the highest growth rates, with a maximum specific growth rate of 1.75 l/d and a minimum doubling time of 9.5 h. These results were even better than those shown here by C. sorokiniana: μmax of 1.25 l/h and minimum doubling time of 13.3 h. Higher specific growth rates and doubling times had been expected for the reference strain, as C. sorokiniana has been considered as one of the photo-autotrophic organisms with specific growth rates of up to 5.76 l/d, and being able to split in less than 3 h [19] under optimal growing conditions. These results point to the fact that native cyanobacteria’s growth rates could be also further improved if their optimal growth conditions were determined. Furthermore, if our native Synechocystis sp. is compared with other species studied for application in CO2 biofixation
systems, it exceeds the growth rates for Chlorellae NTU H-15 and H-25 reported by Chang and Yang,[3] both having μmax = 0.312 l/d under their optimal conditions or the Spirulina sp. in de Morais and Vieria results,[29] showing a μmax of 0.336 l/d. Maximum biomass productivity values (Pmax ) are also given in Table 1 for the different strains. No differences of statistical significance were found in maximum biomass production, which ranged from 0.346 to 0.475 g of biomass per day and litre of culture. One noteworthy difference was the time during culturing when the maximum productivity was attained: this was on day 7 for Synechocystis sp., day 6 for Chlorogonium sp. and C. sorokiniana, and day 2 for Scenedesmus sp. This last strain showed its maximum productivity when light was not yet limited since light started to be limited from day 4. However, Synechocystis sp. showed a relative good capacity to use light in dense cultures, which is a key for sustainable commercial systems. Values of maximum CO2 biofixation (Fmax ), which were calculated from maximum biomass productivity (Pmax ) and biomass carbon content (Equation (5)) in each strain (Table 1), may be seen in Table 1. Since biomass carbon content was around 50% in all strains, it does not constitute a key parameter in the selection of the best strain for CO2 biofixation. Additionally, as can be noted in Table 1, there were no significant differences in the results for Fmax . The accumulated parameters for each strain obtained from experimental batch cultures are presented in Figure 3. The accumulated cell density and Cb allow a comparison of each strain’s behaviour in an overall way, not solely in terms of maximum values. According to these graphs, there are significant differences for accumulated cell density but not for accumulated Cb. This means that by the end of batch cultures all the strains had produced similar amounts of biomass, but Synechocystis sp. and C. sorokiniana had to divide more times than Scenedesmus sp. and Chlorogonium sp. to generate this amount of biomass. A comparison of the outcomes for each strain studied showed that Synechocystis sp. clearly had the best results in terms of the number of cells generated during the period of culture. However, its accumulated Cb, maximum productivity and CO2 biofixation parameters were no better than the other strains under study. This, for example, is due to the smaller size of Synechocystis
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Figure 3. Accumulated cell density (a) and Cb (b) during the seven-day culturing of batches. Different letters for the same parameter indicate statistically different values (p < .05) while equal letters indicate not statistically significant values (p > .05).
sp. cells, which contain smaller amounts of biomass per cell than Scenedesmus sp. Cells. To sum up, according to the results of the present study, although Synechocystis sp. has achieved higher growth rates, cyanobacterium did not fix more carbon dioxide than Scenedesmus sp., Chlorogonium sp. or C. sorokiniana, so all of them could be equally suitable for application in CO2 biofixation systems. Then, a priori, it would seem that results for growth parameters and the analysis of CO2 biofixation were not conclusive for selecting the strain best at CO2 biofixation. Hence, other practical characteristics should be analysed in order to decide the final selection. In the first place, Scenedesmus sp. and Chlorogonium sp. cell sizes are similar, ranging from 5 to 10 μm and their floatability in repose was seen during this study to be worse than what was observed for the cyanobacterium Synechocystis sp. This last strain presented a very considerable resistance to sedimentation. This implies that Synechocystis sp. requires less mixing energy in order to maintain a homogeneous suspension, an essential condition for the appropriate availability of nutrients and light to cells. Furthermore, Synechocystis sp. reached its maximum CO2 biofixation of 0.953 g CO2 /l/d on day 7 of batch culture (Table 1), when the light availability within the culture was at its lowest. Meanwhile, the remaining strains had a considerably lower CO2 biofixation rate compared to their maximum during the last day of culturing: this was 26% lower for C. sorokiniana, 49% for Chlorogonium sp. and 63% for Scenedesmus sp. The fact that Synechocystis sp. developed maximum CO2 biofixation at the end of the culturing period, when light availability was at its lowest, means that this native
cyanobacterium is able to better use light when this is limited than the other strains studied. The explanation for this may be related to its spherical shape and small cell size, giving a larger surface for light reception with the same Cb, as compared to other non-spherical and bigger cells.[30] Synechocystis sp. proved to be able to fix the maximum amount of CO2 when it had reached a dense culture with a Cb from 1.34 to 1.76 g/l (Figure 2(b)). It is important to grow microalgae in high density cultures since high cell density at high productivity is favourable to reduce harvesting costs. In this sense, if culture systems are photobioreactors, Cbs exceeding 1 g/l are usually required.[9, 31,32] From the previous discussion, it may be grasped that quality and practical characteristics, such as better light use and better productivity at high culture densities, permitted a final selection. In terms of the research project’s requirements, C. sorokiniana was excluded by not being a native and local strain. Of the three native strains, comparison of those which showed similar maximum growth and CO2 biofixation parameters, Synechocystis sp. and Scenedesmus sp., Synechocystis sp. is more strongly recommended for application in CO2 biofixation systems by reason of presenting maximum productivity at the highest densities. Acknowledgements The authors thank Ingeniería Idom Internacional S.A. (Spain) and Junta de Castilla y León (project LE017A09) for financially supporting this work. The authors also express their thanks to Ms María Cuaresma, PhD student, of the University of Huelva in Spain for providing them with C. sorokiniana samples. Also,
Environmental Technology Marta Otero acknowledges the support from the Spanish Ministry of Science and Innovation (RYC-2010-05634). [16]
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under CO2 enriched atmosphere. Energ Convers Manag. 1992;33:545–552. Maeda K, Owada M, Kimura N, Omata K, Karube I. CO2 fixation from the flue gas on coal-fired thermal power plant by microalgae. Energ Convers Manag. 1995;36:717–720. Yue L, Chen W. Isolation and determination of cultural characteristics of a new highly CO2 tolerant freshwater microalgae. Energ Convers Manag. 2005;46:1868–1876. Yanagi M, Watanabe Y, Saiki H. CO2 fixation by Chlorella sp. HA-1 and its utilization. Energ Convers Manag. 1995;36(6–9):713–716. Sakai N, Sakamoto Y, Kishimoto N, Chihara M, Karube I. Chlorella strains from hot springs tolerant to high temperature and high CO2 . Energ Convers Manag. 1995;36:693– 696. Lee JS, Kim DK, Lee JP, Park SC, Koh JH, Cho HS, Kim SW. Effects of SO2 and NO on growth of Chlorella sp. KR-1. Bioresour Technol. 2002;82:1–4. Jeong ML, Gillis JM, Hwang JY. Carbon dioxide mitigation by microalgal photosynthesis. Bull Kor Chem Soc. 2003;24:173–176. Greque M, Vieira JA. Isolation and selection of microalgae from coal-fired thermo-electric power plant for biological fixation of carbon dioxide. Energ Convers Manage. 2007;48:2169–2173. Mann JE, Myers J. On pigments, growth and photosynthesis of Phaeodactylum tricornutum. J Phycol. 1968;4:349–355. American Society for Testing and Materials (ASTM). D 5373, standard test methods for instrumental determination of carbon, hydrogen, and nitrogen in laboratory samples of coal and coke. 2002. Andersen RA. Algal culturing techniques. Burlington, MA: Elsevier Academic Press; 2005. Wahidin S, Idris A, Shaleh SRM. The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp. Bioresour Technol. 2013;129:7–11. Kajiwara S, Yamada H, Ohkuni N, Ohtaguchi K. Design of the bioreactor for carbon dioxide fixation by Synechococcus PCC7942. Energ Convers Manag. 1996;38:S529–S532. Schimidell W, Lima AU, Aquarone E, Borzani W. Industrial biotechnology. Sao Paulo: Edgard Blücher LTDA; 2001. de Morais MG, Vieria JA. Biofixation of carbon dioxide by Spirulina sp. and Scenedesmus obliquus cultivated in a three-stage serial tubular photobioreactor. J Biotecnol. 2007;129:439–445. Spektorova LV, Goronkova OI, Nosova LP, Albitskaya ON, Danilova GY. High density culture of marine microalgae – promising items for mariculture. III. Mass culture of Monochrysis lathery Droop. Aquaculture. 1986;55:231– 240. Ugwu CU, Ogbona JC, Tanaka H. Characterization of light utilization and biomass yields of Chlorella sorokiniana in inclined tubular photobioreactors equipped with static mixers. Process Biochem. 2005;40:3406–3411. García-Malea MC, Acién FG, Fernández JM, Cerón MC, Molina E. Continuous production of green cells of Haematococcus pluvialis: modeling of the irradiance effect. Enzyme Microb Tech. 2006;38:981–989.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation a
bc
a
a
L. Martínez , M. Otero , A. Morán & A.I. García a
Natural Resources Institute, University of Leon, Avda. Portugal 42, León 24071, Spain
b
Department of Applied Chemistry and Physics, University of León, Campus de Vegazana, León 24071, Spain c
CESAM & Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Accepted author version posted online: 28 May 2013.Published online: 20 Jun 2013.
To cite this article: L. Martínez, M. Otero, A. Morán & A.I. García (2013) Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation, Environmental Technology, 34:24, 3137-3143, DOI: 10.1080/09593330.2013.808238 To link to this article: http://dx.doi.org/10.1080/09593330.2013.808238
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Environmental Technology, 2013 Vol. 34, No. 24, 3137–3143, http://dx.doi.org/10.1080/09593330.2013.808238
Selection of native freshwater microalgae and cyanobacteria for CO2 biofixation L. Martíneza , M. Oterob,c , A. Morána and A.I. Garcíaa∗ a Natural
Resources Institute, University of Leon, Avda. Portugal 42, León 24071, Spain; b Department of Applied Chemistry and Physics, University of León, Campus de Vegazana, León 24071, Spain; c CESAM & Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
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(Received 9 November 2012; final version received 18 May 2013 ) One of the technologies available for coping with problems related to the rise in atmospheric concentrations of carbon dioxide is CO2 biofixation with microalgae or cyanobacteria. The selection of native strains that grow well at the specific site where the technology is to be used will increase the success possibilities of such a technology. Thus, with the aim of finding a suitable local variety for use in a CO2 biofixation system, three recently isolated freshwater strains, Scenedesmus sp., Chlorogonium sp. and Synechocystis sp. were studied. Chlorella sorokiniana was used as a control strain. All the strains were grown under the same culture conditions for seven days of batch culture, and various growth and CO2 biofixation parameters were determined. Synechocystis sp. showed the highest specific growth rate at 1.75 per day (l/d). Results for CO2 biofixation ranged between 0.650 and 0.953 g of carbon dioxide per litre per day (g CO2 /l/d), but differences among native strains were noted, although they were not statistically significant. However, Synechocystis sp. was selected as the most suitable strain for CO2 biofixation, owing to its good capacity to use light in dense cultures, an essential requirement for sustainable commercial systems. Keywords: Synechocystis sp.; cyanobacterium; fresh water microalgae; CO2 biofixation; Chlorella sorokiniana
1. Introduction The global warming is a worldwide issue primarily attributed to the increase of the greenhouse gases (GHGs) in the atmosphere, especially CO2 . The use of biotechnological processes, such as photosynthetic CO2 biofixation, may be viable for reducing emissions of this GHG. However, among photosynthetic organisms, only microalgae and cyanobacteria are capable of direct CO2 reduction of the flue gases of thermo-electric power plants, thanks to their unique characteristics: (1) they are able to assimilate CO2 at higher concentrations than normal in the atmosphere; indeed, their productivity improves considerably in the presence of high CO2 concentrations [1]; (2) they show higher photosynthetic efficiency than crops and trees,[2] (3) they can be grown in controlled systems such as closed photobioreactors and open ponds [3,4]; and (4) they can be cultivated in areas not suitable for agriculture, such as brackish, marine and residual waters, as well as on clayey and saline lands. Moreover, their considerable requirements for nitrogen (N) and phosphorus (P) as nutrients allow these elements to be reduced when wastewaters are used, with the benefit of a reduction in the costs of inorganic nutrients.[3] Existing power plants use air for combustion and generate a flue gas at atmospheric pressure, whose concentration of CO2 is typically less than 15%.[5] The success of ∗ Corresponding
author. Email: [email protected]
© 2013 Taylor & Francis
applying a CO2 biofixation technology based on microalgae for the reduction of CO2 from these gases depends on the isolation and selection of native freshwater microalgae and cyanobacteria with a strong potential for CO2 biofixation.[6] Native strains are most likely to be successful at the specific site where the technology is to be used. However, the fact of working with native strains implies that the best culture conditions for optimum growth and maximum CO2 biofixation are unknown. Furthermore, these conditions can vary considerably from one strain to another, as they can even belong to different domains. Generally, optimum growth conditions for microalgae and cyanobacteria fall within certain ranges of temperature, pH and concentration of CO2 in air, as emerges from the bibliography published on the subject.[1] According to some studies, the optimum pH for growth tends to be higher than 6 and to fall between 7 and 9.[7–9] With regard to the concentration of CO2 in the air, although some strains reach their optimum growth rates at CO2 concentrations above 5%, including Chlorella sp. RK-1,[10] or Chlorococcum littorale,[11] it has been observed that microalgae and cyanobacteria tend to develop their highest growth rates when there is between 1% and 5% of CO2 in the air.[12] Higher CO2 concentrations negatively affect microalgae growth rates,[12] although microalgae strains capable of tolerating up to 20% CO2 have been cultured under batch
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conditions.[13] Regarding the temperature, the best range for growth is usually from 25◦ C to 35◦ C, but sometimes can be as high as 40◦ C.[3,14–17] Most of the strains investigated for suitability for cultivation under flue gases belonged to the Chlorella genus.[10,16–22] Chlorella are small microalgae (between 2 and 10 μm in size), with cells spherical in shape, and relatively very high maximum growth rates, for instance 0.13 l/h for Chlorella sp. KR-1,[20] or 0.24 l/h for Chlorella sorokiniana.[19] The reported maximum biomass productivity for Chlorella sp. KR-1 was 1.66 g of biomass/l/d (125 ml reactors, 450 μE/m2 /s) [20] and 1.1 g of biomass/l/d for C. sorokiniana (40 l flat reactor, 150 μE/m2 /s).[19] These biomass productivities equate to CO2 biofixation of 2.74 g/l/d and 1.81 g/l/d, respectively, based on 45% C in dry biomass. The aim of the research work presented here was to evaluate the capacity of native strains for their application in a CO2 biofixation system for the flue gases of local power plants, in comparison with C. sorokiniana, which, as it was above reported, is known to have a good CO2 biofixation capacity.
2. Material and methods In order to obtain native microalgae strain, 20 water samples from different origins (sewage treatment plants, manure treatment plants, natural ponds, springs, etc.) were collected and taken to the laboratory where they were cultured in Mann and Myers medium [23] under stirring. Culture under environment temperature, artificial light (200 μE/m2 /s2 ) and a photoperiod of 12/12 h was maintained until microalgae proliferation. If microalgae proliferation did not occur after 15 days under these conditions, the corresponding sample was discarded. With the aim of selecting CO2 resistant strains, inoculums of each of the samples that resulted in microalgae proliferation were cultured by triplicate in the same conditions as above but under a pure CO2 flow (0.5 l/min/l) during five days. In parallel, inoculums of the same samples were cultured by triplicate under an air flow (0.5 l/min/l), this air containing 0.038% of CO2 . Those strains which were grown under the pure CO2 flow were then isolated by the streak plate method. Isolated strains were identified by the Culture collection of Algae and Protozoa, England as Scenedesmus sp., Chlorogonium sp. and Synechocystis sp. These native microalgae and cyanobacteria strains were all cultured under the same culture conditions. Simultaneously, a control strain was cultured under identical conditions. The selected control strain was C. sorokiniana, which was kindly provided by the Chemistry and Materials Science Department of the University of Huelva (Spain). Although the best growth conditions are known for C. sorokiniana, it was cultivated under the same conditions as native strains,
in order to make it feasible to compare the CO2 biofixation results obtained by each strain. After isolation, the control and the native strains were adapted to experimental conditions by sparging the culture medium with 5% CO2 in air under the same temperature and light intensity for three days as an adaptation period. Then, each strain was grown in triplicate in 0.5 l bubble-column photobioreactors installed inside a controlled temperature chamber, as shown in the schematic diagram represented in Figure 1. Photobioreactors were inoculated with biomass suspension taken from the adaptation cultures at their exponential growth phase. The exact volume of suspended biomass was added to ensure a biomass concentration (Cb) of 0.01 g dry weight/l after bringing the volume to 0.5 l with Mann and Myers medium.[23] The temperature in the chamber was adjusted to 25◦ C. As a result, the temperature in the reactors during the period without illumination (night) was 25◦ C and reached 30◦ C ± 1◦ C when the lights were on (day). Homogeneous illumination was provided from the top by a set of six lamps (Phillips TLD 36W, France), using an 8/16 h photoperiod. A mean light intensity (photosynthetically active radiation) of 300 μE/m2 /s was measured on the reactor walls. Sparging air, which came from a small pump installed inside the chamber, was humidified, regulated and filtered through a 0.22 μm filter (Millex® -FG50, Millipore) before being mixed with CO2 . Commercialquality CO2 (N48, 99% purity, Air Liquide, Spain) was semi-automatically controlled, filtered and finally mixed with air for an inlet flow of 5% CO2 in air. The CO2 was not added continuously, but in pulses, with the aim of controlling pH as well as having the necessary carbon source for well-balanced growth. The frequency and duration of CO2 pulses were checked and adjusted daily to minimize pH deviations from the initial pH, which was set at 7. In this way, pH was kept between 7 and 8 along the culture duration. When CO2 was not being injected, an air flow rate of 0.4 l/min/l was sparged through each reactor. The addition of CO2 (at 0.02 l/min/l) did not substantially modify that flow. The above culture conditions were selected on a practical basis, while the pH was set according to the optimum pH data published in the literature.[7–9] Batch cultures were maintained during seven days. Along this time, culture samples (10 ml) were taken daily for 4 h after the lights were switched on, at the same time as pH and temperature were being measured. Changes in the growth of each strain were followed by determining cellular density (N) and Cb. Cb was determined as dry weight by filtering 10 ml of culture through a 0.45 μm Whatman filter (Whatman GF/A, Germany), washed with 20 ml 0.5 N HCl to dissolve precipitated salts. The filter was dried at 105◦ C for 24 h. Cellular density was determined by counting cells using a Thoma counting chamber (Brand GmbH, Germany). The carbon content of each strain was determined in accordance with the D-5373 ASTM method.[24]
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Fluorescent lamps 74 mm
0,5 l-bubble column photobioreactor
260 mm
CO2 controller
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Gas mixing chamber
CO2 flow Electrovalve meter
Gas distributor
Millipore 0.22 µm filter
CO2 cylinder
Air flow meter
Air pump
Figure 1.
Air humidifier
Laboratory-scale experimental setup used for this study.
2.1.
Kinetic parameters defining microbial growth and CO2 biofixation With the aim of comparing growth rates and CO2 biofixation among the strains studied, the following parameters were used. • Specific growth rate (μi , l/h or l/d) for consecutive days of culture (from day i−1 to day i) was calculated as follows [25,26]: μ=
Ln(Ni ) − Ln(Ni−1 ) , ti − ti−1
(1)
where Ni is the cellular density, in cells/ml, at time ti . Time ti was expressed in hours or days, as specified. • Specific growth rate can be also determined as a function of Cb, by replacing cellular density values (N ) with Cb values in Equation (1). • Maximum specific growth rate (μmax , l/h or l/d): was the highest obtained within the seven days of the culture period. • Doubling time (dt , in hours): was the time required for cell division. It can be calculated from specific growth rate for time i (μi , l/h), as described by the following expression [26]: dt,i = Ln(2)/μi .
(2)
• Minimum doubling time (dt,min , in hours) was as determined from μmax (l/h): dt,min =
Ln(2) . μmax
(3)
• Accumulated cellular density (in cells/ml): was the number of cells per millilitre generated during the seven days of the culture period. It was calculated as the difference between cellular density at the end of the culturing period and cellular density at its start. • Accumulated Cb (in g/l): was the quantity of biomass that had been generated by the growth of microalgae or cyanobacteria since the beginning of the culture. It was expressed as grams of biomass per litre of culture. • Daily biomass productivity (Pi , in g/l/d): was the biomass per litre of culture generated during consecutive days: Pi =
(Cbi − Cbi−1 ) , (ti − ti−1 )
(4)
where Cbi , is Cb on day i. Maximum daily productivity (Pmax ) was the highest rate recorded during the seven days of the culture period. • CO2 biofixation (Fi ). This parameter was calculated from biomass productivity and carbon content (%C) for each strain, in accordance with the following
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44 , 12
(5)
where Fi is CO2 biofixation for day i and Pi is biomass productivity on day i, in batch cultures. Maximum CO2 biofixation (Fmax ) was calculated taking Pmax as productivity.
3.
Results and discussion
The selected culture conditions, mostly resembling natural conditions, were to a large extent favourable for the considered aquatic phototrophic micro-organisms here but not optimal for any of them.
Log (cellular densityx107)
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2.2. Statistical analysis Results in the present study were evaluated by one-way analysis of variance of kinetic parameters. Significant differences between parameters for each strain were determined by the Tukey test, at a level of significance of 95% (p = .05).
Growth curves, representing cellular density (N) vs. time and Cb vs. time may be seen in Figure 2. Each point of the curves represents mean values from three replicates and the error bars, standard deviations between replicates. Cellular density was recorded on a logarithmic scale to ensure correct noting of the exponential growth phase. That phase extends along the first segment of higher slope, all the strains showing an early exponential growth phase, which lasted until the third day of culturing, as can be noted in Figure 2(a). After that, growth rates became slower moving towards the stationary phase. This slowdown must be related with the biomass increase, which makes it difficult for light to penetrate into the culture, owing to the self-shading effect. The absence of lag phase observed in all the growth curves indicates good adaptation to the culture conditions imposed,[28] especially to CO2 injection.[22] This gas was bubbled through at the highest concentration allowing uninhibited growth, as mentioned previously. The results revealed that strains studied needed only three days of prior cultivation with 5% CO2 in the air in order to become adapted to these conditions and not to develop any perceptible lag phase.
Synechocystis
Scenedesmus
C. sorokiniana
Chlorogonium
2.0 1.5 1.0 0.5 0.0
0
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time (d) Synechocystis sp.
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Figure 2. Growth curves for the four strains studied. These are shown in terms of cell density (a) and Cb (b). Each data point indicates the mean and standard deviation of three replicates.
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Table 1. Maximum specific growth rates (μmax ), minimum doubling times (dt,min ), maximum productivity (Pmax ), maximum CO2 biofixation (Fmax ) and carbon content for each strain studied. Strain Synechocystis sp. Scenedesmus sp. C. sorokiniana Chlorogonium sp.
μmax (l/d)
Day of culturing
dt,min (h)
Pmax (g biomass/l/d)
Day of culturing
Fmax (g CO2 /l/d)
Carbon (% db)
1.75 a 1.44 ab 1.25 ab 1.08 b
2 2 1 3
9.5 a 11.5 ab 13.3 ab 15.4 b
0.475 ± 0.101 a 0.403 ± 0.075 a 0.364 ± 0.050 a 0.346 ± 0.005 a
7 2 6 6
0.953 ± 0.100 a 0.768 ± 0.139 a 0.670 ± 0.093 a 0.650 ± 0.055 a
49.8 50.7 50.2 49.9
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Note: Different letters for the same parameters indicate statistically different values (p < .05) while equal letters indicate not statistically significant values (p > .05).
Data for cell density indicate that the cyanobacterium Synechocystis sp. presented the highest values over the entire growth curve, while the microalgae Scenedesmus sp. had the lowest (Figure 2). However, this is not true when representing the concentration of biomass vs. time (Figure 2(b)). This apparent inconsistency finds its explanation in the cell size for each strain. Microscopic observations showed that Synechocystis sp. cells are around 4–6 μm in size. However, an adult Scenedesmus sp. cell can be three or even four times bigger than an adult cyanobacterium cell. C. sorokiniana and Chlorogonium sp. have sizes intermediate between Synechocystis sp. and Scenedesmus sp. Thus, despite cell size differences, the biomass produced along time was similar for the four strains (Figure 2). The maximum specific growth rates and the corresponding minimum doubling times are given in Table 1 for each strain. Different letters refer to statistical differences (p < .05) shown by the Tukey test. The maximum specific growth rate for Synechocystis sp., 1.75 l/d, is statistically different and higher than those achieved by the microalgae. Chlorogonium sp. showed the lowest specific growth rate, while there were no statistical differences between Scenedesmus sp. and C. sorokiniana. However, despite the significant differences, all strains showed the same order growth rate under the culture conditions imposed. Regarding the minimum time for cell division, i.e. the doubling time, it varied from 9.5 h for Synechocystis sp. to 15.4 h for Chlorogonium sp. Synechocystis sp. was the strain that showed the highest growth rates, with a maximum specific growth rate of 1.75 l/d and a minimum doubling time of 9.5 h. These results were even better than those shown here by C. sorokiniana: μmax of 1.25 l/h and minimum doubling time of 13.3 h. Higher specific growth rates and doubling times had been expected for the reference strain, as C. sorokiniana has been considered as one of the photo-autotrophic organisms with specific growth rates of up to 5.76 l/d, and being able to split in less than 3 h [19] under optimal growing conditions. These results point to the fact that native cyanobacteria’s growth rates could be also further improved if their optimal growth conditions were determined. Furthermore, if our native Synechocystis sp. is compared with other species studied for application in CO2 biofixation
systems, it exceeds the growth rates for Chlorellae NTU H-15 and H-25 reported by Chang and Yang,[3] both having μmax = 0.312 l/d under their optimal conditions or the Spirulina sp. in de Morais and Vieria results,[29] showing a μmax of 0.336 l/d. Maximum biomass productivity values (Pmax ) are also given in Table 1 for the different strains. No differences of statistical significance were found in maximum biomass production, which ranged from 0.346 to 0.475 g of biomass per day and litre of culture. One noteworthy difference was the time during culturing when the maximum productivity was attained: this was on day 7 for Synechocystis sp., day 6 for Chlorogonium sp. and C. sorokiniana, and day 2 for Scenedesmus sp. This last strain showed its maximum productivity when light was not yet limited since light started to be limited from day 4. However, Synechocystis sp. showed a relative good capacity to use light in dense cultures, which is a key for sustainable commercial systems. Values of maximum CO2 biofixation (Fmax ), which were calculated from maximum biomass productivity (Pmax ) and biomass carbon content (Equation (5)) in each strain (Table 1), may be seen in Table 1. Since biomass carbon content was around 50% in all strains, it does not constitute a key parameter in the selection of the best strain for CO2 biofixation. Additionally, as can be noted in Table 1, there were no significant differences in the results for Fmax . The accumulated parameters for each strain obtained from experimental batch cultures are presented in Figure 3. The accumulated cell density and Cb allow a comparison of each strain’s behaviour in an overall way, not solely in terms of maximum values. According to these graphs, there are significant differences for accumulated cell density but not for accumulated Cb. This means that by the end of batch cultures all the strains had produced similar amounts of biomass, but Synechocystis sp. and C. sorokiniana had to divide more times than Scenedesmus sp. and Chlorogonium sp. to generate this amount of biomass. A comparison of the outcomes for each strain studied showed that Synechocystis sp. clearly had the best results in terms of the number of cells generated during the period of culture. However, its accumulated Cb, maximum productivity and CO2 biofixation parameters were no better than the other strains under study. This, for example, is due to the smaller size of Synechocystis
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Figure 3. Accumulated cell density (a) and Cb (b) during the seven-day culturing of batches. Different letters for the same parameter indicate statistically different values (p < .05) while equal letters indicate not statistically significant values (p > .05).
sp. cells, which contain smaller amounts of biomass per cell than Scenedesmus sp. Cells. To sum up, according to the results of the present study, although Synechocystis sp. has achieved higher growth rates, cyanobacterium did not fix more carbon dioxide than Scenedesmus sp., Chlorogonium sp. or C. sorokiniana, so all of them could be equally suitable for application in CO2 biofixation systems. Then, a priori, it would seem that results for growth parameters and the analysis of CO2 biofixation were not conclusive for selecting the strain best at CO2 biofixation. Hence, other practical characteristics should be analysed in order to decide the final selection. In the first place, Scenedesmus sp. and Chlorogonium sp. cell sizes are similar, ranging from 5 to 10 μm and their floatability in repose was seen during this study to be worse than what was observed for the cyanobacterium Synechocystis sp. This last strain presented a very considerable resistance to sedimentation. This implies that Synechocystis sp. requires less mixing energy in order to maintain a homogeneous suspension, an essential condition for the appropriate availability of nutrients and light to cells. Furthermore, Synechocystis sp. reached its maximum CO2 biofixation of 0.953 g CO2 /l/d on day 7 of batch culture (Table 1), when the light availability within the culture was at its lowest. Meanwhile, the remaining strains had a considerably lower CO2 biofixation rate compared to their maximum during the last day of culturing: this was 26% lower for C. sorokiniana, 49% for Chlorogonium sp. and 63% for Scenedesmus sp. The fact that Synechocystis sp. developed maximum CO2 biofixation at the end of the culturing period, when light availability was at its lowest, means that this native
cyanobacterium is able to better use light when this is limited than the other strains studied. The explanation for this may be related to its spherical shape and small cell size, giving a larger surface for light reception with the same Cb, as compared to other non-spherical and bigger cells.[30] Synechocystis sp. proved to be able to fix the maximum amount of CO2 when it had reached a dense culture with a Cb from 1.34 to 1.76 g/l (Figure 2(b)). It is important to grow microalgae in high density cultures since high cell density at high productivity is favourable to reduce harvesting costs. In this sense, if culture systems are photobioreactors, Cbs exceeding 1 g/l are usually required.[9, 31,32] From the previous discussion, it may be grasped that quality and practical characteristics, such as better light use and better productivity at high culture densities, permitted a final selection. In terms of the research project’s requirements, C. sorokiniana was excluded by not being a native and local strain. Of the three native strains, comparison of those which showed similar maximum growth and CO2 biofixation parameters, Synechocystis sp. and Scenedesmus sp., Synechocystis sp. is more strongly recommended for application in CO2 biofixation systems by reason of presenting maximum productivity at the highest densities. Acknowledgements The authors thank Ingeniería Idom Internacional S.A. (Spain) and Junta de Castilla y León (project LE017A09) for financially supporting this work. The authors also express their thanks to Ms María Cuaresma, PhD student, of the University of Huelva in Spain for providing them with C. sorokiniana samples. Also,
Environmental Technology Marta Otero acknowledges the support from the Spanish Ministry of Science and Innovation (RYC-2010-05634). [16]
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