Bioresource Technology 192 (2015) 321–327
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Chemical absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode with nutrient recycle Gabriel Martins da Rosa, Luiza Moraes, Bruna Barcelos Cardias, Michele da Rosa Andrade Zimmermann de Souza, Jorge Alberto Vieira Costa ⇑ Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil
h i g h l i g h t s Monoethanolamine and the medium recycle provided more growth cycles for Spirulina. Cell growth and CO2 fixation were improved by MEA addition. The Spirulina carbohydrates concentration doubled with monoethanolamine addition.
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 2 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: CO2 capture Monoethanolamine Microalgae Nutrient recycling
a b s t r a c t The chemical absorption of carbon dioxide (CO2) is a technique used for the mitigation of the greenhouse effect. However, this process consumes high amounts of energy to regenerate the absorbent and to separate the CO2. CO2 removal by microalgae can be obtained via the photosynthesis process. The objective of this study was to investigate the cultivation and the macromolecules production by Spirulina sp. LEB 18 with the addition of monoethanolamine (MEA) and CO2. In the cultivation with MEA, were obtained higher results of specific growth rate, biomass productivity, CO2 biofixation, CO2 use efficiency, and lower generation time. Besides this, the carbohydrate concentration obtained at the end of this assay was approximately 96.0% higher than the control assay. Therefore, Spirulina can be produced using medium recycle and the addition of MEA, thereby promoting the reduction of CO2 emissions and showing potential for areas that require higher concentrations of carbohydrates, such as in bioethanol production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The increase in carbon dioxide (CO2) concentration in the atmosphere, resulting from the burning of fossil fuels for energy production is the main cause of global warming and climate change (Basu et al., 2014). In the contemporary period, different strategies are employed in an attempt to reduce and stabilize CO2 concentrations. These strategies include the use of renewable energy, chemical processes, such as chemical absorption (Peng et al., 2012) and biological processes such as photosynthesis microalgae. Microalgae are highlighted due to their ability to capture CO2 and to convert it into oxygen and biomass. This biomass stands out due to its potential to provide renewable energy, ranging from biodiesel, bioethanol, biohydrogen, and biogas. Furthermore, these microorganisms have the ability to synthesize bioactive molecules, such as carotenoids and fatty acids, and other valuable organic ⇑ Corresponding author. E-mail address: [email protected] (J.A.V. Costa). http://dx.doi.org/10.1016/j.biortech.2015.05.020 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
compounds, which can be used in foods, pharmaceuticals, and cosmetics, as well as for composing biomaterials (Ferreira et al., 2013). The microalgae cultivation is similar to other microorganisms such as bacteria (Tebbani et al., 2014). Therefore, there are three main modes of operation employees: discontinuous (or batch), semicontinuous and continuous. The semicontinuous mode is often employed because, according to Ho et al. (2012), it can prevents a low rate of cell division during the early process stages, and it reduces the limitations in relation to the nutrients and light penetration during the later stages. However, as disadvantage, the addition excessive of new culture medium may cause an increase in the osmotic pressure of the medium, which affect the photosynthetic apparatus of microalgae. The increase in the osmotic pressure of the medium also decreases the CO2 solubility, which is governed by the thermodynamic equilibrium for the dissociation of carbonic acid (Green and Perry, 2007). It is therefore believed that the CO2 biofixation by microalgae could be improved if the amount of dissolved gas in the liquid was increased beyond the natural balance of the algal
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culture (Kim et al., 2013). One of the widely used CO2 capturing techniques for flue gas is chemical absorption using alkanolamine solutions. Monoethanolamine (MEA) is the most popular absorbent for this process (Muraleedharan et al., 2012). However, one of the major limitations to the implementation of the CO2 capture technology by amine is the high-energy consumption of the process (Moullec et al., 2014). The integration of chemical and biological processes has previously been investigated. Choi et al. (2012), Kim et al. (2013), Sun et al. (2014) evaluated the use of MEA in microalgae cultures of Scenedesmus genus to increase the CO2 fixation. However, the application of MEA to Spirulina cultivation was not reported. Spirulina has advantages over other microalgae, such as the ease of recovery of the liquid medium, due to the arrangement of trichomes (Tomaselli, 1997), their high nutritional value of biomass, the capacity for adaptation to outdoor systems and for scaling up (Morais et al., 2009), and CO2 biofixation from flue gas (Radmann et al., 2011). In this context, Spirulina cultivation in semicontinuous mode with nutrient recycle and the addition of a chemical absorbent can be an approach for reducing the cost of nutrients and for maximizing CO2 biofixation. Therefore, the current study aimed to evaluate the effect of the addition of monoethanolamine on the growth kinetics and biomass composition of Spirulina in semicontinuous cultivation with CO2 addition and nutrient recycle. 2. Methods 2.1. Microorganism and culture medium The microorganism used for cultivation was the cyanobacterium Spirulina sp. LEB 18 (Morais et al., 2008), which was obtained from the Culture Collection of the Laboratory of Biochemical Engineering at Federal University of Rio Grande (FURG). Zarrouk medium (Zarrouk, 1966) without a carbon source (NaHCO3) was used in the assays. 2.2. Maintenance of the inoculum Spirulina sp. LEB 18 was maintained using CO2 as the carbon source, which replaced the sodium bicarbonate from the Zarrouk medium. This was achieved by decanting the Spirulina inoculum, removing the supernatant (approximately 90% v v1) and recovering the pellet (approximately 10% v v1). The cell pellet was resuspended in Zarrouk medium without a carbon source and was subjected to the new carbon source (CO2) at a daily specific flow 1
1 rate of 0:12 mLCO2 mL1 during the light medium d , for 1 min h period.
2.3. Cultivation conditions The assays were performed in duplicate in 2.0 L vertical tubular photobioreactors with 1.8 L of working volume (Morais and Costa, 2007), in semicontinuous mode and fed with CO2. The chemical absorbent was monoethanolamine (MEA, C2H7NO). Assays with and without (control assay) the addition of MEA were performed. The assays were maintained at 30 °C in a growth chamber under a 12 h light/dark photoperiod. Illumination was provided by 40 W daylight-type fluorescent lamps which produced an illuminance of 41.6 lmolphotons m2 s1. Stirring was carried out by compressed air injection (Morais and Costa, 2007) with specific flow rate of 0.05 vvm (air volume per work volume per min). The assays were carried out for 25 d and initial cellular concentration of 0.20 g L1. The CO2 was added 2 min h1 in the light period 1
ð0:36 mLCO2 mLmedium d Þ. For the CO2 supply occur the stirring
of the cultivation was stopped, 1 min before and 1 min after the CO2 addition, in order to increase the residence time of the carbon source in the liquid medium. The blend concentration was 0.5 g L1 (Reichert et al., 2006), and the volumetric fraction of the medium recycle was 0.5. For each medium recycle, 0.20 mmol L1 of MEA was added. 2.4. Analytical determinations The biomass concentration, pH and alkalinity were monitored daily. When the biomass concentration reached the blend concentration (0.5 g L1), the samples were also taken for the same analyses. 2.4.1. Biomass concentration The biomass concentration was determined spectrophotometry using a standard curve of Spirulina sp. LEB 18. This curve was obtained by measuring the optical density of the Spirulina inoculum in a spectrophotometer (QUIMIS Q798DRM, Diadema – SP – Brazil), at 670 nm, by relating the optical density and dry weight biomass, as performed by Costa et al. (2002). 2.4.2. Alkalinity, pH and concentration of dissolved inorganic carbon The medium alkalinity was determined via potentiometric titration and pH via direct measurements using a digital pH meter (PH-221, Lutron – Taiwan) according to the official method (APHA, 1998). These determinations were also used to calculate the concentration of dissolved inorganic carbon (DIC), following the equations of equilibrium used by Brune and Novak (1981), Rubio et al. (1999). For this calculation, it is assumed that in equilibrium (Eq. (1)) the same all chemical species represented the dissolved total inorganic carbon. k1
k2
CO2ðaqÞ () H2 CO3 () HCO3 () CO2 3
ð1Þ
CO2 concentration in equilibrium is approximately 650 times greater than that of H2CO3. Thus, the sum of the concentrations of the two species is represented by CO⁄2. Eqs. (2) and (3) show the equilibrium constants in Eq. (1). The equilibrium constants values at 30 °C.
K1 ¼
½HCO3 ½Hþ ¼ 106:327 ½CO2
ð2Þ
K2 ¼
þ ½CO2 3 ½H ¼ 1010:29 ½HCO3
ð3Þ
The concentrations of each species (Eqs. (4)–(6)) can be calculated from the ionization fractions and the total concentration of inorganic carbon (TC).
½CO2 ¼ a0 TC
ð4Þ
½HCO3 ¼ a1 TC
ð5Þ
½CO2 3 ¼ a2 TC
ð6Þ
The ionization fractions (a0, a1 and a2) of each chemical species at equilibrium were obtained as a function of pH and ionization constants K1 and K2, as shown in Eqs. (7)–(9).
a0 ¼
a1 ¼
1 1þ
k1 Hþ
1þ
Hþ k1
þ
k1 k2
ð7Þ
þ 2
ðH Þ
1 þ Hk2þ
ð8Þ
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a2 ¼
1þ
1 ðH Þ
þ 2
k1 k2
þ
þ Hk2
ð9Þ
The total concentration of dissolved inorganic carbon (TC) is not measurable , but it can be replaced by the total alkalinity (Eq. (10)).
AlcC ¼ ½HCO3 þ 2½CO2 3 ¼ a1 TC þ 2a2 TC
AlcC a1 2a2
½HCO3 ¼ a1 ½CO2 3 ¼ a2
AlcC
a1 2a2 AlcC
a1 2a2
each growth cycle (Xfi, g L1), the initial biomass concentration of each growth cycle (Xii, g L1), and the culture volume from each blend process (VCi, L).
Gb ¼
X ½ðX f 1 X i1 ÞV C1 þ ðX f 2 X i2 ÞV C2 þ . . . þ ðX fn X in ÞV Cn
ð14Þ
ð10Þ
The concentration of each chemical species in the carbon balance is therefore obtained from Eqs. (11)–(13).
½CO2 ¼ a0
323
ð11Þ
ð12Þ
ð13Þ
Thus, the concentration of DIC is obtained after determining the concentration of each chemical species in equilibrium. 2.5. Cultivation responses From the cell growth outlines of Spirulina sp. LEB 18, the following parameters were determined for each growth cycle: volumetric biomass productivity (PX, mg L1 d1), maximum specific growth rate (lmax, d1), generation time (tg, d), CO2 biofixation rate (RCO2 , mg L1 d1), and CO2 use efficiency (ECO2 ,% w w1). The maximum productivity (Pmax), maximum CO2 biofixation rate (Rmax) and maximum CO2 use efficiency (Emax), were the maximum values each parameter among the cycles growth. Whereas the average value and the standard deviation (S.d.) were obtained through of every one maximum values of each growth cycles. 2.5.1. Biomass volumetric productivity The biomass volumetric productivity was obtained using PX = (Xt X0)/(t t0), where Xt is the biomass concentration (g L1) at time t (d), and X0 is the biomass concentration (g L1) at time t0 (d). 2.5.2. Maximum specific growth rate and generation time The maximum specific growth rate was calculated via linear regression applied to the logarithmic growth rate of each blend process to give the outline ln X (mg L1) versus t (d), where the slope was lmax. From this outline, the generation time (tg) or cell duplication was also calculated according to tg = ln (2)/lmax. 2.5.3. Carbon dioxide biofixation rate The CO2 biofixation rate (RCO2 , mg L1 d1) was calculated according to the following equation: RCO2 ¼ PX xcbm ðM CO2 =M C Þ, from the PX (mg L1 d1), xcbm (carbon fraction in the biomass determined by elemental analysis) and the molecular weights of carbon dioxide and carbon (M CO2 and MC, respectively). 2.5.4. Carbon dioxide use efficiency The CO2 use efficiency (ECO2 , % w w1) was calculated according _ 100, where RCO2 was the daily CO2 to ECO2 ¼ ðRCO2 V work =mÞ biofixation rate (mg L1 d1), Vwork was the useful working volume _ was the daily CO2 feed rate of the photobioreactor (L), and m (mg d1). 2.5.5. Generated biomass The biomass generated (Gb, g) at the end of the assays (Eq. (14)) was calculated according to the final biomass concentration of
2.6. Biomass recovery and characterization The total biomass from each experiment was recovered by centrifugation (Hitachi Himac CR-GIII, Tokyo – Japan) at 15,200g and 20 °C for 15 min, resuspended in distilled water and centrifuged again under the same conditions. This step was repeated once to improve the nutrient removal. Then, the biomass was concentrated to 50 mL in a sterile recipient, frozen at 80 °C, lyophilized, and stored at 20 °C until characterization. 2.6.1. Protein concentration The total proteins concentration of the Spirulina biomass was determined at each growth cycle and at the end of the assays by colorimetric method (Lowry et al., 1951), from thermal and alkaline pretreatment of the microalgal biomass. 2.6.2. Carbohydrate concentration The carbohydrate concentration in the microalgae biomass was determined via the Dubois phenol–sulfuric method using a standard glucose curve (Dubois et al., 1956). 2.6.3. Elemental analysis The lyophilized biomass obtained from each assays was used to determine the elemental concentrations of carbon and nitrogen using an Elemental Analyzer CHNS/Perkin Elmer 2400 Series using acetanilide as the standard. 2.6.4. Lipid content The lipid content was determined using the Folch method (Folch et al., 1957), which is based on the extraction of the nonpolar and polar lipids (at room temperature) using the solvents chloroform:methanol (2:1) and methanol:water (2:1), respectively. 2.7. Theoretical conversion of biomass carbohydrates to gasoline gallon equivalent (GGE) From the carbohydrates concentration present in Spirulina biomass (produced with and without MEA), it was calculated the conversion of this macromolecule in ethanol, expressed in gasoline gallon equivalent (GGE). For conversion of carbohydrates to ethanol was used based calculation 100 g of biomass, constant theoretical stoichiometric conversion of glucose to ethanol of 0.511, the ethanol formation efficiency of 70%, and equivalence of ethanol gallons to GGE of 1.5:1.0. 2.8. Statistical analysis The responses were assessed using analysis of variance followed by Tukey’s test at a 95% confidence level. 3. Results and discussion The growth cycles were characterized by the mean and standard deviation between the two replicates of the assay with MEA and the control (Fig. 1). The addition of MEA led to a 1-day in lag phase growth and eight growth cycles (Fig. 1a). These intervals were considered the exponential growth phase, all with a coefficient of determination (R2) great than 0.94. The control assay
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Fig. 1. The mean biomass concentration outlines of Spirulina sp. LEB 18 cultivated in semicontinuous mode with the addition of MEA (a) and the control sample (b).
presented five growth cycles (Fig. 1b). The first cycle was characterized by a lag phase during the first 3 days and a log phase within 4 days. The maximum cell multiplication was observed during the second, third and fourth growth cycles, with an R2 > 0.98. During the last growth cycle (5th), the log phase was observed during the first 4 days, while the biomass compensation phase occurred after the 20th day (Fig. 1b). The growth period can be affected by the physiological adaptation of the cells caused by changes in the nutrient conditions (Lee and Shen, 2004), such as medium recycle. The existence of a log phase in all of the growth cycles of the assays with MEA showed that the addition of 0.2 mmol L1 of chemical absorbent in each growth cycle combined with nutrient recycle might have been beneficial for the multiplication of the Spirulina cells. However, in both of them conditions of this study, with or without the addition of the chemical absorbent, an excessive deposition of organic matter on the inner side surface of the bioreactors was observed. This deposition may have inhibited the light availability for the microalgae growth due to the shielding property of this organic material (Chen et al., 2013), instead enabling the development of opportunistic organisms. The BG-11 medium (Rippka et al., 1979) without a carbon source and instead containing MEA was previously shown to be capable of converting higher CO2 concentrations to dissolved inorganic carbon (DIC) than the regular BG-11 medium (Choi et al., 2012; Kim et al., 2013). However, the conditions of the present study for the cultivation of Spirulina using Zarrouk showed no difference between the assay containing MEA and the control assay, as the initial and final DIC values were similar (62.4 ± 0.01 and 62.0 ± 0.03 mg L1, 178.4 ± 4.9 and 185.2 ± 0.8 mg L1, respectively). The solubility of the carbon dioxide in the liquid medium decreases with increasing temperature and salt concentrations (Green and Perry, 2007). Zarrouk medium contains a higher concentration of inorganic salts than the BG-11 medium. Therefore, Zarrouk medium offers an higher osmotic pressure than BG-11, limiting gas dissolution even in the presence of a CO2 chemical absorbent. Furthermore, Choi et al. (2012), Kim et al. (2013), in assays containing BG-11 medium, employed concentration of MEA (4.92 mmol L1) that were three times higher than the concentration used in the current work (1.60 mmol L1, in the end of assay with MEA) with Zarrouk medium. The pH values of both of them assays with the addition of MEA and the control were similar throughout the cultivation of the Spirulina in semicontinuous mode, which ranged from 7.1 ± 0.1 to 8.9 ± 0.0 and from 6.9 ± 0.0 to 8.8 ± 0.1 for the assays using the chemical absorbent and for the control assay, respectively.
Table 1 The results of the maximum specific growth rate (lmax), the generation time (tg) and the maximum biomass productivity (Pmax) of the Spirulina cultivation in semicontinuous mode with or without (control) the addition of MEA. Assay with MEA Cycle
lmax (d1)
tg (d)
Pmax (mg L1 d1)
1 2 3 4 5 6 7 8
0.206 ± 0.004 0.240 ± 0.001 0.324 ± 0.008 0.291 ± 0.016 0.256 ± 0.004 0.250 ± 0.005 0.182 ± 0.008 0.190 ± 0.020
3.37 ± 0.07 2.89 ± 0.01 2.14 ± 0.05 2.39 ± 0.13 2.71 ± 0.05 2.77 ± 0.06 3.80 ± 0.16 3.68 ± 0.40
62.1 ± 2.6 41.2 ± 3.3 29.5 ± 1.4 23.3 ± 0.4 19.5 ± 1.0 16.3 ± 0.6 13.6 ± 0.8 5.6 ± 0.9
Maximum Minimum Mean S.d.*
0.324 0.182 0.242a 0.049
Cycle
lmax (d1)
tg (d)
Pmax (mg L1 d1)
1 2 3 4 5
0.191 ± 0.002 0.297 ± 0.009 0.285 ± 0.001 0.248 ± 0.027 0.192 ± 0.025
3.63 ± 0.03 2.34 ± 0.07 2.44 ± 0.01 2.82 ± 0.30 3.62 ± 0.48
44.0 ± 2.5 37.2 ± 0.8 27.8 ± 0.4 21.5 ± 2.0 14.8 ± 0.3
Maximum Minimum Mean S.d.*
0.297 0.191 0.242a 0.050
3.64 2.43 2.97a 0.63
44.0 14.8 29.1b 1.0
3.80 2.14 2.97a 0.60 Assay control
62.1 5.6 26.3a 0.9
The same superscript letters in the same column for the same responses indicate that the averages were not significantly different at the 95% confidence level (p > 0.05). * S.d.: Standard deviation.
The cell growth did not increase proportionally with the increase in the DIC, even after the addition of MEA to each growth cycle. Carbon availability for the microalgae absorption was sufficient, given that the concentration of elemental carbon in biomass has remained constant during the assays (Fig. 3a). Thus, CO2 dissolution was not a limiting factor for the biofixation of the carbon dioxide by Spirulina. Therefore, the mean increase of 31.4% for biomass generated by the assay using MEA (3.35 ± 0.25 g) when compared to the control (2.55 ± 0.11 g) was due to the larger number of growth cycles. The mean values observed for the growth parameters (lmax and tg) with and without the addition of MEA did not differ significantly at the 95.0% confidence level (Table 1). The productivity of the biomass Spirulina presented a similar declining behavior during the growth cycles in both of them
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Fig. 2. The outlines of maximum CO2 biofixation rate (Rmax) (a) and maximum CO2 use efficiency (Emax) (b) obtained in the assay with Spirulina sp. LEB 18 cultivated with addition of MEA (d) and the control assay (s).
Fig. 3. The mean outlines of the carbon, nitrogen (a), protein and carbohydrate (b) concentrations determined in the Spirulina biomass with the addition of MEA and in the control assay.
Table 2 The mean protein, carbohydrate and lipid concentrations of the final dry biomass in the last growth cycle for each condition. Condition
Proteins (% w w1)
Carbohydrates (% w w1)
Lipids (% w w1)
With MEA Control
44.4 ± 6.9a 60.8 ± 4.0b
28.2 ± 3.7a 14.4 ± 1.4b
8.3 ± 1.4a 10.0 ± 1.2a
The same superscript letters in the same column indicate that the averages were not significantly different at the 95% confidence level (p > 0.05).
conditions (Table 1), with mean values that were significantly higher in the control assay (p < 0.001). However, the maximum lmax value and the minimum tg value were observed in the assay with the addition of MEA, which were 13.3% higher and 11.9% lower, respectively, than the values found in the assay without the CO2 absorbent. Reichert et al. (2006) studied the cultivation of the Spirulina sp. Paracas strain in semicontinuous mode, with medium renewal, and found a lower lmax (0.101 ± 0.011 d1) and Pmax (37.0 ± 4.6 mg L1 d1) than the values observed in the current study. This emphasizes that the use of new culture medium may not provide higher growth rates and biomass productivity because the rate of nutrient uptake by the microalgae is limited by its biochemical cell demand.
The maximums of CO2 biofixation rate (Rmax) and CO2 use efficiency (Emax) are important parameters used to evaluate the potential for removal of this greenhouse gas. The Rmax obtained in assay with MEA addition was higher than that obtained in the control assay throughout the experiment time (Fig. 2a). This behavior was the same found to Emax (Fig. 2b), reaching maximum values of 15.8% and 11.1% for assay with MEA and control assay, respectively. In cultures carried out by Radmann et al. (2011) with 1
Spirulina sp. LEB 18 and 3:24 mLCO2 fed mLmeio d , Rmax ¼ 150 1
1
mgCO2 fixed L d was found. Thus, considering the conversion of fed CO2 to the fixed CO2, Radmann et al. (2011) (150/3.24) has a value of 46:3 mgCO2 fixed mg1 CO2 fed , whereas the conversion with MEA in this study (104.0/0.36) it was 6.2 times higher ð289 mgCO2 fixed mg1 CO2 fed Þ. The protein concentration in the Spirulina biomass produced with MEA was higher that the control assay on 1st and 2nd growth cycles, and equal to protein content until the final cycles without addition of MEA (5th cycle) (Fig. 3b). With respect to the biomass produced using MEA, was observed reduction in the protein concentration among third and the eighth cycle (Fig. 3b). The final protein content (44.4 ± 6.9% w w1) in the biomass cultivated using MEA (Table 2) was lower than that normally found in Spirulina sp. LEB 18 biomass (60–70% w w1), as reported by Borges et al. (2013). Nonetheless, the control assay (60.8 ± 4.0% w w1) remained within this protein concentration range.
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Microalgae under conditions of nutrient stress tend to accumulate compounds such as carbohydrates and lipids (Tanzi et al., 2013). The carbohydrate concentration outline throughout the growth cycles (Fig. 3b) in the assays using MEA evidenced that the nutrient recycle proposed in this study may have deprived the Spirulina of at nitrogen source in the culture medium, prioritizing its metabolic route to the formation of reserve compounds. This hypothesis was evidenced by the final carbohydrate concentration in the biomass produced using MEA (8th cycle), which was higher than that observed in the assay without MEA (5th cycle) (Table 2). Following this idea Klok et al. (2013), who found that nitrogen depletion during the cultivation of microalgae leads to the wasting of light by the cell due to energy dissipation as heat and light in the form of fluorescence. Therefore, microalgae can lose efficiency during the photosynthetic process, leading to a decrease in the biomass productivity (Table 1) and nitrogen compounds, such as proteins (Fig. 3b and Table 2). The lipid content of the biomass produced with MEA (8.3 ± 1.4% w w1) and without MEA (10.0 ± 1.2% w w1) was higher than that found by Borges et al. (2013) (5.0% w w1) and Morais et al. (2009) (3.3% w w1) using the same strain as the present study. These authors cultivated Spirulina in open reactors that, according to Chisti (2007), may lead to lower biomass productivity, but this cannot explain the low lipid levels in relation to the values found in the present experiments. Therefore, as was observed with the carbohydrate concentration, the Spirulina may have increased the lipid concentration due to the scarcity or loss of certain nutrients, due to the medium recycle in both of them of the studied conditions. The results found for the three macromolecules in the Spirulina biomass indicate that the addition of MEA to each growth cycle did not exert a significant effect on the lipid concentration (p = 0.1140), it significantly increased the carbohydrate content (p = 0.00007) and decreased the protein content (p = 0.00007). According to Hu (2004), the effect of nutritional deficiency of phosphorus and nitrogen is evidenced with lower chlorophyll concentrations and higher carbohydrate content in biomass. Subsequently, it is believed that the a reduction in chlorophyll is related to lower protein content because, according to Masojídek et al. (2004), the green pigments are lipophilic compounds that are associated with protein complexes. In this scenario, it is believed that there was a synergistic effect of nutrients recycle with the addition of monoethanolamine on the concentrations of protein and carbohydrate, i.e., biomass with higher carbohydrates concentration and lower protein content. Due to growing demand for biofuels, microalgae are gaining wide attention as an alternative renewable source of biomass for production of bioethanol, which is grouped under ‘‘Third generation biofuels’’ (Nigam and Singh, 2011). In this perspective, it was observed that the carbohydrates content in 100 g of Spirulina sp. LEB 18 biomass, cultivated with MEA, it has the potential of producing of 12.8 mL of ethanol and 0.0023 GGE. Whereas the amount of carbohydrates in 100 g of biomass produced in the control assay has potential of producing of 6.6 mL of ethanol and 0.0012 GGE.
4. Conclusions The addition of monoethanolamine (MEA) and medium recycle has promoted more growth cycles and 31.4% more biomass generated. This addition of MEA also led to higher results of maximum growth parameters and CO2 biofixation by Spirulina sp. LEB 18. Spirulina biomass cultivated with MEA has shown a protein concentration lower than the biomass produced on control assay, but the carbohydrate content was almost 96.0% higher. In addition,
has verified that the Spirulina biomass, in the conditions used, have potential for bioprocesses that require higher concentrations of carbohydrates, such as bioethanol production. Acknowledgements The authors thank Eletrobras-CGTEE (Centrais Elétricas Brasileiras S.A.-Companhia de Geração Térmica de Energia Elétrica) for the financial support conduct to this work. References APHA, 1998. American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA-AWWAWPCF, Washington. Basu, S., Roy, A.S., Mohanty, K., Ghosha, A.K., 2014. CO2 biofixation and carbonic anhydrase activity in Scenedesmus obliquus SA1 cultivated in large scale open system. Bioresour. Technol. 164, 323–330. Borges, J.A., Rosa, G.M., Meza, L.H.R., Henrard, A.A., Souza, M.R.A.Z., Costa, J.A.V., 2013. Spirulina sp. LEB-18 culture using effluent from the anaerobic digestion. Braz. J. Chem. Eng. 30 (2), 277–287. Brune, D.E., Novak, J.T., 1981. 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Blackwell Publishing Ltd., Oxford, pp. 83–94. Kim, G., Choi, W., Lee, C.H., Lee, K., 2013. Enhancement of dissolved inorganic carbon and carbon fixation by green alga Scenedesmus sp in the presence of alkanolamine CO2 absorbents. Biochem. Eng. J. 78, 18–23. Klok, A.J., Martens, D.E., Wijffels, R.H., Lamers, P.P., 2013. Simultaneous growth and neutral lipid accumulation in microalgae. Bioresour. Technol. 134, 233–243. Lee, Y.-K., Shen, H., 2004. Basic culturing techniques. In: Richmond, A. (Ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd., Oxford, pp. 40–56. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 (1), 265–275. Masojídek, J., Koblízek, M., Torzillo, G., 2004. Photosynthesis in Microalgae. In: Richmond, A. (Ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd., Oxford, pp. 20–39. Morais, M.G., Costa, J.A., 2007. Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors. Biotechnol. Lett. 29 (9), 1349–1352. Morais, M.G., Reichert, C.C., Dalcanton, F., Durante, A.J., Marins, L.F., Costa, J.A.V., 2008. Isolation and characterization of a new Arthrospira strain. Z. Naturforsch. C. 63, 6. Morais, M.G., Radmann, E.M., Andrade, M.R., Teixeira, G.G., Brusch, L.R.F., Costa, J.A.V., 2009. Pilot scale semicontinuous production of Spirulina biomass in southern Brazil. Aquaculture 294 (1–2), 60–64. Moullec, Y.L., Neveuxa, T., Azkib, A.A., Chikukwac, A., Hoff, K.A., 2014. Process modifications for solvent-based post-combustion CO2 capture. Int. J. Greenh. Gas Control. 31, 96–112. Muraleedharan, R., Mondal, A., Mandal, B., 2012. Absorption of carbon dioxide into aqueous blends of 2-amino-2-hydroxymethyl-1,3-propanediol and monoethanolamine. Sep. Purif. Technol. 94, 92–96. 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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Chemical absorption and CO2 biofixation via the cultivation of Spirulina in semicontinuous mode with nutrient recycle Gabriel Martins da Rosa, Luiza Moraes, Bruna Barcelos Cardias, Michele da Rosa Andrade Zimmermann de Souza, Jorge Alberto Vieira Costa ⇑ Laboratory of Biochemical Engineering, College of Chemistry and Food Engineering, Federal University of Rio Grande, Rio Grande, RS, Brazil
h i g h l i g h t s Monoethanolamine and the medium recycle provided more growth cycles for Spirulina. Cell growth and CO2 fixation were improved by MEA addition. The Spirulina carbohydrates concentration doubled with monoethanolamine addition.
a r t i c l e
i n f o
Article history: Received 26 February 2015 Received in revised form 2 May 2015 Accepted 8 May 2015 Available online 14 May 2015 Keywords: CO2 capture Monoethanolamine Microalgae Nutrient recycling
a b s t r a c t The chemical absorption of carbon dioxide (CO2) is a technique used for the mitigation of the greenhouse effect. However, this process consumes high amounts of energy to regenerate the absorbent and to separate the CO2. CO2 removal by microalgae can be obtained via the photosynthesis process. The objective of this study was to investigate the cultivation and the macromolecules production by Spirulina sp. LEB 18 with the addition of monoethanolamine (MEA) and CO2. In the cultivation with MEA, were obtained higher results of specific growth rate, biomass productivity, CO2 biofixation, CO2 use efficiency, and lower generation time. Besides this, the carbohydrate concentration obtained at the end of this assay was approximately 96.0% higher than the control assay. Therefore, Spirulina can be produced using medium recycle and the addition of MEA, thereby promoting the reduction of CO2 emissions and showing potential for areas that require higher concentrations of carbohydrates, such as in bioethanol production. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The increase in carbon dioxide (CO2) concentration in the atmosphere, resulting from the burning of fossil fuels for energy production is the main cause of global warming and climate change (Basu et al., 2014). In the contemporary period, different strategies are employed in an attempt to reduce and stabilize CO2 concentrations. These strategies include the use of renewable energy, chemical processes, such as chemical absorption (Peng et al., 2012) and biological processes such as photosynthesis microalgae. Microalgae are highlighted due to their ability to capture CO2 and to convert it into oxygen and biomass. This biomass stands out due to its potential to provide renewable energy, ranging from biodiesel, bioethanol, biohydrogen, and biogas. Furthermore, these microorganisms have the ability to synthesize bioactive molecules, such as carotenoids and fatty acids, and other valuable organic ⇑ Corresponding author. E-mail address: [email protected] (J.A.V. Costa). http://dx.doi.org/10.1016/j.biortech.2015.05.020 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
compounds, which can be used in foods, pharmaceuticals, and cosmetics, as well as for composing biomaterials (Ferreira et al., 2013). The microalgae cultivation is similar to other microorganisms such as bacteria (Tebbani et al., 2014). Therefore, there are three main modes of operation employees: discontinuous (or batch), semicontinuous and continuous. The semicontinuous mode is often employed because, according to Ho et al. (2012), it can prevents a low rate of cell division during the early process stages, and it reduces the limitations in relation to the nutrients and light penetration during the later stages. However, as disadvantage, the addition excessive of new culture medium may cause an increase in the osmotic pressure of the medium, which affect the photosynthetic apparatus of microalgae. The increase in the osmotic pressure of the medium also decreases the CO2 solubility, which is governed by the thermodynamic equilibrium for the dissociation of carbonic acid (Green and Perry, 2007). It is therefore believed that the CO2 biofixation by microalgae could be improved if the amount of dissolved gas in the liquid was increased beyond the natural balance of the algal
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culture (Kim et al., 2013). One of the widely used CO2 capturing techniques for flue gas is chemical absorption using alkanolamine solutions. Monoethanolamine (MEA) is the most popular absorbent for this process (Muraleedharan et al., 2012). However, one of the major limitations to the implementation of the CO2 capture technology by amine is the high-energy consumption of the process (Moullec et al., 2014). The integration of chemical and biological processes has previously been investigated. Choi et al. (2012), Kim et al. (2013), Sun et al. (2014) evaluated the use of MEA in microalgae cultures of Scenedesmus genus to increase the CO2 fixation. However, the application of MEA to Spirulina cultivation was not reported. Spirulina has advantages over other microalgae, such as the ease of recovery of the liquid medium, due to the arrangement of trichomes (Tomaselli, 1997), their high nutritional value of biomass, the capacity for adaptation to outdoor systems and for scaling up (Morais et al., 2009), and CO2 biofixation from flue gas (Radmann et al., 2011). In this context, Spirulina cultivation in semicontinuous mode with nutrient recycle and the addition of a chemical absorbent can be an approach for reducing the cost of nutrients and for maximizing CO2 biofixation. Therefore, the current study aimed to evaluate the effect of the addition of monoethanolamine on the growth kinetics and biomass composition of Spirulina in semicontinuous cultivation with CO2 addition and nutrient recycle. 2. Methods 2.1. Microorganism and culture medium The microorganism used for cultivation was the cyanobacterium Spirulina sp. LEB 18 (Morais et al., 2008), which was obtained from the Culture Collection of the Laboratory of Biochemical Engineering at Federal University of Rio Grande (FURG). Zarrouk medium (Zarrouk, 1966) without a carbon source (NaHCO3) was used in the assays. 2.2. Maintenance of the inoculum Spirulina sp. LEB 18 was maintained using CO2 as the carbon source, which replaced the sodium bicarbonate from the Zarrouk medium. This was achieved by decanting the Spirulina inoculum, removing the supernatant (approximately 90% v v1) and recovering the pellet (approximately 10% v v1). The cell pellet was resuspended in Zarrouk medium without a carbon source and was subjected to the new carbon source (CO2) at a daily specific flow 1
1 rate of 0:12 mLCO2 mL1 during the light medium d , for 1 min h period.
2.3. Cultivation conditions The assays were performed in duplicate in 2.0 L vertical tubular photobioreactors with 1.8 L of working volume (Morais and Costa, 2007), in semicontinuous mode and fed with CO2. The chemical absorbent was monoethanolamine (MEA, C2H7NO). Assays with and without (control assay) the addition of MEA were performed. The assays were maintained at 30 °C in a growth chamber under a 12 h light/dark photoperiod. Illumination was provided by 40 W daylight-type fluorescent lamps which produced an illuminance of 41.6 lmolphotons m2 s1. Stirring was carried out by compressed air injection (Morais and Costa, 2007) with specific flow rate of 0.05 vvm (air volume per work volume per min). The assays were carried out for 25 d and initial cellular concentration of 0.20 g L1. The CO2 was added 2 min h1 in the light period 1
ð0:36 mLCO2 mLmedium d Þ. For the CO2 supply occur the stirring
of the cultivation was stopped, 1 min before and 1 min after the CO2 addition, in order to increase the residence time of the carbon source in the liquid medium. The blend concentration was 0.5 g L1 (Reichert et al., 2006), and the volumetric fraction of the medium recycle was 0.5. For each medium recycle, 0.20 mmol L1 of MEA was added. 2.4. Analytical determinations The biomass concentration, pH and alkalinity were monitored daily. When the biomass concentration reached the blend concentration (0.5 g L1), the samples were also taken for the same analyses. 2.4.1. Biomass concentration The biomass concentration was determined spectrophotometry using a standard curve of Spirulina sp. LEB 18. This curve was obtained by measuring the optical density of the Spirulina inoculum in a spectrophotometer (QUIMIS Q798DRM, Diadema – SP – Brazil), at 670 nm, by relating the optical density and dry weight biomass, as performed by Costa et al. (2002). 2.4.2. Alkalinity, pH and concentration of dissolved inorganic carbon The medium alkalinity was determined via potentiometric titration and pH via direct measurements using a digital pH meter (PH-221, Lutron – Taiwan) according to the official method (APHA, 1998). These determinations were also used to calculate the concentration of dissolved inorganic carbon (DIC), following the equations of equilibrium used by Brune and Novak (1981), Rubio et al. (1999). For this calculation, it is assumed that in equilibrium (Eq. (1)) the same all chemical species represented the dissolved total inorganic carbon. k1
k2
CO2ðaqÞ () H2 CO3 () HCO3 () CO2 3
ð1Þ
CO2 concentration in equilibrium is approximately 650 times greater than that of H2CO3. Thus, the sum of the concentrations of the two species is represented by CO⁄2. Eqs. (2) and (3) show the equilibrium constants in Eq. (1). The equilibrium constants values at 30 °C.
K1 ¼
½HCO3 ½Hþ ¼ 106:327 ½CO2
ð2Þ
K2 ¼
þ ½CO2 3 ½H ¼ 1010:29 ½HCO3
ð3Þ
The concentrations of each species (Eqs. (4)–(6)) can be calculated from the ionization fractions and the total concentration of inorganic carbon (TC).
½CO2 ¼ a0 TC
ð4Þ
½HCO3 ¼ a1 TC
ð5Þ
½CO2 3 ¼ a2 TC
ð6Þ
The ionization fractions (a0, a1 and a2) of each chemical species at equilibrium were obtained as a function of pH and ionization constants K1 and K2, as shown in Eqs. (7)–(9).
a0 ¼
a1 ¼
1 1þ
k1 Hþ
1þ
Hþ k1
þ
k1 k2
ð7Þ
þ 2
ðH Þ
1 þ Hk2þ
ð8Þ
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a2 ¼
1þ
1 ðH Þ
þ 2
k1 k2
þ
þ Hk2
ð9Þ
The total concentration of dissolved inorganic carbon (TC) is not measurable , but it can be replaced by the total alkalinity (Eq. (10)).
AlcC ¼ ½HCO3 þ 2½CO2 3 ¼ a1 TC þ 2a2 TC
AlcC a1 2a2
½HCO3 ¼ a1 ½CO2 3 ¼ a2
AlcC
a1 2a2 AlcC
a1 2a2
each growth cycle (Xfi, g L1), the initial biomass concentration of each growth cycle (Xii, g L1), and the culture volume from each blend process (VCi, L).
Gb ¼
X ½ðX f 1 X i1 ÞV C1 þ ðX f 2 X i2 ÞV C2 þ . . . þ ðX fn X in ÞV Cn
ð14Þ
ð10Þ
The concentration of each chemical species in the carbon balance is therefore obtained from Eqs. (11)–(13).
½CO2 ¼ a0
323
ð11Þ
ð12Þ
ð13Þ
Thus, the concentration of DIC is obtained after determining the concentration of each chemical species in equilibrium. 2.5. Cultivation responses From the cell growth outlines of Spirulina sp. LEB 18, the following parameters were determined for each growth cycle: volumetric biomass productivity (PX, mg L1 d1), maximum specific growth rate (lmax, d1), generation time (tg, d), CO2 biofixation rate (RCO2 , mg L1 d1), and CO2 use efficiency (ECO2 ,% w w1). The maximum productivity (Pmax), maximum CO2 biofixation rate (Rmax) and maximum CO2 use efficiency (Emax), were the maximum values each parameter among the cycles growth. Whereas the average value and the standard deviation (S.d.) were obtained through of every one maximum values of each growth cycles. 2.5.1. Biomass volumetric productivity The biomass volumetric productivity was obtained using PX = (Xt X0)/(t t0), where Xt is the biomass concentration (g L1) at time t (d), and X0 is the biomass concentration (g L1) at time t0 (d). 2.5.2. Maximum specific growth rate and generation time The maximum specific growth rate was calculated via linear regression applied to the logarithmic growth rate of each blend process to give the outline ln X (mg L1) versus t (d), where the slope was lmax. From this outline, the generation time (tg) or cell duplication was also calculated according to tg = ln (2)/lmax. 2.5.3. Carbon dioxide biofixation rate The CO2 biofixation rate (RCO2 , mg L1 d1) was calculated according to the following equation: RCO2 ¼ PX xcbm ðM CO2 =M C Þ, from the PX (mg L1 d1), xcbm (carbon fraction in the biomass determined by elemental analysis) and the molecular weights of carbon dioxide and carbon (M CO2 and MC, respectively). 2.5.4. Carbon dioxide use efficiency The CO2 use efficiency (ECO2 , % w w1) was calculated according _ 100, where RCO2 was the daily CO2 to ECO2 ¼ ðRCO2 V work =mÞ biofixation rate (mg L1 d1), Vwork was the useful working volume _ was the daily CO2 feed rate of the photobioreactor (L), and m (mg d1). 2.5.5. Generated biomass The biomass generated (Gb, g) at the end of the assays (Eq. (14)) was calculated according to the final biomass concentration of
2.6. Biomass recovery and characterization The total biomass from each experiment was recovered by centrifugation (Hitachi Himac CR-GIII, Tokyo – Japan) at 15,200g and 20 °C for 15 min, resuspended in distilled water and centrifuged again under the same conditions. This step was repeated once to improve the nutrient removal. Then, the biomass was concentrated to 50 mL in a sterile recipient, frozen at 80 °C, lyophilized, and stored at 20 °C until characterization. 2.6.1. Protein concentration The total proteins concentration of the Spirulina biomass was determined at each growth cycle and at the end of the assays by colorimetric method (Lowry et al., 1951), from thermal and alkaline pretreatment of the microalgal biomass. 2.6.2. Carbohydrate concentration The carbohydrate concentration in the microalgae biomass was determined via the Dubois phenol–sulfuric method using a standard glucose curve (Dubois et al., 1956). 2.6.3. Elemental analysis The lyophilized biomass obtained from each assays was used to determine the elemental concentrations of carbon and nitrogen using an Elemental Analyzer CHNS/Perkin Elmer 2400 Series using acetanilide as the standard. 2.6.4. Lipid content The lipid content was determined using the Folch method (Folch et al., 1957), which is based on the extraction of the nonpolar and polar lipids (at room temperature) using the solvents chloroform:methanol (2:1) and methanol:water (2:1), respectively. 2.7. Theoretical conversion of biomass carbohydrates to gasoline gallon equivalent (GGE) From the carbohydrates concentration present in Spirulina biomass (produced with and without MEA), it was calculated the conversion of this macromolecule in ethanol, expressed in gasoline gallon equivalent (GGE). For conversion of carbohydrates to ethanol was used based calculation 100 g of biomass, constant theoretical stoichiometric conversion of glucose to ethanol of 0.511, the ethanol formation efficiency of 70%, and equivalence of ethanol gallons to GGE of 1.5:1.0. 2.8. Statistical analysis The responses were assessed using analysis of variance followed by Tukey’s test at a 95% confidence level. 3. Results and discussion The growth cycles were characterized by the mean and standard deviation between the two replicates of the assay with MEA and the control (Fig. 1). The addition of MEA led to a 1-day in lag phase growth and eight growth cycles (Fig. 1a). These intervals were considered the exponential growth phase, all with a coefficient of determination (R2) great than 0.94. The control assay
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Fig. 1. The mean biomass concentration outlines of Spirulina sp. LEB 18 cultivated in semicontinuous mode with the addition of MEA (a) and the control sample (b).
presented five growth cycles (Fig. 1b). The first cycle was characterized by a lag phase during the first 3 days and a log phase within 4 days. The maximum cell multiplication was observed during the second, third and fourth growth cycles, with an R2 > 0.98. During the last growth cycle (5th), the log phase was observed during the first 4 days, while the biomass compensation phase occurred after the 20th day (Fig. 1b). The growth period can be affected by the physiological adaptation of the cells caused by changes in the nutrient conditions (Lee and Shen, 2004), such as medium recycle. The existence of a log phase in all of the growth cycles of the assays with MEA showed that the addition of 0.2 mmol L1 of chemical absorbent in each growth cycle combined with nutrient recycle might have been beneficial for the multiplication of the Spirulina cells. However, in both of them conditions of this study, with or without the addition of the chemical absorbent, an excessive deposition of organic matter on the inner side surface of the bioreactors was observed. This deposition may have inhibited the light availability for the microalgae growth due to the shielding property of this organic material (Chen et al., 2013), instead enabling the development of opportunistic organisms. The BG-11 medium (Rippka et al., 1979) without a carbon source and instead containing MEA was previously shown to be capable of converting higher CO2 concentrations to dissolved inorganic carbon (DIC) than the regular BG-11 medium (Choi et al., 2012; Kim et al., 2013). However, the conditions of the present study for the cultivation of Spirulina using Zarrouk showed no difference between the assay containing MEA and the control assay, as the initial and final DIC values were similar (62.4 ± 0.01 and 62.0 ± 0.03 mg L1, 178.4 ± 4.9 and 185.2 ± 0.8 mg L1, respectively). The solubility of the carbon dioxide in the liquid medium decreases with increasing temperature and salt concentrations (Green and Perry, 2007). Zarrouk medium contains a higher concentration of inorganic salts than the BG-11 medium. Therefore, Zarrouk medium offers an higher osmotic pressure than BG-11, limiting gas dissolution even in the presence of a CO2 chemical absorbent. Furthermore, Choi et al. (2012), Kim et al. (2013), in assays containing BG-11 medium, employed concentration of MEA (4.92 mmol L1) that were three times higher than the concentration used in the current work (1.60 mmol L1, in the end of assay with MEA) with Zarrouk medium. The pH values of both of them assays with the addition of MEA and the control were similar throughout the cultivation of the Spirulina in semicontinuous mode, which ranged from 7.1 ± 0.1 to 8.9 ± 0.0 and from 6.9 ± 0.0 to 8.8 ± 0.1 for the assays using the chemical absorbent and for the control assay, respectively.
Table 1 The results of the maximum specific growth rate (lmax), the generation time (tg) and the maximum biomass productivity (Pmax) of the Spirulina cultivation in semicontinuous mode with or without (control) the addition of MEA. Assay with MEA Cycle
lmax (d1)
tg (d)
Pmax (mg L1 d1)
1 2 3 4 5 6 7 8
0.206 ± 0.004 0.240 ± 0.001 0.324 ± 0.008 0.291 ± 0.016 0.256 ± 0.004 0.250 ± 0.005 0.182 ± 0.008 0.190 ± 0.020
3.37 ± 0.07 2.89 ± 0.01 2.14 ± 0.05 2.39 ± 0.13 2.71 ± 0.05 2.77 ± 0.06 3.80 ± 0.16 3.68 ± 0.40
62.1 ± 2.6 41.2 ± 3.3 29.5 ± 1.4 23.3 ± 0.4 19.5 ± 1.0 16.3 ± 0.6 13.6 ± 0.8 5.6 ± 0.9
Maximum Minimum Mean S.d.*
0.324 0.182 0.242a 0.049
Cycle
lmax (d1)
tg (d)
Pmax (mg L1 d1)
1 2 3 4 5
0.191 ± 0.002 0.297 ± 0.009 0.285 ± 0.001 0.248 ± 0.027 0.192 ± 0.025
3.63 ± 0.03 2.34 ± 0.07 2.44 ± 0.01 2.82 ± 0.30 3.62 ± 0.48
44.0 ± 2.5 37.2 ± 0.8 27.8 ± 0.4 21.5 ± 2.0 14.8 ± 0.3
Maximum Minimum Mean S.d.*
0.297 0.191 0.242a 0.050
3.64 2.43 2.97a 0.63
44.0 14.8 29.1b 1.0
3.80 2.14 2.97a 0.60 Assay control
62.1 5.6 26.3a 0.9
The same superscript letters in the same column for the same responses indicate that the averages were not significantly different at the 95% confidence level (p > 0.05). * S.d.: Standard deviation.
The cell growth did not increase proportionally with the increase in the DIC, even after the addition of MEA to each growth cycle. Carbon availability for the microalgae absorption was sufficient, given that the concentration of elemental carbon in biomass has remained constant during the assays (Fig. 3a). Thus, CO2 dissolution was not a limiting factor for the biofixation of the carbon dioxide by Spirulina. Therefore, the mean increase of 31.4% for biomass generated by the assay using MEA (3.35 ± 0.25 g) when compared to the control (2.55 ± 0.11 g) was due to the larger number of growth cycles. The mean values observed for the growth parameters (lmax and tg) with and without the addition of MEA did not differ significantly at the 95.0% confidence level (Table 1). The productivity of the biomass Spirulina presented a similar declining behavior during the growth cycles in both of them
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Fig. 2. The outlines of maximum CO2 biofixation rate (Rmax) (a) and maximum CO2 use efficiency (Emax) (b) obtained in the assay with Spirulina sp. LEB 18 cultivated with addition of MEA (d) and the control assay (s).
Fig. 3. The mean outlines of the carbon, nitrogen (a), protein and carbohydrate (b) concentrations determined in the Spirulina biomass with the addition of MEA and in the control assay.
Table 2 The mean protein, carbohydrate and lipid concentrations of the final dry biomass in the last growth cycle for each condition. Condition
Proteins (% w w1)
Carbohydrates (% w w1)
Lipids (% w w1)
With MEA Control
44.4 ± 6.9a 60.8 ± 4.0b
28.2 ± 3.7a 14.4 ± 1.4b
8.3 ± 1.4a 10.0 ± 1.2a
The same superscript letters in the same column indicate that the averages were not significantly different at the 95% confidence level (p > 0.05).
conditions (Table 1), with mean values that were significantly higher in the control assay (p < 0.001). However, the maximum lmax value and the minimum tg value were observed in the assay with the addition of MEA, which were 13.3% higher and 11.9% lower, respectively, than the values found in the assay without the CO2 absorbent. Reichert et al. (2006) studied the cultivation of the Spirulina sp. Paracas strain in semicontinuous mode, with medium renewal, and found a lower lmax (0.101 ± 0.011 d1) and Pmax (37.0 ± 4.6 mg L1 d1) than the values observed in the current study. This emphasizes that the use of new culture medium may not provide higher growth rates and biomass productivity because the rate of nutrient uptake by the microalgae is limited by its biochemical cell demand.
The maximums of CO2 biofixation rate (Rmax) and CO2 use efficiency (Emax) are important parameters used to evaluate the potential for removal of this greenhouse gas. The Rmax obtained in assay with MEA addition was higher than that obtained in the control assay throughout the experiment time (Fig. 2a). This behavior was the same found to Emax (Fig. 2b), reaching maximum values of 15.8% and 11.1% for assay with MEA and control assay, respectively. In cultures carried out by Radmann et al. (2011) with 1
Spirulina sp. LEB 18 and 3:24 mLCO2 fed mLmeio d , Rmax ¼ 150 1
1
mgCO2 fixed L d was found. Thus, considering the conversion of fed CO2 to the fixed CO2, Radmann et al. (2011) (150/3.24) has a value of 46:3 mgCO2 fixed mg1 CO2 fed , whereas the conversion with MEA in this study (104.0/0.36) it was 6.2 times higher ð289 mgCO2 fixed mg1 CO2 fed Þ. The protein concentration in the Spirulina biomass produced with MEA was higher that the control assay on 1st and 2nd growth cycles, and equal to protein content until the final cycles without addition of MEA (5th cycle) (Fig. 3b). With respect to the biomass produced using MEA, was observed reduction in the protein concentration among third and the eighth cycle (Fig. 3b). The final protein content (44.4 ± 6.9% w w1) in the biomass cultivated using MEA (Table 2) was lower than that normally found in Spirulina sp. LEB 18 biomass (60–70% w w1), as reported by Borges et al. (2013). Nonetheless, the control assay (60.8 ± 4.0% w w1) remained within this protein concentration range.
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Microalgae under conditions of nutrient stress tend to accumulate compounds such as carbohydrates and lipids (Tanzi et al., 2013). The carbohydrate concentration outline throughout the growth cycles (Fig. 3b) in the assays using MEA evidenced that the nutrient recycle proposed in this study may have deprived the Spirulina of at nitrogen source in the culture medium, prioritizing its metabolic route to the formation of reserve compounds. This hypothesis was evidenced by the final carbohydrate concentration in the biomass produced using MEA (8th cycle), which was higher than that observed in the assay without MEA (5th cycle) (Table 2). Following this idea Klok et al. (2013), who found that nitrogen depletion during the cultivation of microalgae leads to the wasting of light by the cell due to energy dissipation as heat and light in the form of fluorescence. Therefore, microalgae can lose efficiency during the photosynthetic process, leading to a decrease in the biomass productivity (Table 1) and nitrogen compounds, such as proteins (Fig. 3b and Table 2). The lipid content of the biomass produced with MEA (8.3 ± 1.4% w w1) and without MEA (10.0 ± 1.2% w w1) was higher than that found by Borges et al. (2013) (5.0% w w1) and Morais et al. (2009) (3.3% w w1) using the same strain as the present study. These authors cultivated Spirulina in open reactors that, according to Chisti (2007), may lead to lower biomass productivity, but this cannot explain the low lipid levels in relation to the values found in the present experiments. Therefore, as was observed with the carbohydrate concentration, the Spirulina may have increased the lipid concentration due to the scarcity or loss of certain nutrients, due to the medium recycle in both of them of the studied conditions. The results found for the three macromolecules in the Spirulina biomass indicate that the addition of MEA to each growth cycle did not exert a significant effect on the lipid concentration (p = 0.1140), it significantly increased the carbohydrate content (p = 0.00007) and decreased the protein content (p = 0.00007). According to Hu (2004), the effect of nutritional deficiency of phosphorus and nitrogen is evidenced with lower chlorophyll concentrations and higher carbohydrate content in biomass. Subsequently, it is believed that the a reduction in chlorophyll is related to lower protein content because, according to Masojídek et al. (2004), the green pigments are lipophilic compounds that are associated with protein complexes. In this scenario, it is believed that there was a synergistic effect of nutrients recycle with the addition of monoethanolamine on the concentrations of protein and carbohydrate, i.e., biomass with higher carbohydrates concentration and lower protein content. Due to growing demand for biofuels, microalgae are gaining wide attention as an alternative renewable source of biomass for production of bioethanol, which is grouped under ‘‘Third generation biofuels’’ (Nigam and Singh, 2011). In this perspective, it was observed that the carbohydrates content in 100 g of Spirulina sp. LEB 18 biomass, cultivated with MEA, it has the potential of producing of 12.8 mL of ethanol and 0.0023 GGE. Whereas the amount of carbohydrates in 100 g of biomass produced in the control assay has potential of producing of 6.6 mL of ethanol and 0.0012 GGE.
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