Utilization of Carbon Dioxide from Coal-Firing Flue Gas for Cultivation of Spirulina platensis

CO2 emission from burning coal has been used as a carbon source for growing Cyanobacterium Spirulina platensis in order to minimize the cost of biomass production, and currently to carry out CO2 bioremediation. This article presents the results of feeding S. platensis in laboratory conditions with 2 formulas including Pure CO2 and Flue gas CO2 upon using modified Zarrouk’s medium with 1.6 g / L NaHCO3 and 2g / L Na2CO3. Pure CO2 with 1.2% concentrations taken from 99% vol of industrial CO2 and CO2 gas (1.2%) received from the flue gas through the Modular system of Exhausted Gas Treatment (MEGT). Growth of the Cyanobacterium using CO2 Flue gas is equivalent to CO2 -Pure. On this basis, S. platensis has been cultivated outdoor in an 25 m 2 pond using CO2 gas (1.2%) from the tunnel brick factory emissions after suitable cleaning. The experiment in an outdoor pond system of 25 m 2 indicated that the yield of biomass is of 10g/m 2 d with high-protein content (62.58 ± 2.34%) and fatty acids of high nutritional value (8.72 ± 0.14%), such as Omega 6 and Omega 3 reaching 14.74 ± 0.42% and 26.05 ± 0.64% of total fatty acid content, respectively. The quality of Spirulina cultured by CO2 gas meets the requirements for functional foods according to Vietnam national food standards. The article also presents the results of biomass productivity and chemical composition of the Cyanobacterium in different culture conditions.


Introduction
CO 2 -anthropogenic carbon dioxide represents the most important greenhouse gases (GHGs) that contribute to approximately 77% of the global atmospheric temperature increase [1,2]. The increase of CO 2 concentration in the atmosphere, mainly due to burning fossil fuels like coal, oil, gas and the forest destroying raises the deep concern about the climate change, and thereby, induces great challenges to the global sustainable development [3,4].
Burning coal generates more carbon dioxide than any other widely used fuel, including burning oil and gas that may harm the environment. Due to a rapid increase of the demand for the fossil fuels, there is a need for developing methods that allow continuous use of them through environmental friendly pathway along with reducing carbon dioxide emissions. In fact, there have been many efforts to reduce CO 2 emissions from burning fossil fuels. Overall, the current methods are focusing on CO 2 separation from emission sources and then trying to remove or capture it [5]. Some other technologies such as chemical absorption and membrane separation, were also considered [6]. However, these methods can significantly reduce CO 2 concentration, they can not solve the problem of sustainable development [7]. Nowadays, in order to meet the demand for sustainable industrial development, it is highly desirable that exhaust emissions are treated thoroughly and sustainably through CO 2 recovery for use in photosynthesis. The recovery of CO 2 for microalgae culture is a novel pathway that has been studied and delivered in the reports. Lopes et al., 2008 indicated that as much as 40% of the carbon dioxide on the Earth can be absorbed by photosynthesis, in which microalgae or cyanobacteria make a great contribution with high species diversity and wide distribution in the ecological system [8]. So, photosynthesis by microalgae is an effective way to utilize CO 2 sources [9].
The selected culture strains have a considerable impact on biological fixation of CO 2 by level of temperature, SO x , NO x and CO 2 from flue gas [10]. Richmond mentioned that, cellular contents of Spirulina platensis were not changed by varying environmental conditions, compared with eukaryotic microalgae [11]. This alga is an excellent candidate for producing single cell protein due to its high protein content and nutritional value. Study on S. platensis due to potential of biomass production under high CO 2 concentration in flue gas is a good solution for CO 2 biofixation and for decreasing atmospheric CO 2 [12,13].
Our objective is to assess the possibility of using CO 2 from coal combustion emissions for the growth of Spirulina platensis.

CO 2 Source
Pure CO 2 with 1.2% concentrations taken from 99% vol of industrial CO 2 . CO 2 received from the flue gas through the Modular system of Exhausted Gas Treatment (MEGT) described is the page [14].

Cyanobacterium and Cultivation Medium
Spirulina platensis strain, classified as Arthrospira (Spirulina) platensis used for the experiments, was supplied from the Collection of Microalgae and Cyanobacteria of Institute of Environmental Technology, Vietnam Academy of Science and Technology.
Culture medium: The medium for the microalgal growth is Zarrouk's medium modified by reducing NaHCO 3 to 1,36 g/L and by adding Na 2 CO 3 to 2g/L [15].

Experimental Design
Laboratory Cultivation: Spirulina platensis cyanobacteria is cultivated in glass columns with a volume of 1 liter (inner diameter of 60 mm, height of 412 mm) which are maintained at a temperature of 27-32°C and illuminated by cold fluorescent light with intensity of 5,000 lux, and lighting time of 8 hours/day ( Fig. 1.a). The liquid columns of Cyanobacterium are continuously bubbled with CO 2 -Flue gas (1.2 vol% CO 2 ) or CO 2 -Pure as control experiments (1.2 vol.% CO 2 ), at a rate of 50 L/min regulated by various valves. The pH of the medium is continuously controlled over time by pH equipment. Distilled water is added daily to eliminate evaporation effects during incubation. Spirulina platensis pond parameters have an area of 25 m 2 with culture depth of 0.25 m. This pond used 1.2% CO 2 from coal-fired flue gas which was cleaned via MEGT [14]. The experiments were carried out in 5 months ( Fig. 1.b). The conditions for the Cyanobacterium culture: the average outdoor light intensity was about 25,000 lux, the temperature was in the range of 27 -32°C.
The pH of the suspension is maintained at 8.5 -9.5 and water is also added daily to eliminate evaporation effects. The pond was aerated by the paddle wheel system [16] in order to maintain moving speed of the suspension of about 18 cms -1 . The samples containing the Spirulina suspension were collected every two days for OD measurement at wavelength of 445nm using spectrophotometer UV-Vis 2450, Shimadzu, Japan. Each month, the fresh biomass of the Spirulina was collected for quality analysis.

Sampling and Analysis
Samples were collected for biomass growth analysis (OD) and biomass quality analysis (lipids, fatty acids, total protein, fiber (%), carbohydrates, polysaccharide, ash, moisture and some important elements).
Lipids and fatty acids were analyzed according to the methods of Bligh and Dyer 1959 [17]. Total protein was determined by Kjeldahl method, multiplying by 6.25. Fiber, carbohydrates, ash, moisture were determined by the method of analysis AOAC 2000 [18]. Arsenic, Cd, Pb and Hg concentrations in Spirulina samples were measured using a Atomic Absorption Spectroscopy AA-6800, Shimadzu, Japan [19].

Data Analysis
All the data in mean and standard deviation were performed using Microsoft excel for Windows.

Growth of S. platensis in Two Formulas: Pure -CO 2 and Flue Gas -CO 2 at the Laboratory Scale
In the growth process, Spirulina platensis can use inorganic C sources under forms of CO 2 , NaHCO 3 or Na 2 CO 3 but the primary and most appropriate source is still HCO 3 - [13]. The supplementation of CO 2 to the algae culture medium does not only provide C source but also control pH of the suspension. Fig. 2 presents the results of Spirulina platensis growth rate in 2 different formulas at the laboratory scale. After 21 days of experiment, Spirulina platensis increased biomass both in the 2 experimental formulas. Difference in Spirulina platensis biomass between two formulas (Pure CO 2 and FG CO 2 ) is negligible. However, in the last week of the experiment, the growth of Spirulina platensis in FG-CO 2 formula was slightly better than in Pure-CO 2 formula. The achieved results may be explained that there is a small amount of NO x as nutrient for algae besides CO 2 in the coal burning emissions [20].
In addition to assessing efficiency of the above 2 sources of CO 2 on the growth of S. platensis, we also analyzed the nutritional composition of the biomass of this Cyanobacterium (Table 1). The research results presented in Table 1 show that there is no difference in chemical composition of the biomass between two formulas and the use of CO 2 from coal-fired emissions for Spirulina platensis cultivation has been proved to be advantageous, and could be applied in large scale.

Growth and Productivity of Spirulina platensis in Culture Conditions at Dan Phuong, Hanoi
Optical density (OD) measurement for Spirulina platensis growth was applied in our study. The OD 445nm and dry biomass of the Spirulina platensis were determined every two days. The results in Fig. 3 demonstrated the variation of OD 445nm of the culture suspension. On the first two days S. platensis grew slowly, the OD 445nm value increased from 0.21 to 0.35. After ten days, S. platensis grew rapidly from 0.34 -0.35 to 1.09 -1.11. The highest level of OD 445nm in this experimental process reached up to 1.67 -1.73 when the biomass harvest was performed for maintaining the algal OD relatively constant.

Fig. 3. Spirulina platensis' growth at outdoor conditions.
Carbon dioxide is well adsorbed inside the S. platensis culture medium with pH> 8.5. During the photosynthesis, alkaline medium is normally created through the metabolic processes by phototrophic microorganisms participating in the transport of hydroxide ion (OH -) outwards its cell through catalytic reaction by carbohydrate anhydrase. As a result, the medium with phototrophic organisms as Spirulina platensis displays a strong alkaline property that helps them adsorb CO 2 with high efficiency [21]. Therefore, there have been a lot of studies taking into account the microalgae using CO 2 for nutritive biomass production. Cheng et al., (2006) has cultured Chlorella vulgaris in photobioreactor presenting that its growth rate is good in the medium with 1% CO 2 [22]. Aphanothece microscopica Nägeli (RSMan92) was cultured in tubular photobioreactors with different concentration of the carbon dioxide (3, 15, 25, 50 and 62%), light intensity (960, 3000, 6000, 9000 and 11000 lux), and temperature (21.5, 25, 30, 35 and 38.5°C) in order to determine the optimum condition, the highest CO 2 absorption processes for this microalgae strain [8]. In the study of Sydney et al. (2010), Botryococcus braunii presented the highest CO 2 fixation rate, followed by Spirulina platensis, Dunaliella tertiolecta, Chlorella vulgaris (as 496.98, 318.61, 272.4, and 251.64 mg l -1 d -1 , respectively) [23].
In this research, flue-gas emissions from coal combustion contained CO 2 with the amount around of 1.2%. Using such a gas source for the algae production pond with the aeration time of 6 to 8 hr d -1 made the pH of their medium not to raise sharply, and keeps the pH unchanged in the range of 8.5-9.6. Additionally, using a modified Zarrouk's medium (the content of bicarbonate reduced to 1.6 gl -1 NaHCO 3 and adding 2 gl -1 carbonate -Na 2 CO 3 ) in which CO 2 extracted from coal-fired gas gave good results in Spirulina growth during the 180-day experiment.
For assessing the growth and productivity of S. platensis using CO 2 in the outdoor conditions, the analysis of chemical composition in the Cyanobacterium biomass was also carried out ( Table 2 and 3). The obtained results presented in Table 2 indicated that S. platensis was rich in protein, reaching up to 62.69% dry weight while the lipid content did not exceed 9%. Moreover, the Spirulina also contained fatty acids having high nutritional value, such as Omega -6 and Omega -3 which reached 14.74% and 26.05% of total fatty acid content, respectively (Table 3). As shown in Fig. 3, during the experimental production of the Spirulina at the outdoor conditions, the Cyanobacterium was harvested when OD 445 values achieved about 1.6 with the productivity of about 10g/m 2 d. This showed that the using CO 2 from coal-fired flue gas for culturing Spirulina platensis is feasible for mass culture.
The quality of Spirulina cultured by CO 2 gas from coal-fired flue gas of the Tuynel Brick Factory is good and equivalent to that of Siam Algae Company (SAC) assessed by Japan Food Researcher Laboratories [24]. Heavy metal concentrations, including Pb, Cd, As, Hg, and the others (  [25,26]. This is an important basis for using Spirulina as a nutritive food or functional food source for humans. In the context of global climate change, Spirulina platensis not only makes a positive contribution to reducing greenhouse gases -CO 2 but could be also good biomass for different purposes.

Conclusion
At the laboratory scale, biomass growth and quality of Spirulina platensis in 2 formulas (Pure CO 2 and Flue gas CO 2 ) are equivalent. The experiment in an outdoor pond system of 25 m 2 indicated that the yield of biomass is of 10g/m 2 d with high-protein content of 62.58 ± 2.34% and fatty acids of high nutritional value (8.72 ± 0.14%), such as Omega -6 and Omega -3 reaching 14.74% ± 0.42 and 26.05 ± 0.64% of total fatty acid content, respectively. The obtained results allowed evaluating the potential of using CO 2 from coal combustion emissions for S. platensis culture with cost effective way for carbon sources and also for environment protection.

Nomenclature
GHGs: Greenhouse Gases MEGT: Modular system of Exhausted Gas Treatment OD: Optical Density VNNTR: Viet Nam National technical regulation