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Performance evaluation of a green process for microalgal CO2 sequestration in closed photobioreactor using flue gas generated in-situ
Accepted Manuscript Performance evaluation of green microalgal CO2 sequestration in closed photobioreactor using in situ generated flue gas Geetanjali Yadav, Ankush Karemore, Sukanta Kumar Dash, Ramkrishna Sen PII: DOI: Reference:
S0960-8524(15)00538-6 http://dx.doi.org/10.1016/j.biortech.2015.04.040 BITE 14881
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
29 January 2015 14 April 2015 15 April 2015
Please cite this article as: Yadav, G., Karemore, A., Dash, S.K., Sen, R., Performance evaluation of green microalgal CO2 sequestration in closed photobioreactor using in situ generated flue gas, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.04.040
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Performance evaluation of green microalgal CO2 sequestration in closed photobioreactor using in situ generated flue gas Geetanjali Yadava, Ankush Karemorea, Sukanta Kumar Dashb, Ramkrishna Sena* a
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302 India Department of Mechanical Engineering, Indian Institute of Technology Kharagpur, West Bengal 721302 India
b
*Corresponding author Dr. Ramkrishna Sen Professor Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India. Phone (O): +913222283752, Fax: +913222278707 Email: [email protected]
Abstract In the present study, carbon-dioxide capture from in-situ generated flue gas was carried out using Chlorella sp. in bubble column photobioreactors to develop a cost effective process for
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concomitant carbon sequestration and biomass production. Firstly, a comparative analysis of CO2 sequestration with varying concentrations of CO2 in air-CO2 and air-flue gas mixtures was performed. Chlorella sp. was found to be tolerant to 5% CO2 concentration. Subsequently, inhibitory effect of pure flue gas was minimized using various strategies like use of high initial cell density and photobioreactors in series. The final biofixation efficiency was improved by 54% using the adopted strategies. Further, sequestered microalgal biomass was analyzed for various biochemical constituents for their use in food, feed or biofuel applications. Keywords Microalgae, CO2 fixation, photobioreactor-in-series, coal-fired flue gas 1. Introduction The anthropogenic activities such as excessive use of fossil fuel reserves, deforestation and intensive industrialization has led to unceasing rise in greenhouse gas emissions. In recent times, the CO2 concentration in the atmosphere has reached an alarming level of 400 ppm (Tans, 2015). It is envisaged that CO2 levels greater than 450 ppm could be destructive to global climate (Hansen et al., 2007). Therefore, demand for effective carbon-dioxide (CO2) mitigation technologies is the need of hour. Of all the post combustion CO2 capture technologies viz. chemical and physical absorption, geological and oceanic storage, and biological fixation; microalgal mediated CO2 fixation offers several advantages such as faster growth rates and higher CO2 fixation rates (10-50 times more) than terrestrial plants. Hence, microalgae are regarded as prime candidates for biological fixation of CO2 (Yoo et al., 2010; Cheng et al., 2013). As a result of CO2 fixation, microalgal biomass accumulates significant amounts of lipids, carbohydrates, proteins and other valuable compounds, such as pigments and vitamins, which
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can be used as active ingredients in nutraceutical, food and feed supplements or in the production of biofuels. (Francisco et al., 2010; Cheah et al., 2015). An effective microalgal cultivation system is designed in order to achieve high surface area per volume ratio and good hydrodynamics to give more surface area for the light penetration and gaseous CO2 transfer (Cheah et al., 2015). An efficient CO2-fixation system must ensure good mixing, high gas-liquid transfer rates, and even distribution of light. Broadly, microalgal cultivation is done in two types of systems, open and closed. Usually open microalgal cultivation is carried out in raceway ponds which are low cost, ease in operation and maintenance but suffer from poor light penetration, contamination and poor biomass productivity. On the other hand, closed system includes different configuration of photobioreactors (PBRs) (Air-lift, flat plate, tubular) and generally have better mass transfer rates, higher productivity and better control over process parameters than open ponds. Due to their simplicity, robustness and relatively high process efficiency, closed system especially bubble column reactors are widely used (Kumar et al., 2012). Various strains of microalgae have been utilized for CO2 fixation. Chlorella sp. is widely studied strain which has been used in several industrial applications and found to be a fast growing microalgal strain capable of fixing CO2 from flue gases (Van Den Hende et al., 2012). It contains high amount of protein (51–58%), carbohydrate (12–17%) and lipid (14–22%) that could be used for various applications like health food, nutritional supplements, animal feed, biofuels etc. (Becker, 1994; Cheah et al., 2015). Flue gas is inexpensive and rich source of CO2, approximately 400 times more concentrated than atmospheric CO2 and thus can be exploited for microalgal mediated CO2 fixation (Cheah et al., 2015). However, the major constraints are due to the presence of toxic compounds such as NOx, SOx and CO which are inhibitory for microalgal growth and biomass productivity (Kumar et al., 2014), primarily due to acidification of the
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growth medium (Van Den Hende et al., 2012). Flue gas from various sources has been used to cultivate microalgae for CO2 fixation (Van Den Hende et al., 2012, Kumar et al., 2014). The direct use of flue gas is detrimental for microalgal growth and generally requires expensive pretreatments processes. The efficiency of CO2 fixation from flue gas by microalgae generally found to be less than 50% (Cheng et al., 2013). In order to treat flue gas by microalgae in a costeffective manner, there is need to develop effective strategies which will not only reduce the cost of its pretreatment but also increase CO2 fixation. Therefore, in this study, strategies were developed to directly use coal-fired flue gas for the cultivation of microalgae and simultaneous flue gas remediation. Firstly, a comparative study was conducted to evaluate the performance of Chlorella sp. for CO2 fixation using pure CO2 and waste flue gas. Subsequently, inhibitory effect of pure flue gas was minimized using various strategies like use of high initial cell density and photobioreactors in series. Further, commercial potential of the sequestered microalgal biomass was assessed for their use in food, feed or biofuel applications.
2. Material and methods 2.1. Strain and culture medium The strain Chlorella sp. (Chlorophyta, Chlorophyceae) obtained in this experiment was procured from National Environmental Engineering Research Institute, Nagpur, India (Fulke et al., 2010). The inoculum was grown on modified Bold Basal’s medium (Nichols and Bold,1965) and has the following composition (mg L-1): NaNO3 (750), CaCl2 · 2H2O (12.5), MgSO4 · 7H2O (150), FeSO4 (6.27), K2HPO4 (62.4), KH2PO4 (225), NaCl (0.341), H3BO3 (5), MnSO4 (0.72), ZnSO4 ·7H2O (17.64), KOH (15.5), NaCl (12.5), CuSO4 · 7H2O (1.06), NaMoO3 (0.6), CoCl2 (0.2). The
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strain was maintained in liquid culture (100 mL) in an Erlenmeyer’s flask (150 ml), at 28 ± 2 °C, under intermittent agitation and continuous illumination (24 µ mol m−2 s−1) and an atmosphere of air 0.04% CO2 (v/v). Initial pH of the medium was adjusted to 6.8 ± 0.1. All chemicals used are of analytical grade and procured from Merck®, India. All experiments were performed in triplicates and expressed as mean with standard deviation (S.D). 2.2. Photobioreactor, Experimental set-up and batch culture conditions Batch experiments were performed in bubble column glass photobioreactors (BCR) (length, 33. cm; inner diameter, 4.5 cm; working volume, 500 mL). The average temperature during batch experiment was maintained at 28 ± 2 °C. Chlorella sp. was cultured in three bubble column PBRs connected in series and labeled sequentially as PBR 1st, PBR 2nd and PBR 3rd. Flue gas was fed into the sparger of PBR 1st and effluent from this PBR was fed to PBR 2nd and similarly the effluent from this PBR was fed to PBR 3rd (Supplementary Fig A.1 and A.2). The initial pH of medium was noted to be 6.8 and the rate of flow of gas into the reactor was maintained at 0.5 vvm. Carbon-dioxide or flue gas was fed only during the light period and its supply was stopped during the dark period, however air was fed continuously. The light intensity falling from one side of reactor through cool white fluorescent tubes was measured using quantum meter (HTC LX 102 Lux Meter) to be approximately 106.6 µ moles m-2 s-1 during the entire batch experiment in a 12:12 hr (light/dark) photoperiod and. Ambient air or CO2 enriched air was supplied through an aquarium sparger (Φdiameter = 1.0 cm) located at the bottom of the BCR. The flue gas produced from burning of coal in the flue gas generator is mixed with air to create different v/v mixtures of air-flue gas before it is filtered using syringe filter and injected into the culture photobioreactors. The concentrations of air-pure CO2 as well as air-flue gas mixture were adjusted and set to desired concentrations of 2.5%, 5%, 7.5%, 10% and 0.04%
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(ambient air) of the total mixture. A single batch culture was incubated for 7 days. Samples were removed once every 24 hours to determine biomass concentration and CO2 fixation rate and twice every 24 hours to check the culture pH. Sample pH was determined using a pH meter (Deluxe pH Meter, Model LT-10). The effect of inoculum density was investigated by varying the cell density of initial inoculum viz. 0.06 g L-1, 0.1 g L-1, 0.15 g L-1 and 0.2 g L-1 respectively. Subsequently, the best inoculum density was selected to carry out the photobioreactor-in-series experiment to minimize the toxic effects of flue gas components in sequential reactors (Supplementary Fig A.2). 2.3.Flue Gas Analysis The flue gas used in the present study was obtained after burning of coal procured from Kolaghat Thermal Power Station (KTPS), West Bengal India. An indigenously designed and fabricated flue gas generator was used for burning the coal and collecting in-situ generated flue gas. The flue gas generator was equipped with three-chambered double layered jacket in which water was circulated to cool down the temperature of flue gas from 120° C to approximately 45 °C. The raw flue gas was collected from the upper position of stack and passed through a vacuum precipitator to remove the suspended particulate matter by a compressor pump (1HP). The flue gas was then transferred through pipelines to storage gas tank (volume: 41.3 liters, pressure: 10 kg cm-2) before feeding it to the microalgal PBR. The flue gas was analyzed by a portable on-line Flue gas analyzer (Model: Indus FGA 53X). A schematic illustration of the flue-gas based cultivation system for Chlorella sp. is shown in Supplementary Fig. A.2.The flue gas was analyzed by a portable on-line Flue gas analyzer (Model: Indus FGA 53X). The typical composition of the flue gas was 10 ± 2% (v/v) carbon dioxide, 0.554% (v/v) carbon-monoxide,
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8.33% (v/v) oxygen, 61 ppm nitrogen oxides, 0.3% (v/v) sulphur oxides, 9 ppm other hydrocarbons. 2.4.Biomass concentration, Biomass productivity and Nitrate consumption profile Biomass concentration (dry weight per liter) of microalgal cells was measured by taking optical density at 750 nm (O.D750nm) in Agilent Cary 60 Scan UV/Visible spectrophotometer (Griffiths et al., 2011). Dry cell weight (dwt) was calculated using a calibration plot between Dwt and O.D. Dry weight biomass (g L-1) = 0.44 × O.D750nm. Sampling was done every 24 hours to determine the cell concentration for further algal growth calculation. Each sample was diluted to give an absorbance in the range of 0.1–0.5 if the optical density was greater than 1.0. This is due to the fact that probability of obtaining erroneous result becomes higher as the cell density crosses O.D value > 1. The maximum biomass concentration achieved in culture was designated as Xmax (gL1
). The overall biomass productivity Px (g biomass L-1d-1) in the batch experiment was calculated
using the following equation: ∆
= ∆
(1)
Where ∆ and ∆ are the total amount of biomass and total cultivation time (days) respectively. The nitrate concentration was calculated following the method of Armstrong, (1963) given in Eq. (2). = . − 2#. $% &
(2)
2.5.Determination of maximal CO2 biofixation rate To produce 1 g of dry microalgal biomass, approximately 1.83 g of CO2 is required to be fixed assuming 50% of carbon is fixed in the microalgal biomass (Cheah et al., 2015). Maximal CO2 biofixation rate F (g d-1) was calculated from Eq. (3), as described by Pegallapati and Nirmalakhandan, (2013).
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' ( ) * , , = -
(3)
Where, ‘a’ is the carbon dioxide fixed by unit biomass (considering 50% of carbon in the 00
biomass) a = 0.5 )= 1.833 g CO2 (g dry cell)-1); and V is the culture volume (= 0.5 L), is the volumetric productivity. 2.6.Analysis of lipid, carbohydrate and protein content The total lipids were extracted from freeze-dried microalgal biomass using a modified Folch method (Folch et al., 1957). Firstly, freeze-dried algal biomass was transferred into a chloroform/ methanol (2:1, v/v) mixture and homogenized for 10 minutes using pestle and mortar. This was followed by centrifugation at 300 rpm for 5 minutes. The lower chloroform phase containing lipids was collected and dried overnight inside oven at 60° C to remove the chloroform. The difference of final and initial weight gives the estimation for lipid content (%). Carbohydrates were extracted from lyophilized microalgal biomass following acid hydrolysis (NREL, 2011). The dried lyophilized microalgal biomass was subjected to a two-step acid hydrolysis. In the first step, approximately 50 mg biomass was dissolved in 0.5 ml of 72% H2SO4 for 30 min in 30° C water bath. Further, the concentration of H2SO4 was reduced and brought to 4% by the addition of distilled water. In the next step, this mixture was incubated at high temperature of 121° C (preferably by autoclaving) for 1 hour. After the required incubation the samples are neutralized using sodium carbonate till the effervescence ceased and pH reaches to a value of approximately 7. The samples are then centrifuged at 3500 rpm for 6 minutes and the supernatant is used for phenol-sulfuric acid assay to analyze the carbohydrate concentration (Dubois et al., 1956), which was determined by measuring the absorbance at 490 nm (Agilent Cary 60 Scan UV/Visible spectrophotometer).
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Proteins were extracted using alkali hydrolysis using 0.5N NaOH and incubation at 80 °C for 10 minutes. The supernatant after centrifugation is used for Bradford’s assay. The protein concentration was measured by taking O.D at 595nm. Results and discussion
3.1. Use of CO2 and flue gas for growing microalgae Microalgae utilize CO2, nutrients and light for its growth via autotrophic metabolism. Thus, CO2 fixation could be positively correlated with increased biomass productivity. In general, microalgae can tolerate various concentrations of CO2 (10–50%) for its growth (Cheah et al., 2015). Chlorella sp. normally exhibits satisfactory growth with CO2 concentrations ranging from 1% to 18% v/v (Hulatt and Thomas, 2011) but it can also grow at extreme conditions of up to 50% CO2 concentration (Maeda et al., 1995). CO2 feeding into the microalgal culture system leads to mass transfer of CO2 gas into aqueous media, followed by its solubilization in different forms such as CO2, H2CO3, HCO3− and CO32− (depending upon pH of the medium), and finally its uptake by microalgae through the process of photosynthesis (Van Den Hende et al., 2012).
3.1.1. Growth of Chlorella sp. using varying concentrations of CO2 To investigate the effect of varying CO2 concentrations on growth, Chlorella sp. was incubated with different concentrations of CO2 viz. 0.04%, 2.5%, 5%, 7.5% and 10% for 7 days at 28 ±2 °C. The batch experiments were carried out till nitrate was completely exhausted. The time course profile of growth of Chlorella sp. is illustrated in Fig. 1 (a). The most suitable CO2 concentration for microalgal growth was found to be 5%, above which the growth started declining as shown in Fig. 1(a). The maximum biomass concentration and productivity of 2.2045 ± 0.12 g L-1 and 0.314 ± 0.004 g L-1d-1was obtained at 5% CO2 level. Previous reported studies
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also showed that CO2 concentration above 5% could be inhibitory to microalgal growth and CO2 biofixation (Chiu et al., 2008, Chiu et al., 2009; de Morais and Costa, 2007, Cheah et al. 2015). Effect of increasing CO2 concentrations on pH change was monitored as illustrated in Fig. 1(b). It was found that increasing the CO2 concentrations in culture medium resulted in decrease of pH from 6.8 to 6.4. The pH was controlled for initial 36 h with the addition of potassium hydroxide solution. Addition of pH solution during the initial stage of growth and for the brief period of time helps in achieving stable microalgal growth (Lee et al., 2000). Later, as the microalgal growth progressed towards exponential phase, the pH stabilized in the range of 9-10. However, when air was used for aeration, no pH drop was observed. This is due to very low concentration of CO2 (0.04%, v/v) in air and poor mass transfer (Tans, 2015). Also, at very low CO2 concentrations, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), a key enzyme of Calvin cycle mainly performs its oxygenase activity, resulting in poor CO2 fixation. On the contrary, when there is sufficient CO2 in the culture, carboxylase activity of RuBisCO catalyses CO2 fixation which in turn leads to expression of another important enzyme i.e. carbonic anhydrase (CA). CA increases the alkalinity in medium by transporting hydroxide ions outside the cell while keeping the H+ ions within the thylakoid membranes (Kumar et al. 2012). Increasing the CO2 concentration upto 10% led to significant decline in pH initially, though later, due to increase in biomass growth, pH improved to alkaline range due to the normal photosynthetic activity (Hulatt and Thomas, 2011). This may also be due to tolerance of Chlorella sp. to the elevated levels of CO2. Nitrate consumption by microalgae also results in rise of culture pH during photosynthesis (Hulatt and Thomas, 2011). The Xmax and Px values gradually decreased with the increase in CO2 concentration above 5% (i.e. at 7.5% and 10% CO2, v/v) (Table 1). Chiu et al. (2008) also reported that microalgal cell
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growth was significantly reduced when Chlorella sp. cultures were treated with high concentration of CO2 gas. The average rate of CO2 fixation in cultures at 0.04%, 2.5%, 5%, 7.5% and 10% CO2 in the photobioreactor for 0.5 L working volume was 196 ± 10 mg CO2-1day-1, 221± 40 mg CO2 -1day-1, 287± 7 mg CO2 -1day-1, 274 ± 30 mg CO2 -1day-1 and 249 ± 11 mg CO21
day-1 respectively. The most efficient CO2 fixation and sequestration rates were also obtained at
5% CO2 concentrations above which it started declining. Nitrate was utilized for the production of biomass; Table 1 shows residual nitrate concentration at the end of 7 days of batch experiment. It was observed that a decreasing trend of nitrate consumption was obtained with increasing biomass concentration (Chen et al., 2012). 3.1.2. Growth of Chlorella sp. using varying concentrations of actual flue gas Coal-fired flue gas contains upto 10-15% CO2 (v/v) and numerous compounds like H2O, O2, N2, nitrogen oxides (NOx), sulphur oxides (SOx), unburned hydrocarbons (CxHy), CO, heavy metals, halogen acids and particulate matter (Van Den Hende et al., 2012). The growth of Chlorella sp. with addition of actual coal-fired flue-gas at different concentration was investigated. The flue gas was diluted with air to vary its CO2 concentration in the range of 2.5%, 5%, 7.5% and 10% (v/v). Effect of various concentration of flue gas on biomass growth and pH profiles is shown in Fig.2 (a) and (b). Flue gas feeding into microalgal culture resulted in decline of the medium pH value from 6.8 to 5.9. Potassium hydroxide solution was added to maintain the pH value for initial 48 h. Flue gas contains acidic gases such as SOx, NOx, and CO2, which decreases the medium pH upon dissolution (Kumar et al., 2014). It is generally reported that direct use of flue gas results in slower microalgal growth rate (Richmond and Zou, 1999). This may be due to time taken by the microalgae to adapt the harsh conditions (Kumar et al., 2014). The Chlorella sp. showed a lag
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phase of one day and later adapted itself with time to produce biomass. The study by Kastanek et al. (2010) reported a lag time of 3 days for growing Chlorella sp. with flue gas. Subsequently, pH increased via normal photosynthetic process and reached alkaline range of 9-10 as shown in Fig 2 (b). The biomass concentration of Chlorella sp. aerated with flue gas at different concentrations; 0.04%, 2.5%, 5%, 7.5% and pure flue gas (~10%) CO2, v/v were 1.502 ± 0.011 g L-1, 1.601 ± 0.025 g L-1, 1.915 ± 0.12 g L-1, 1.8065 ± 0.12 g L-1 and 1.4 ± 0.015 g L-1 respectively. Comparatively, the biomass concentration and productivity of microalga grown using 5% CO2 in flue gas was approximately 13% lower than that obtained in 5% pure CO2 feeding (Table 1). Kumar et al. (2014) used Chlorella sorokiniana to sequester CO2 from flue gas and also observed 5% CO2 as the most optimum concentration. However, significant improvement in overall biomass production by about 21% and 36% was achieved at 5% flue gas and 5% CO2 (v/v), respectively, with respect to air. The maximum CO2 sequestrations and biofixation rate of 0.974 ± 0.131 g L-1 and 250 ± 13 mg CO2-1 day-1was also obtained for 5% CO2 concentration in flue gas. The greater CO2 fixation rates achieved with dilution of flue gas could be due to decreased concentration of CO2 and toxic gases. Further, upon using pure flue gas, the microalgal growth as well as fixation rates reduced due to the presence of inhibitory compounds in the flue gas (Van Den Hende et al., 2012). Since, direct use of flue gas feeding into microalgal cultures resulted in lowest CO2 fixation rate of 175 ± 10 mg CO2-1 day-1 ; there is a need to search for alternative methods which can directly use the flue gas efficiently for higher biomass production as well as CO2 fixation. 3.2 Overcoming flue gas inhibition using different techniques
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Although direct use of flue gas is a cheaper technique to sequester carbon-dioxide by microalgae, the presence of toxic compounds like SOx, NOx in addition to high concentrations of CO2 (~1015%, v/v) imposes extreme conditions on microalgal growth (Van Den Hende et al., 2012). Besides, the presence of acidic gases in flue gas not only affects the culture pH substantially but also prolongs the lag phase of microalgal growth (Kumar et al., 2014). Several flue gas pretreatment strategies have been devised to scrub off the high concentration of inhibitory compounds from flue gas; viz. passing through water to remove oils and dust particle, use of scrubbers (Kumar et al., 2014), use of chemical solvents like euchlorine scrubbing solution (Deshwal and Lee, 2009), BioDeNOx process (Kumaraswamy et al., 2005), etc. In our study, the harmful effects caused by direct use of flue gas was attenuated by adopting following strategies; variation in initial cell density and ‘photobioreactor-in-series’ for concomitant biomass production and CO2 fixation. 3.2.1 Effect of flue gas on microalgal culture at different initial cell density The effect of actual flue gas containing 10% CO2 (v/v) was determined on microalgal growth at different initial cell densities (t=0 h). It is reported that inoculum density influences the productivity and economics of bioprocesses (Sen and Swaminathan, 2004; Chiu et al., 2008) and has positive effect on subsequent biomass production (Richmond et al., 2003; Chen et al., 2012). Experiments were conducted to find a suitable inoculum concentration that will overcome the initial lag phase as observed earlier in Fig. 2(a) during batch growth of Chlorella sp. with flue gas (10% CO2 in flue gas). The study was conducted with Chlorella sp. using four different initial inoculum densities comprising of 0.06 g L-1, 0.1 g L-1, 0.15 g L-1 and 0.2 g L-1 concentration. The most suited initial cell density was observed to be 0.15 g L-1 resulting in highest biomass concentration and CO2 fixation rate of 2.13 ± 0.01 g L-1 and 0.259 ± 0.01 g CO2-
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day-1, respectively. It is evident that the microalgal cultures with lower initial inoculum densities
could not adapt well to direct exposure of flue gas and hence resulted in lower biomass yield, biomass productivity, and CO2 sequestration and fixation rate as illustrated in Table 2. Nevertheless, with the higher inoculum densities of 0.15 g L-1 and 0.2 g L-1, there was no significant difference in the biomass yield. However, it is worth noting from Fig.3 (a) that inoculum density of 0.2 g L-1 reached the stationary phase earlier on 5th day of incubation than inoculum density of 0.15 g L-1. Hence, lower productivity in this case could be due to the phenomenon of ‘autoshading’ resulting in poor light penetration (Kenekar and Deodhar, 2013). The initial medium pH of 6.8 decreased gradually with flue gas feeding as shown in Fig. 3(b). The pH value of culture inoculated with different initial inoculum densities of 0.06 g L-1, 0.1 g L1
, 0.15 g L-1 and 0.2 g L-1 was dropped to about 5.5, 5.9, 6.0 and 6.2, respectively. High-density
cultures have shown better ability to combat the harsh conditions put forward by toxic flue gas elements and therefore double its population faster (Chiu et al., 2008). It has been reported that regulating pH and increasing the inoculating cell density are effective ways to prevent growth inhibition exerted by flue gas (Lee et al. 2002). Nitrate concentration decreased for all four variable initial inoculum densities at the end of entire batch experiment (Table 2). The present study stands out in its approach by avoiding the prolonged lag phase and inhibitory effect encountered of flue gas on microalgal growth by using optimal inoculum density. Chlorella sp. grew well in a high-density culture and achieved better CO2 fixation rates (Table 2). The result is in accordance with previous studies where high CO2 and flue gas tolerance of microalgae was dependent on cell density (Lee et al., 2002, Yoshihara et al., 1996 and Chiu et al 2008). Thus, it can be inferred that direct flue gas could be utilized to grow microalgae for CO2
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biofixation. This optimal inoculum density was further used in subsequent experiment of ‘PBRin-series’. 3.2.2 Use of sequential photobioreactors for CO2 fixation from flue gas To employ inexpensive strategies for direct usage of flue gas, another innovative approach of ‘photobioreactor-in-series’ was investigated for microalgal cultivation. Photobioreactor systems with three-staged bubble column reactors connected in series were labeled PBR 1st, PBR 2nd and PBR 3rd. The inoculum density of 0.15 gL-1was selected to carry out the batch experiment in all the three PBRs. There was no significant difference in the biomass yield observed in PBR 1st and PBR 2nd. However, biomass concentration in PBR 2nd was slightly higher than PBR 1st with the value of 2.225 g L-1 and 2.13 g L-1, respectively (Table 2). Since PBR 1st received direct supply of flue gas, high concentrations of CO2 as well as inhibitory compounds present in the flue gas resulted in lesser biomass growth due to lowering of pH as compared to PBR 2nd. Upon feeding the flue gas into serially connected photobioreactors, consumption of some CO2 might have occurred during the process of photosynthesis and dissolution of some toxic gases in the culture medium of the preceding reactor which resulted in better productivities of PBR 2nd. Similar observation was reported by Kumar et al. (2014), by using the reactor-in-series strategy to fix CO2 from industrial flue gas and observed major reduction in CO2 (4.1%, v/v) along with other hydrocarbons in the serially connected PBRs. Previously, de Morais and Costa, (2007) have used three-stage serial tubular photo-bioreactors to assess the growth of Scenedesmus obliquus and Spirulina sp. with respect to CO2 fixation in different concentrations of CO2 and found an increase in biomass production as well as CO2 fixation towards the downstream reactors. PBR 3rd, located at the end of serially connected PBRs, showed lower biomass growth of 1.5 g L-1, which might be due to lesser gas flow rate and also could be due to further dilution
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of CO2 concentration in flue gas (Lee et al., 2000; Morais and Costa, 2007). Another reason might be due to simultaneous decrease in CO2 concentration and increase in oxygen build up in the downstream reactors (Suzuki and Ikawa, 1984). 3.3 Analysis of carbohydrate, protein and lipid content CO2 present in flue gas can be used to steer the biochemical composition of microalgae like lipid and fatty acids, proteins, polysaccharides and pigments (Van Den Hende et al., 2012; Cheng et al., 2014; Tsuzuki et al., 1990). The impact of CO2 concentration on the synthesis of major metabolites such as lipid, protein and carbohydrates content were measured (Table 3). Algal cells grown with 5% and 10% CO2 accumulated about 12.72% and 5.52% of total lipids respectively. In contrast, when grown in air, Chlorella sp. accumulated approximately 15.84% of total lipids. Cheng et al. (2014) also reported decrease in total lipids accumulation in Chlorella vulgaris when grown at 2% CO2 concentration as compared to air. The reason for difference in lipid percentage could be either strain specificity or tolerance of microalgae to elevated CO2 concentrations (Cheng et al., 2014). As summarized in Table 3, the decreasing trend of lipid content was obtained with increasing CO2 concentrations both with pure CO2 as well as flue gas grown microalgae. Similar trend were also reported by Elvira-Antonio et al. (2013) by using different sodium bicarbonate concentration as a measure of dissolved CO2 in the culture medium. Furthermore, flue gas treated samples received much lesser lipid accumulations at elevated CO2 concentrations (Table 3). This could be due to the presence of different flue gas components which could have directed the microalgal biochemical machinery to synthesize other valuable components like carbohydrates, proteins, pigments etc. (Kumar et al., 2014; Yoo et al., 2010; Tsuzuki et al., 1990).
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Unlike the decreasing trends observed in case of lipids accumulation, carbohydrate and protein contents of the microalgal cultures increased upon CO2 or flue gas feeding. The increase in carbohydrate and protein contents was significant (Table 3). Cheng et al. (2014) also reported that cell wall carbohydrate content in C. vulgaris, C. sorokiniana and C. variabilis increased significantly in CO2 enriched conditions due to overall increase in the uronic acid fractions. At high CO2 concentrations, there was substantial increase in total protein and carbohydrate content from 58.39–59.38% and 20.4–24.48%, respectively, in 10% pure CO2 and actual flue gas. Generally, microalgae having higher CO2 fixation ability at elevated CO2 concentrations, Chlorella sp., Scenedesmus sp. and Spirulina sp., are found to contain high amount of important metabolites (Anjos et al., 2013; Tanadul et al., 2014; Cheah et al., 2015). Significant differences were observed in the biochemical composition of microalgal cells under varying CO2 concentrations in flue gas and hence it should be utilized for various applications in food, feed or biofuel. 4. Conclusions A comparative analysis of microalgal CO2 sequestration using different concentrations of pure and flue gas CO2 was accomplished. Significant improvement in overall biomass production by about 21-36% was achieved at 5% CO2 (v/v). Considering the fact that direct supply of flue gas inhibits microalgal growth, reactor strategies involving the use of PBR-in-series with high initial (t= 0 h) biomass density worked well in improving CO2 fixation rate, total carbohydrate and protein contents. Thus, this study offers the proof of the concept of mitigating flue gas CO2 for the production of algal biomass, carbohydrates and proteins for various potential applications. Acknowledgements
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Geetanjali Yadav acknowledges West Bengal Government-Department of Science and Technology (Project Grant No. 560 (SANC.)/ST/P/S&T/5G-5/2011; Date: 21-11-2011) for providing fellowship support. The authors are thankful to Dr. Sukhendu Kumar Das for his valuable suggestions. The authors are also grateful to Dr. Tapan Chakrabarti of National Environmental Engineering Research Institute, Nagpur, India for giving Chlorella sp. as a gift. GY also acknowledges the help of Mr. Lakshmikanta Dolai with flue gas set-up operation.
References 1. Anjos, M., Fernandes, B.D., Vicente, A.A., Teixeira, J.A., Dragone, G. 2013. Optimization of CO2 bio-mitigation by Chlorella vulgaris. Bioresour Technol. 139, 149–154. 2. Becker, E.W., 1994. Microalgae: biotechnology and microbiology. In: Baddiley J, Carey NH, Higgins IJ, Potter WG (eds) Cambridge studies in biotechnology. Cambridge University Press, New York, p 178. 3. Chen, Y., Wang, J., Liu, T., Gao, L., 2012. Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp. J. Appl. Phycol. 24, 1623–1627. 4. Cheng, J., Huang, Y., Feng, J., Sun, J., Zhou, J., Cen, K., 2013. Improving CO2 fixation efficiency by optimizing Chlorella PY-ZU1 culture conditions in sequential bioreactors. Bioresour. Technol. 144, 321–327. 5. Cheng, Y.S., Labavitch, J.M., Vander, G.J.S., 2014. Elevated CO2 concentration impacts cell wall polysaccharide composition of green microalgae of the genus Chlorella. Appl. Microbiol. 60 (1), 1-7.
19
6. Cheah, W.Y., Show, P.L., Chang, J.S., Ling, T.C., Juan, J.C., 2015. Biosequestration of atmospheric CO2 and flue gas-containing CO2 by microalgae. Bioresour. Technol. 184, 190– 201. 7. Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99 (9), 3389-3396. 8. Chiu, S.Y. Kao C.Y., Tsai, M.T. Ong, S.C. Chen, C.H. Lin C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838. 3. Deshwal, B.R., Lee, H.K., 2009. Mass transfer in the absorption of SO2 and NOx using aqueous euchlorine scrubbing solution. J. Environ. Sci. 21, 155–161. 4. de Morais, M.G., Costa, J.A.V., 2007. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energy Convers. Manage. 48, 2169–2173. 5. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. 6. Elvira-Antonio, N., Ruíz-Marín, A., Canedo-López, Y., 2013. Effect of nitrogen content and CO2 consumption rate by adding sodium carbonate in the lipid content of Chlorella vulgaris and Neochloris oleoabundans. Int. J. Envir. Protect. 3(10), 13-19. 7. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol Chem. 226, 497-509.
20
8. Francisco, É.C., Neves, D.B., Jacob‐Lopes, E., Franco, T.T., 2010. Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality. J. Chem. Technol. Biotechnol. 85, 395–403. 9. Fulke, A.B., Mudliar, S.N., Yadav, R, Shekh., A, Srinivasan. N., 2010. Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella vulgaris. Bioresour. Technol. 101(21), 8473-8476. 10. Griffiths, M.J, Garcin, C., van Hille, R.P., Harrison, S.T., 2011. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J. Microbiol Meth. 85(2), 119-123. 11. Hansen, J., Sato, M., Ruedy, R., Kharech, P., Lacis, A., Miller, R., 2007. Dangerous humanmade interference with climate: a GISS model study. Atmos. Chem. Phys.7, 2287–312. 12. Van Den Hende, S., Han, V., Boon, N., 2012. Flue gas compounds and microalgae: (Bio-) chemical interactions leading to biotechnological opportunities. Biotech. Adv. 30(6), 14051424. 13. Hulatt, C. J., Thomas, D. N., 2011. Productivity, carbon dioxide uptake and net energy return of microalgal bubble column photobioreactors. Bioresour. Technol. 102, 5775–5787. 14. Kenekar, A. A., Deodhar, M. A., 2013. Effect of varying physicochemical parameters on the productivity and phycobiliprotein content of indigenous isolate Geitlerinema sulphureum. Biotechnol. 12 (3), 146-154. 15. Kastanek, F., Sabata, S., Solcova, O., Maleterova, Y., Kastanek, P., Branyikova, I., Kuthan, K., Zachleder, V., 2010. In-field experimental verification of cultivation of microalgae Chlorella sp. using the flue gas from a cogeneration unit as a source of carbon dioxide. Waste Manage. Res. 28 (11), 961–966.
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16. Kumar, K., Das, D., 2012. Growth characteristics of Chlorella sorokiniana in airlift and bubble column photobioreactors. Bioresour. Technol. 116, 307–313. 17. Kumar, K., Banerjee, D., Das, D., 2014. Carbon dioxide sequestration from industrial flue gas by Chlorella sorokiniana. Bioresour. Technol. 152, 225–233. 18. Kumaraswamy, R., Dongen, U.V., Kuenen, J.G., Wiebe, A., Loosdrecht, M.C.M.V., Muyzer, G., 2005. Characterization of microbial communities removing nitrogen oxides from flue gas: the BioDeNOx Process. Appl. Environ. Microbiol. 71 (10), 6345–6352. 19. Lee, J. N., Lee, J. S., Shin, C. S., Park, S. C., Kim, S. W., 2000. Methods to enhance tolerances of Chlorella KR-1 to toxic compounds in flue gas. Appl. Biochem. Biotechnol. 84–86 (1-9), 329-342. 20. Lee, J.S., Kim, D.K., Lee, J.P., Park, S.C., Koh, J.H., Cho, H.S., Kim, S.W., 2002. Effects of SO2 and NO on growth of Chlorella sp. KR-1. Bioresour. Technol. 82 (1) 1–4 21. Maeda, K., Owada, M., Kimura, N., Omata, K., Karube, I., 1995. CO2 fixation from the fluegas on coal-fired thermal power-plant by microalgae. Energy Convers. Manage. 36, 717–20. 22. National Renewable Energy Laboratory 2011. www.nrel.gov/biomass/analytical_procedures.html 23. Pegallapati, A. K., Nirmalakhandan, N., 2013. Internally illuminated photobioreactor for algal cultivation under carbon dioxide-supplementation: Performance evaluation. Renw. Energy. 56, 129-135. 24. Richmond A, Zou N., 1999. Efficient utilization of high photon irradiance for mass production of photoautotrophic micro-organisms. J. Appl. Phycol. 11,123–127.
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25. Sen, R., Swaminathan, T., 2004. Response surface modeling and optimization to elucidate and analyze the effects of inoculum age and size on surfactin production. Biochem. Engg. J.21(2),141–148. 26. Suzuki, K., Ikawa, T., 1984. Effect of Oxygen on Photosynthetic 14CO2Fixation in Chroomonas sp. (Cryptophyta) I. Some Characteristics of the Oxygen Effect. Plant Cell Physiol. 25(3), 367-375. 27. Tans, P., "Trends in Carbon Dioxide". NOAA/ESRL. Retrieved 2015-03-05. 28. Tanadul, O.U.M, VanderGheynst, J. S., Beckles, D. M. Powell, A. L.T., Labavitch, J.M., 2014. The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock. Biotechnol. Bioengg. 111 (7), 1323-31. 29. Tsuzuki, M., Ohnuma, E., Sato, N., Takaku, T., Kawaguchi, A., 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93, 851– 856. 30. Yoo, C., Jun, S.Y., Lee, J.Y., Ahn, C.Y., Oh, H.M., 2010. Selection of microalgae for lipid production under high level of carbon dioxide. Bioresour. Technol. 101, 71–74. 31. Yoshihara, K., Nagase, H., Eguchi, K., Hirata, K., and Miyamoto, K., 1996. Biological elimination of nitric oxide and carbon dioxide from flue gas by marine microalga NOA-113 cultivated in a long tubular photobioreactor. J. Ferment. Bioeng. 82, 351–354. Figure Captions Fig. 1 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with different concentrations of CO2. Each data indicates the mean ± SD and were measured from three independent cultures. External illumination of light was provided with 106.6 µmolm−2 s−2 in a 12:12 light: dark photoperiod
23
Fig. 2 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with different concentrations of CO2 in flue gas. Each data indicates the mean ± SD and were measured from three independent cultures.
Fig. 3 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with pure flue gas with different initial cell density. Each data indicates the mean ± SD and were measured from three independent cultures.
Fig. 4 Time-course of growth and pH profile for Chlorella sp. using actual flue gas sparged to three-stage serially connected bubble column reactor.
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25
26
27
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Table 1 Biomass concentration, Productivity and CO2 sequestration of CO2 and Flue gas cultivated algae in different concentrations of CO2 Growth Biomass Biomass CO2 Carbon Residual conditions concentration Productivity sequestration dioxide Nitrate content (g L-1)b (g L-1d-1) (g L-1) fixation rate (mg L-1) -1 (mg CO2 day 1 ) (For 0.5L)c CO2-air ratio
Aira
1.502 ± 0.011
0.214 ± 0.002
0.712 ± 0.001
196 ± 5
24.66
2.5
1.7 ± 0.034
0.242 ±0.11
0.799 ± 0.04
221± 4
-
5
2.2045 ± 0.12
0.314 ± 0.004
1.19 ± 0.077
287± 7
-
7.5
2.097 ± 0.03
0.295 ± 0.045
1.111 ± 0.003
274 ± 3
-
10
1.902 ± 0.13
0.271 ±0. 067
0.970 ± 0. 011
249 ± 11
-
Air
1.502 ± 0.011
0.214 ± 0.002
0.712 ± 0.001
196 ± 5
24.66
2.5
1.601 ± 0.025
0.228 ± 0.033
0.762 ± 0.055
208 ± 5
2.1
5
1.915 ±0.12
0.273 ± 0.012
0.974 ± 0.131
250 ± 13
-
7.5
1.8065 ±0.12
0.258 ±0.023
0.865 ± 0.102
236 ± 10
8.122
Pure FG
1.4 ± 0.015
0.191 ±0.11
0.7 ± 0.0133
175 ± 10
112.11
Flue gas-air ratio
a
CO2 in air is assumed as 0.04%.
b
Each data indicates the mean ± SD and were measured from three independent cultures.
c
Carbon fixation rates were calculated for 0.5 L working volume of microalgal culture.
- Not detectable
29
Table 2 Biomass productivity, CO2 sequestration ,CO2 fixation rate and residual nitrate concentration in experiments with different inoculum density and reactor in series for growing Chlorella sp. with actual flue gas Growth Biomass Biomass CO2 Carbon Residual Nitrate conditions concentration Productivity sequestration dioxide content (g L-1)a (g L-1d-1) (g L-1) fixation rate (g (mg L-1) CO2-1day-1) (For 0.5L)b Inoculum Density 1X
1.4 ± 0.014
0.192 ±0.011
0.705 ± 0.054
176± 10
11.2 ± 1.87
2X
1.981 ± 0.02
0.268 ± 0.124
0.990± 0.012
246± 32
-
3X
2.13 ± 0.01
0.282 ± 0.015
1.065 ± 0.044
259 ± 10
-
4X
2.014 ± 0.27
0.259 ±0. 071
1.007 ± 0. 001
237± 8
1.11 ± 0.0012
Reactor 1
2.13 ± 0.02
0.282 ± 0.12
1.065 ± 0.011
259 ± 7
102.88 ± 0.0003
Reactor 2
2.225 ±0.12
0.296 ± 0.044
1.112 ± 0.09
271 ± 11
-
Reactor 3
1.501 ±0.112
0.193 ±0.053
0.75 ± 0.011
170 ± 65
233.55 ± 0.0054
Reactor in series
a
Each data indicates the mean ± SD and were measured from three independent cultures.
b
Carbon fixation rates were calculated for 0.5 L working volume of microalgal culture.
- Not detectable
30
Table 3 Effect of flue gas on Lipid, Carbohydrate and Protein production in Chlorella sp. cultures Growth Biomass Protein (%) Carbohydrate Lipids (%) conditions concentration (%) (gL-1)b
Aira
1.5 ± 0.01
51.3 ± 2.13
12.24 ± 1.2
15.84 ± 1.4
5%
2.2 ± 0.12
55.7 ± 1.5
14.02 ± 0.86
12.72 ± 1.7
10%
1.9 ± 0.13
59.38 ± 2.01
20.4 ± 1.7
5.52 ± 0.85
5%
1.92 ±0.12
56.8 ± 3.02
19.88 ± 1.43
8.65 ± 0.97
Pure FG
1.41 ±0.02
58.39 ± 1.3
24.48 ± 1.3
4.45 ± 1.02
Flue Gas
a b
CO2 in air is assumed as 0.04%. Each data indicates the mean ± SD and were measured from three independent cultures.
31
Appendix A. Supplementary material
Fig A.1 Schematic diagram of CO2 fixation process in bubble column reactors for batch microalgal cultivation.
32
Fig A.2 Experimental set–up of three-stage photobioreactors-in-series utilizing actual flue gas. Reactors were continuously supplied with flue gas or pure CO2 during light cycle and air during dark cycles. External illumination of light was provided with 106.6 µmolm−2 s−2 in a 12:12 light: dark photoperiod
33
Highlights: 1. CO2 fixation from industrial flue gas using Chlorella sp. 2. Chlorella sp. tolerates gas stream with 5% of CO2 v/v. 3. Different strategies to minimize toxic effect of flue gas compounds were evaluated. 4. Biofixation efficiency was improved by 54%.
S0960-8524(15)00538-6 http://dx.doi.org/10.1016/j.biortech.2015.04.040 BITE 14881
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
29 January 2015 14 April 2015 15 April 2015
Please cite this article as: Yadav, G., Karemore, A., Dash, S.K., Sen, R., Performance evaluation of green microalgal CO2 sequestration in closed photobioreactor using in situ generated flue gas, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.04.040
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Performance evaluation of green microalgal CO2 sequestration in closed photobioreactor using in situ generated flue gas Geetanjali Yadava, Ankush Karemorea, Sukanta Kumar Dashb, Ramkrishna Sena* a
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302 India Department of Mechanical Engineering, Indian Institute of Technology Kharagpur, West Bengal 721302 India
b
*Corresponding author Dr. Ramkrishna Sen Professor Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India. Phone (O): +913222283752, Fax: +913222278707 Email: [email protected]
Abstract In the present study, carbon-dioxide capture from in-situ generated flue gas was carried out using Chlorella sp. in bubble column photobioreactors to develop a cost effective process for
2
concomitant carbon sequestration and biomass production. Firstly, a comparative analysis of CO2 sequestration with varying concentrations of CO2 in air-CO2 and air-flue gas mixtures was performed. Chlorella sp. was found to be tolerant to 5% CO2 concentration. Subsequently, inhibitory effect of pure flue gas was minimized using various strategies like use of high initial cell density and photobioreactors in series. The final biofixation efficiency was improved by 54% using the adopted strategies. Further, sequestered microalgal biomass was analyzed for various biochemical constituents for their use in food, feed or biofuel applications. Keywords Microalgae, CO2 fixation, photobioreactor-in-series, coal-fired flue gas 1. Introduction The anthropogenic activities such as excessive use of fossil fuel reserves, deforestation and intensive industrialization has led to unceasing rise in greenhouse gas emissions. In recent times, the CO2 concentration in the atmosphere has reached an alarming level of 400 ppm (Tans, 2015). It is envisaged that CO2 levels greater than 450 ppm could be destructive to global climate (Hansen et al., 2007). Therefore, demand for effective carbon-dioxide (CO2) mitigation technologies is the need of hour. Of all the post combustion CO2 capture technologies viz. chemical and physical absorption, geological and oceanic storage, and biological fixation; microalgal mediated CO2 fixation offers several advantages such as faster growth rates and higher CO2 fixation rates (10-50 times more) than terrestrial plants. Hence, microalgae are regarded as prime candidates for biological fixation of CO2 (Yoo et al., 2010; Cheng et al., 2013). As a result of CO2 fixation, microalgal biomass accumulates significant amounts of lipids, carbohydrates, proteins and other valuable compounds, such as pigments and vitamins, which
3
can be used as active ingredients in nutraceutical, food and feed supplements or in the production of biofuels. (Francisco et al., 2010; Cheah et al., 2015). An effective microalgal cultivation system is designed in order to achieve high surface area per volume ratio and good hydrodynamics to give more surface area for the light penetration and gaseous CO2 transfer (Cheah et al., 2015). An efficient CO2-fixation system must ensure good mixing, high gas-liquid transfer rates, and even distribution of light. Broadly, microalgal cultivation is done in two types of systems, open and closed. Usually open microalgal cultivation is carried out in raceway ponds which are low cost, ease in operation and maintenance but suffer from poor light penetration, contamination and poor biomass productivity. On the other hand, closed system includes different configuration of photobioreactors (PBRs) (Air-lift, flat plate, tubular) and generally have better mass transfer rates, higher productivity and better control over process parameters than open ponds. Due to their simplicity, robustness and relatively high process efficiency, closed system especially bubble column reactors are widely used (Kumar et al., 2012). Various strains of microalgae have been utilized for CO2 fixation. Chlorella sp. is widely studied strain which has been used in several industrial applications and found to be a fast growing microalgal strain capable of fixing CO2 from flue gases (Van Den Hende et al., 2012). It contains high amount of protein (51–58%), carbohydrate (12–17%) and lipid (14–22%) that could be used for various applications like health food, nutritional supplements, animal feed, biofuels etc. (Becker, 1994; Cheah et al., 2015). Flue gas is inexpensive and rich source of CO2, approximately 400 times more concentrated than atmospheric CO2 and thus can be exploited for microalgal mediated CO2 fixation (Cheah et al., 2015). However, the major constraints are due to the presence of toxic compounds such as NOx, SOx and CO which are inhibitory for microalgal growth and biomass productivity (Kumar et al., 2014), primarily due to acidification of the
4
growth medium (Van Den Hende et al., 2012). Flue gas from various sources has been used to cultivate microalgae for CO2 fixation (Van Den Hende et al., 2012, Kumar et al., 2014). The direct use of flue gas is detrimental for microalgal growth and generally requires expensive pretreatments processes. The efficiency of CO2 fixation from flue gas by microalgae generally found to be less than 50% (Cheng et al., 2013). In order to treat flue gas by microalgae in a costeffective manner, there is need to develop effective strategies which will not only reduce the cost of its pretreatment but also increase CO2 fixation. Therefore, in this study, strategies were developed to directly use coal-fired flue gas for the cultivation of microalgae and simultaneous flue gas remediation. Firstly, a comparative study was conducted to evaluate the performance of Chlorella sp. for CO2 fixation using pure CO2 and waste flue gas. Subsequently, inhibitory effect of pure flue gas was minimized using various strategies like use of high initial cell density and photobioreactors in series. Further, commercial potential of the sequestered microalgal biomass was assessed for their use in food, feed or biofuel applications.
2. Material and methods 2.1. Strain and culture medium The strain Chlorella sp. (Chlorophyta, Chlorophyceae) obtained in this experiment was procured from National Environmental Engineering Research Institute, Nagpur, India (Fulke et al., 2010). The inoculum was grown on modified Bold Basal’s medium (Nichols and Bold,1965) and has the following composition (mg L-1): NaNO3 (750), CaCl2 · 2H2O (12.5), MgSO4 · 7H2O (150), FeSO4 (6.27), K2HPO4 (62.4), KH2PO4 (225), NaCl (0.341), H3BO3 (5), MnSO4 (0.72), ZnSO4 ·7H2O (17.64), KOH (15.5), NaCl (12.5), CuSO4 · 7H2O (1.06), NaMoO3 (0.6), CoCl2 (0.2). The
5
strain was maintained in liquid culture (100 mL) in an Erlenmeyer’s flask (150 ml), at 28 ± 2 °C, under intermittent agitation and continuous illumination (24 µ mol m−2 s−1) and an atmosphere of air 0.04% CO2 (v/v). Initial pH of the medium was adjusted to 6.8 ± 0.1. All chemicals used are of analytical grade and procured from Merck®, India. All experiments were performed in triplicates and expressed as mean with standard deviation (S.D). 2.2. Photobioreactor, Experimental set-up and batch culture conditions Batch experiments were performed in bubble column glass photobioreactors (BCR) (length, 33. cm; inner diameter, 4.5 cm; working volume, 500 mL). The average temperature during batch experiment was maintained at 28 ± 2 °C. Chlorella sp. was cultured in three bubble column PBRs connected in series and labeled sequentially as PBR 1st, PBR 2nd and PBR 3rd. Flue gas was fed into the sparger of PBR 1st and effluent from this PBR was fed to PBR 2nd and similarly the effluent from this PBR was fed to PBR 3rd (Supplementary Fig A.1 and A.2). The initial pH of medium was noted to be 6.8 and the rate of flow of gas into the reactor was maintained at 0.5 vvm. Carbon-dioxide or flue gas was fed only during the light period and its supply was stopped during the dark period, however air was fed continuously. The light intensity falling from one side of reactor through cool white fluorescent tubes was measured using quantum meter (HTC LX 102 Lux Meter) to be approximately 106.6 µ moles m-2 s-1 during the entire batch experiment in a 12:12 hr (light/dark) photoperiod and. Ambient air or CO2 enriched air was supplied through an aquarium sparger (Φdiameter = 1.0 cm) located at the bottom of the BCR. The flue gas produced from burning of coal in the flue gas generator is mixed with air to create different v/v mixtures of air-flue gas before it is filtered using syringe filter and injected into the culture photobioreactors. The concentrations of air-pure CO2 as well as air-flue gas mixture were adjusted and set to desired concentrations of 2.5%, 5%, 7.5%, 10% and 0.04%
6
(ambient air) of the total mixture. A single batch culture was incubated for 7 days. Samples were removed once every 24 hours to determine biomass concentration and CO2 fixation rate and twice every 24 hours to check the culture pH. Sample pH was determined using a pH meter (Deluxe pH Meter, Model LT-10). The effect of inoculum density was investigated by varying the cell density of initial inoculum viz. 0.06 g L-1, 0.1 g L-1, 0.15 g L-1 and 0.2 g L-1 respectively. Subsequently, the best inoculum density was selected to carry out the photobioreactor-in-series experiment to minimize the toxic effects of flue gas components in sequential reactors (Supplementary Fig A.2). 2.3.Flue Gas Analysis The flue gas used in the present study was obtained after burning of coal procured from Kolaghat Thermal Power Station (KTPS), West Bengal India. An indigenously designed and fabricated flue gas generator was used for burning the coal and collecting in-situ generated flue gas. The flue gas generator was equipped with three-chambered double layered jacket in which water was circulated to cool down the temperature of flue gas from 120° C to approximately 45 °C. The raw flue gas was collected from the upper position of stack and passed through a vacuum precipitator to remove the suspended particulate matter by a compressor pump (1HP). The flue gas was then transferred through pipelines to storage gas tank (volume: 41.3 liters, pressure: 10 kg cm-2) before feeding it to the microalgal PBR. The flue gas was analyzed by a portable on-line Flue gas analyzer (Model: Indus FGA 53X). A schematic illustration of the flue-gas based cultivation system for Chlorella sp. is shown in Supplementary Fig. A.2.The flue gas was analyzed by a portable on-line Flue gas analyzer (Model: Indus FGA 53X). The typical composition of the flue gas was 10 ± 2% (v/v) carbon dioxide, 0.554% (v/v) carbon-monoxide,
7
8.33% (v/v) oxygen, 61 ppm nitrogen oxides, 0.3% (v/v) sulphur oxides, 9 ppm other hydrocarbons. 2.4.Biomass concentration, Biomass productivity and Nitrate consumption profile Biomass concentration (dry weight per liter) of microalgal cells was measured by taking optical density at 750 nm (O.D750nm) in Agilent Cary 60 Scan UV/Visible spectrophotometer (Griffiths et al., 2011). Dry cell weight (dwt) was calculated using a calibration plot between Dwt and O.D. Dry weight biomass (g L-1) = 0.44 × O.D750nm. Sampling was done every 24 hours to determine the cell concentration for further algal growth calculation. Each sample was diluted to give an absorbance in the range of 0.1–0.5 if the optical density was greater than 1.0. This is due to the fact that probability of obtaining erroneous result becomes higher as the cell density crosses O.D value > 1. The maximum biomass concentration achieved in culture was designated as Xmax (gL1
). The overall biomass productivity Px (g biomass L-1d-1) in the batch experiment was calculated
using the following equation: ∆
= ∆
(1)
Where ∆ and ∆ are the total amount of biomass and total cultivation time (days) respectively. The nitrate concentration was calculated following the method of Armstrong, (1963) given in Eq. (2). = . − 2#. $% &
(2)
2.5.Determination of maximal CO2 biofixation rate To produce 1 g of dry microalgal biomass, approximately 1.83 g of CO2 is required to be fixed assuming 50% of carbon is fixed in the microalgal biomass (Cheah et al., 2015). Maximal CO2 biofixation rate F (g d-1) was calculated from Eq. (3), as described by Pegallapati and Nirmalakhandan, (2013).
8
' ( ) * , , = -
(3)
Where, ‘a’ is the carbon dioxide fixed by unit biomass (considering 50% of carbon in the 00
biomass) a = 0.5 )= 1.833 g CO2 (g dry cell)-1); and V is the culture volume (= 0.5 L), is the volumetric productivity. 2.6.Analysis of lipid, carbohydrate and protein content The total lipids were extracted from freeze-dried microalgal biomass using a modified Folch method (Folch et al., 1957). Firstly, freeze-dried algal biomass was transferred into a chloroform/ methanol (2:1, v/v) mixture and homogenized for 10 minutes using pestle and mortar. This was followed by centrifugation at 300 rpm for 5 minutes. The lower chloroform phase containing lipids was collected and dried overnight inside oven at 60° C to remove the chloroform. The difference of final and initial weight gives the estimation for lipid content (%). Carbohydrates were extracted from lyophilized microalgal biomass following acid hydrolysis (NREL, 2011). The dried lyophilized microalgal biomass was subjected to a two-step acid hydrolysis. In the first step, approximately 50 mg biomass was dissolved in 0.5 ml of 72% H2SO4 for 30 min in 30° C water bath. Further, the concentration of H2SO4 was reduced and brought to 4% by the addition of distilled water. In the next step, this mixture was incubated at high temperature of 121° C (preferably by autoclaving) for 1 hour. After the required incubation the samples are neutralized using sodium carbonate till the effervescence ceased and pH reaches to a value of approximately 7. The samples are then centrifuged at 3500 rpm for 6 minutes and the supernatant is used for phenol-sulfuric acid assay to analyze the carbohydrate concentration (Dubois et al., 1956), which was determined by measuring the absorbance at 490 nm (Agilent Cary 60 Scan UV/Visible spectrophotometer).
9
Proteins were extracted using alkali hydrolysis using 0.5N NaOH and incubation at 80 °C for 10 minutes. The supernatant after centrifugation is used for Bradford’s assay. The protein concentration was measured by taking O.D at 595nm. Results and discussion
3.1. Use of CO2 and flue gas for growing microalgae Microalgae utilize CO2, nutrients and light for its growth via autotrophic metabolism. Thus, CO2 fixation could be positively correlated with increased biomass productivity. In general, microalgae can tolerate various concentrations of CO2 (10–50%) for its growth (Cheah et al., 2015). Chlorella sp. normally exhibits satisfactory growth with CO2 concentrations ranging from 1% to 18% v/v (Hulatt and Thomas, 2011) but it can also grow at extreme conditions of up to 50% CO2 concentration (Maeda et al., 1995). CO2 feeding into the microalgal culture system leads to mass transfer of CO2 gas into aqueous media, followed by its solubilization in different forms such as CO2, H2CO3, HCO3− and CO32− (depending upon pH of the medium), and finally its uptake by microalgae through the process of photosynthesis (Van Den Hende et al., 2012).
3.1.1. Growth of Chlorella sp. using varying concentrations of CO2 To investigate the effect of varying CO2 concentrations on growth, Chlorella sp. was incubated with different concentrations of CO2 viz. 0.04%, 2.5%, 5%, 7.5% and 10% for 7 days at 28 ±2 °C. The batch experiments were carried out till nitrate was completely exhausted. The time course profile of growth of Chlorella sp. is illustrated in Fig. 1 (a). The most suitable CO2 concentration for microalgal growth was found to be 5%, above which the growth started declining as shown in Fig. 1(a). The maximum biomass concentration and productivity of 2.2045 ± 0.12 g L-1 and 0.314 ± 0.004 g L-1d-1was obtained at 5% CO2 level. Previous reported studies
10
also showed that CO2 concentration above 5% could be inhibitory to microalgal growth and CO2 biofixation (Chiu et al., 2008, Chiu et al., 2009; de Morais and Costa, 2007, Cheah et al. 2015). Effect of increasing CO2 concentrations on pH change was monitored as illustrated in Fig. 1(b). It was found that increasing the CO2 concentrations in culture medium resulted in decrease of pH from 6.8 to 6.4. The pH was controlled for initial 36 h with the addition of potassium hydroxide solution. Addition of pH solution during the initial stage of growth and for the brief period of time helps in achieving stable microalgal growth (Lee et al., 2000). Later, as the microalgal growth progressed towards exponential phase, the pH stabilized in the range of 9-10. However, when air was used for aeration, no pH drop was observed. This is due to very low concentration of CO2 (0.04%, v/v) in air and poor mass transfer (Tans, 2015). Also, at very low CO2 concentrations, ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), a key enzyme of Calvin cycle mainly performs its oxygenase activity, resulting in poor CO2 fixation. On the contrary, when there is sufficient CO2 in the culture, carboxylase activity of RuBisCO catalyses CO2 fixation which in turn leads to expression of another important enzyme i.e. carbonic anhydrase (CA). CA increases the alkalinity in medium by transporting hydroxide ions outside the cell while keeping the H+ ions within the thylakoid membranes (Kumar et al. 2012). Increasing the CO2 concentration upto 10% led to significant decline in pH initially, though later, due to increase in biomass growth, pH improved to alkaline range due to the normal photosynthetic activity (Hulatt and Thomas, 2011). This may also be due to tolerance of Chlorella sp. to the elevated levels of CO2. Nitrate consumption by microalgae also results in rise of culture pH during photosynthesis (Hulatt and Thomas, 2011). The Xmax and Px values gradually decreased with the increase in CO2 concentration above 5% (i.e. at 7.5% and 10% CO2, v/v) (Table 1). Chiu et al. (2008) also reported that microalgal cell
11
growth was significantly reduced when Chlorella sp. cultures were treated with high concentration of CO2 gas. The average rate of CO2 fixation in cultures at 0.04%, 2.5%, 5%, 7.5% and 10% CO2 in the photobioreactor for 0.5 L working volume was 196 ± 10 mg CO2-1day-1, 221± 40 mg CO2 -1day-1, 287± 7 mg CO2 -1day-1, 274 ± 30 mg CO2 -1day-1 and 249 ± 11 mg CO21
day-1 respectively. The most efficient CO2 fixation and sequestration rates were also obtained at
5% CO2 concentrations above which it started declining. Nitrate was utilized for the production of biomass; Table 1 shows residual nitrate concentration at the end of 7 days of batch experiment. It was observed that a decreasing trend of nitrate consumption was obtained with increasing biomass concentration (Chen et al., 2012). 3.1.2. Growth of Chlorella sp. using varying concentrations of actual flue gas Coal-fired flue gas contains upto 10-15% CO2 (v/v) and numerous compounds like H2O, O2, N2, nitrogen oxides (NOx), sulphur oxides (SOx), unburned hydrocarbons (CxHy), CO, heavy metals, halogen acids and particulate matter (Van Den Hende et al., 2012). The growth of Chlorella sp. with addition of actual coal-fired flue-gas at different concentration was investigated. The flue gas was diluted with air to vary its CO2 concentration in the range of 2.5%, 5%, 7.5% and 10% (v/v). Effect of various concentration of flue gas on biomass growth and pH profiles is shown in Fig.2 (a) and (b). Flue gas feeding into microalgal culture resulted in decline of the medium pH value from 6.8 to 5.9. Potassium hydroxide solution was added to maintain the pH value for initial 48 h. Flue gas contains acidic gases such as SOx, NOx, and CO2, which decreases the medium pH upon dissolution (Kumar et al., 2014). It is generally reported that direct use of flue gas results in slower microalgal growth rate (Richmond and Zou, 1999). This may be due to time taken by the microalgae to adapt the harsh conditions (Kumar et al., 2014). The Chlorella sp. showed a lag
12
phase of one day and later adapted itself with time to produce biomass. The study by Kastanek et al. (2010) reported a lag time of 3 days for growing Chlorella sp. with flue gas. Subsequently, pH increased via normal photosynthetic process and reached alkaline range of 9-10 as shown in Fig 2 (b). The biomass concentration of Chlorella sp. aerated with flue gas at different concentrations; 0.04%, 2.5%, 5%, 7.5% and pure flue gas (~10%) CO2, v/v were 1.502 ± 0.011 g L-1, 1.601 ± 0.025 g L-1, 1.915 ± 0.12 g L-1, 1.8065 ± 0.12 g L-1 and 1.4 ± 0.015 g L-1 respectively. Comparatively, the biomass concentration and productivity of microalga grown using 5% CO2 in flue gas was approximately 13% lower than that obtained in 5% pure CO2 feeding (Table 1). Kumar et al. (2014) used Chlorella sorokiniana to sequester CO2 from flue gas and also observed 5% CO2 as the most optimum concentration. However, significant improvement in overall biomass production by about 21% and 36% was achieved at 5% flue gas and 5% CO2 (v/v), respectively, with respect to air. The maximum CO2 sequestrations and biofixation rate of 0.974 ± 0.131 g L-1 and 250 ± 13 mg CO2-1 day-1was also obtained for 5% CO2 concentration in flue gas. The greater CO2 fixation rates achieved with dilution of flue gas could be due to decreased concentration of CO2 and toxic gases. Further, upon using pure flue gas, the microalgal growth as well as fixation rates reduced due to the presence of inhibitory compounds in the flue gas (Van Den Hende et al., 2012). Since, direct use of flue gas feeding into microalgal cultures resulted in lowest CO2 fixation rate of 175 ± 10 mg CO2-1 day-1 ; there is a need to search for alternative methods which can directly use the flue gas efficiently for higher biomass production as well as CO2 fixation. 3.2 Overcoming flue gas inhibition using different techniques
13
Although direct use of flue gas is a cheaper technique to sequester carbon-dioxide by microalgae, the presence of toxic compounds like SOx, NOx in addition to high concentrations of CO2 (~1015%, v/v) imposes extreme conditions on microalgal growth (Van Den Hende et al., 2012). Besides, the presence of acidic gases in flue gas not only affects the culture pH substantially but also prolongs the lag phase of microalgal growth (Kumar et al., 2014). Several flue gas pretreatment strategies have been devised to scrub off the high concentration of inhibitory compounds from flue gas; viz. passing through water to remove oils and dust particle, use of scrubbers (Kumar et al., 2014), use of chemical solvents like euchlorine scrubbing solution (Deshwal and Lee, 2009), BioDeNOx process (Kumaraswamy et al., 2005), etc. In our study, the harmful effects caused by direct use of flue gas was attenuated by adopting following strategies; variation in initial cell density and ‘photobioreactor-in-series’ for concomitant biomass production and CO2 fixation. 3.2.1 Effect of flue gas on microalgal culture at different initial cell density The effect of actual flue gas containing 10% CO2 (v/v) was determined on microalgal growth at different initial cell densities (t=0 h). It is reported that inoculum density influences the productivity and economics of bioprocesses (Sen and Swaminathan, 2004; Chiu et al., 2008) and has positive effect on subsequent biomass production (Richmond et al., 2003; Chen et al., 2012). Experiments were conducted to find a suitable inoculum concentration that will overcome the initial lag phase as observed earlier in Fig. 2(a) during batch growth of Chlorella sp. with flue gas (10% CO2 in flue gas). The study was conducted with Chlorella sp. using four different initial inoculum densities comprising of 0.06 g L-1, 0.1 g L-1, 0.15 g L-1 and 0.2 g L-1 concentration. The most suited initial cell density was observed to be 0.15 g L-1 resulting in highest biomass concentration and CO2 fixation rate of 2.13 ± 0.01 g L-1 and 0.259 ± 0.01 g CO2-
14 1
day-1, respectively. It is evident that the microalgal cultures with lower initial inoculum densities
could not adapt well to direct exposure of flue gas and hence resulted in lower biomass yield, biomass productivity, and CO2 sequestration and fixation rate as illustrated in Table 2. Nevertheless, with the higher inoculum densities of 0.15 g L-1 and 0.2 g L-1, there was no significant difference in the biomass yield. However, it is worth noting from Fig.3 (a) that inoculum density of 0.2 g L-1 reached the stationary phase earlier on 5th day of incubation than inoculum density of 0.15 g L-1. Hence, lower productivity in this case could be due to the phenomenon of ‘autoshading’ resulting in poor light penetration (Kenekar and Deodhar, 2013). The initial medium pH of 6.8 decreased gradually with flue gas feeding as shown in Fig. 3(b). The pH value of culture inoculated with different initial inoculum densities of 0.06 g L-1, 0.1 g L1
, 0.15 g L-1 and 0.2 g L-1 was dropped to about 5.5, 5.9, 6.0 and 6.2, respectively. High-density
cultures have shown better ability to combat the harsh conditions put forward by toxic flue gas elements and therefore double its population faster (Chiu et al., 2008). It has been reported that regulating pH and increasing the inoculating cell density are effective ways to prevent growth inhibition exerted by flue gas (Lee et al. 2002). Nitrate concentration decreased for all four variable initial inoculum densities at the end of entire batch experiment (Table 2). The present study stands out in its approach by avoiding the prolonged lag phase and inhibitory effect encountered of flue gas on microalgal growth by using optimal inoculum density. Chlorella sp. grew well in a high-density culture and achieved better CO2 fixation rates (Table 2). The result is in accordance with previous studies where high CO2 and flue gas tolerance of microalgae was dependent on cell density (Lee et al., 2002, Yoshihara et al., 1996 and Chiu et al 2008). Thus, it can be inferred that direct flue gas could be utilized to grow microalgae for CO2
15
biofixation. This optimal inoculum density was further used in subsequent experiment of ‘PBRin-series’. 3.2.2 Use of sequential photobioreactors for CO2 fixation from flue gas To employ inexpensive strategies for direct usage of flue gas, another innovative approach of ‘photobioreactor-in-series’ was investigated for microalgal cultivation. Photobioreactor systems with three-staged bubble column reactors connected in series were labeled PBR 1st, PBR 2nd and PBR 3rd. The inoculum density of 0.15 gL-1was selected to carry out the batch experiment in all the three PBRs. There was no significant difference in the biomass yield observed in PBR 1st and PBR 2nd. However, biomass concentration in PBR 2nd was slightly higher than PBR 1st with the value of 2.225 g L-1 and 2.13 g L-1, respectively (Table 2). Since PBR 1st received direct supply of flue gas, high concentrations of CO2 as well as inhibitory compounds present in the flue gas resulted in lesser biomass growth due to lowering of pH as compared to PBR 2nd. Upon feeding the flue gas into serially connected photobioreactors, consumption of some CO2 might have occurred during the process of photosynthesis and dissolution of some toxic gases in the culture medium of the preceding reactor which resulted in better productivities of PBR 2nd. Similar observation was reported by Kumar et al. (2014), by using the reactor-in-series strategy to fix CO2 from industrial flue gas and observed major reduction in CO2 (4.1%, v/v) along with other hydrocarbons in the serially connected PBRs. Previously, de Morais and Costa, (2007) have used three-stage serial tubular photo-bioreactors to assess the growth of Scenedesmus obliquus and Spirulina sp. with respect to CO2 fixation in different concentrations of CO2 and found an increase in biomass production as well as CO2 fixation towards the downstream reactors. PBR 3rd, located at the end of serially connected PBRs, showed lower biomass growth of 1.5 g L-1, which might be due to lesser gas flow rate and also could be due to further dilution
16
of CO2 concentration in flue gas (Lee et al., 2000; Morais and Costa, 2007). Another reason might be due to simultaneous decrease in CO2 concentration and increase in oxygen build up in the downstream reactors (Suzuki and Ikawa, 1984). 3.3 Analysis of carbohydrate, protein and lipid content CO2 present in flue gas can be used to steer the biochemical composition of microalgae like lipid and fatty acids, proteins, polysaccharides and pigments (Van Den Hende et al., 2012; Cheng et al., 2014; Tsuzuki et al., 1990). The impact of CO2 concentration on the synthesis of major metabolites such as lipid, protein and carbohydrates content were measured (Table 3). Algal cells grown with 5% and 10% CO2 accumulated about 12.72% and 5.52% of total lipids respectively. In contrast, when grown in air, Chlorella sp. accumulated approximately 15.84% of total lipids. Cheng et al. (2014) also reported decrease in total lipids accumulation in Chlorella vulgaris when grown at 2% CO2 concentration as compared to air. The reason for difference in lipid percentage could be either strain specificity or tolerance of microalgae to elevated CO2 concentrations (Cheng et al., 2014). As summarized in Table 3, the decreasing trend of lipid content was obtained with increasing CO2 concentrations both with pure CO2 as well as flue gas grown microalgae. Similar trend were also reported by Elvira-Antonio et al. (2013) by using different sodium bicarbonate concentration as a measure of dissolved CO2 in the culture medium. Furthermore, flue gas treated samples received much lesser lipid accumulations at elevated CO2 concentrations (Table 3). This could be due to the presence of different flue gas components which could have directed the microalgal biochemical machinery to synthesize other valuable components like carbohydrates, proteins, pigments etc. (Kumar et al., 2014; Yoo et al., 2010; Tsuzuki et al., 1990).
17
Unlike the decreasing trends observed in case of lipids accumulation, carbohydrate and protein contents of the microalgal cultures increased upon CO2 or flue gas feeding. The increase in carbohydrate and protein contents was significant (Table 3). Cheng et al. (2014) also reported that cell wall carbohydrate content in C. vulgaris, C. sorokiniana and C. variabilis increased significantly in CO2 enriched conditions due to overall increase in the uronic acid fractions. At high CO2 concentrations, there was substantial increase in total protein and carbohydrate content from 58.39–59.38% and 20.4–24.48%, respectively, in 10% pure CO2 and actual flue gas. Generally, microalgae having higher CO2 fixation ability at elevated CO2 concentrations, Chlorella sp., Scenedesmus sp. and Spirulina sp., are found to contain high amount of important metabolites (Anjos et al., 2013; Tanadul et al., 2014; Cheah et al., 2015). Significant differences were observed in the biochemical composition of microalgal cells under varying CO2 concentrations in flue gas and hence it should be utilized for various applications in food, feed or biofuel. 4. Conclusions A comparative analysis of microalgal CO2 sequestration using different concentrations of pure and flue gas CO2 was accomplished. Significant improvement in overall biomass production by about 21-36% was achieved at 5% CO2 (v/v). Considering the fact that direct supply of flue gas inhibits microalgal growth, reactor strategies involving the use of PBR-in-series with high initial (t= 0 h) biomass density worked well in improving CO2 fixation rate, total carbohydrate and protein contents. Thus, this study offers the proof of the concept of mitigating flue gas CO2 for the production of algal biomass, carbohydrates and proteins for various potential applications. Acknowledgements
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Geetanjali Yadav acknowledges West Bengal Government-Department of Science and Technology (Project Grant No. 560 (SANC.)/ST/P/S&T/5G-5/2011; Date: 21-11-2011) for providing fellowship support. The authors are thankful to Dr. Sukhendu Kumar Das for his valuable suggestions. The authors are also grateful to Dr. Tapan Chakrabarti of National Environmental Engineering Research Institute, Nagpur, India for giving Chlorella sp. as a gift. GY also acknowledges the help of Mr. Lakshmikanta Dolai with flue gas set-up operation.
References 1. Anjos, M., Fernandes, B.D., Vicente, A.A., Teixeira, J.A., Dragone, G. 2013. Optimization of CO2 bio-mitigation by Chlorella vulgaris. Bioresour Technol. 139, 149–154. 2. Becker, E.W., 1994. Microalgae: biotechnology and microbiology. In: Baddiley J, Carey NH, Higgins IJ, Potter WG (eds) Cambridge studies in biotechnology. Cambridge University Press, New York, p 178. 3. Chen, Y., Wang, J., Liu, T., Gao, L., 2012. Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp. J. Appl. Phycol. 24, 1623–1627. 4. Cheng, J., Huang, Y., Feng, J., Sun, J., Zhou, J., Cen, K., 2013. Improving CO2 fixation efficiency by optimizing Chlorella PY-ZU1 culture conditions in sequential bioreactors. Bioresour. Technol. 144, 321–327. 5. Cheng, Y.S., Labavitch, J.M., Vander, G.J.S., 2014. Elevated CO2 concentration impacts cell wall polysaccharide composition of green microalgae of the genus Chlorella. Appl. Microbiol. 60 (1), 1-7.
19
6. Cheah, W.Y., Show, P.L., Chang, J.S., Ling, T.C., Juan, J.C., 2015. Biosequestration of atmospheric CO2 and flue gas-containing CO2 by microalgae. Bioresour. Technol. 184, 190– 201. 7. Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S., 2008. Reduction of CO2 by a high-density culture of Chlorella sp. in a semicontinuous photobioreactor. Bioresour. Technol. 99 (9), 3389-3396. 8. Chiu, S.Y. Kao C.Y., Tsai, M.T. Ong, S.C. Chen, C.H. Lin C.S., 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresour. Technol. 100, 833–838. 3. Deshwal, B.R., Lee, H.K., 2009. Mass transfer in the absorption of SO2 and NOx using aqueous euchlorine scrubbing solution. J. Environ. Sci. 21, 155–161. 4. de Morais, M.G., Costa, J.A.V., 2007. Isolation and selection of microalgae from coal fired thermoelectric power plant for biofixation of carbon dioxide. Energy Convers. Manage. 48, 2169–2173. 5. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., 1956. Calorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. 6. Elvira-Antonio, N., Ruíz-Marín, A., Canedo-López, Y., 2013. Effect of nitrogen content and CO2 consumption rate by adding sodium carbonate in the lipid content of Chlorella vulgaris and Neochloris oleoabundans. Int. J. Envir. Protect. 3(10), 13-19. 7. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol Chem. 226, 497-509.
20
8. Francisco, É.C., Neves, D.B., Jacob‐Lopes, E., Franco, T.T., 2010. Microalgae as feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality. J. Chem. Technol. Biotechnol. 85, 395–403. 9. Fulke, A.B., Mudliar, S.N., Yadav, R, Shekh., A, Srinivasan. N., 2010. Bio-mitigation of CO2, calcite formation and simultaneous biodiesel precursors production using Chlorella vulgaris. Bioresour. Technol. 101(21), 8473-8476. 10. Griffiths, M.J, Garcin, C., van Hille, R.P., Harrison, S.T., 2011. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J. Microbiol Meth. 85(2), 119-123. 11. Hansen, J., Sato, M., Ruedy, R., Kharech, P., Lacis, A., Miller, R., 2007. Dangerous humanmade interference with climate: a GISS model study. Atmos. Chem. Phys.7, 2287–312. 12. Van Den Hende, S., Han, V., Boon, N., 2012. Flue gas compounds and microalgae: (Bio-) chemical interactions leading to biotechnological opportunities. Biotech. Adv. 30(6), 14051424. 13. Hulatt, C. J., Thomas, D. N., 2011. Productivity, carbon dioxide uptake and net energy return of microalgal bubble column photobioreactors. Bioresour. Technol. 102, 5775–5787. 14. Kenekar, A. A., Deodhar, M. A., 2013. Effect of varying physicochemical parameters on the productivity and phycobiliprotein content of indigenous isolate Geitlerinema sulphureum. Biotechnol. 12 (3), 146-154. 15. Kastanek, F., Sabata, S., Solcova, O., Maleterova, Y., Kastanek, P., Branyikova, I., Kuthan, K., Zachleder, V., 2010. In-field experimental verification of cultivation of microalgae Chlorella sp. using the flue gas from a cogeneration unit as a source of carbon dioxide. Waste Manage. Res. 28 (11), 961–966.
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16. Kumar, K., Das, D., 2012. Growth characteristics of Chlorella sorokiniana in airlift and bubble column photobioreactors. Bioresour. Technol. 116, 307–313. 17. Kumar, K., Banerjee, D., Das, D., 2014. Carbon dioxide sequestration from industrial flue gas by Chlorella sorokiniana. Bioresour. Technol. 152, 225–233. 18. Kumaraswamy, R., Dongen, U.V., Kuenen, J.G., Wiebe, A., Loosdrecht, M.C.M.V., Muyzer, G., 2005. Characterization of microbial communities removing nitrogen oxides from flue gas: the BioDeNOx Process. Appl. Environ. Microbiol. 71 (10), 6345–6352. 19. Lee, J. N., Lee, J. S., Shin, C. S., Park, S. C., Kim, S. W., 2000. Methods to enhance tolerances of Chlorella KR-1 to toxic compounds in flue gas. Appl. Biochem. Biotechnol. 84–86 (1-9), 329-342. 20. Lee, J.S., Kim, D.K., Lee, J.P., Park, S.C., Koh, J.H., Cho, H.S., Kim, S.W., 2002. Effects of SO2 and NO on growth of Chlorella sp. KR-1. Bioresour. Technol. 82 (1) 1–4 21. Maeda, K., Owada, M., Kimura, N., Omata, K., Karube, I., 1995. CO2 fixation from the fluegas on coal-fired thermal power-plant by microalgae. Energy Convers. Manage. 36, 717–20. 22. National Renewable Energy Laboratory 2011. www.nrel.gov/biomass/analytical_procedures.html 23. Pegallapati, A. K., Nirmalakhandan, N., 2013. Internally illuminated photobioreactor for algal cultivation under carbon dioxide-supplementation: Performance evaluation. Renw. Energy. 56, 129-135. 24. Richmond A, Zou N., 1999. Efficient utilization of high photon irradiance for mass production of photoautotrophic micro-organisms. J. Appl. Phycol. 11,123–127.
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25. Sen, R., Swaminathan, T., 2004. Response surface modeling and optimization to elucidate and analyze the effects of inoculum age and size on surfactin production. Biochem. Engg. J.21(2),141–148. 26. Suzuki, K., Ikawa, T., 1984. Effect of Oxygen on Photosynthetic 14CO2Fixation in Chroomonas sp. (Cryptophyta) I. Some Characteristics of the Oxygen Effect. Plant Cell Physiol. 25(3), 367-375. 27. Tans, P., "Trends in Carbon Dioxide". NOAA/ESRL. Retrieved 2015-03-05. 28. Tanadul, O.U.M, VanderGheynst, J. S., Beckles, D. M. Powell, A. L.T., Labavitch, J.M., 2014. The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock. Biotechnol. Bioengg. 111 (7), 1323-31. 29. Tsuzuki, M., Ohnuma, E., Sato, N., Takaku, T., Kawaguchi, A., 1990. Effects of CO2 concentration during growth on fatty acid composition in microalgae. Plant Physiol. 93, 851– 856. 30. Yoo, C., Jun, S.Y., Lee, J.Y., Ahn, C.Y., Oh, H.M., 2010. Selection of microalgae for lipid production under high level of carbon dioxide. Bioresour. Technol. 101, 71–74. 31. Yoshihara, K., Nagase, H., Eguchi, K., Hirata, K., and Miyamoto, K., 1996. Biological elimination of nitric oxide and carbon dioxide from flue gas by marine microalga NOA-113 cultivated in a long tubular photobioreactor. J. Ferment. Bioeng. 82, 351–354. Figure Captions Fig. 1 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with different concentrations of CO2. Each data indicates the mean ± SD and were measured from three independent cultures. External illumination of light was provided with 106.6 µmolm−2 s−2 in a 12:12 light: dark photoperiod
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Fig. 2 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with different concentrations of CO2 in flue gas. Each data indicates the mean ± SD and were measured from three independent cultures.
Fig. 3 Time-course profiles of (a) growth and (b) pH of Chlorella sp. supplemented with pure flue gas with different initial cell density. Each data indicates the mean ± SD and were measured from three independent cultures.
Fig. 4 Time-course of growth and pH profile for Chlorella sp. using actual flue gas sparged to three-stage serially connected bubble column reactor.
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Table 1 Biomass concentration, Productivity and CO2 sequestration of CO2 and Flue gas cultivated algae in different concentrations of CO2 Growth Biomass Biomass CO2 Carbon Residual conditions concentration Productivity sequestration dioxide Nitrate content (g L-1)b (g L-1d-1) (g L-1) fixation rate (mg L-1) -1 (mg CO2 day 1 ) (For 0.5L)c CO2-air ratio
Aira
1.502 ± 0.011
0.214 ± 0.002
0.712 ± 0.001
196 ± 5
24.66
2.5
1.7 ± 0.034
0.242 ±0.11
0.799 ± 0.04
221± 4
-
5
2.2045 ± 0.12
0.314 ± 0.004
1.19 ± 0.077
287± 7
-
7.5
2.097 ± 0.03
0.295 ± 0.045
1.111 ± 0.003
274 ± 3
-
10
1.902 ± 0.13
0.271 ±0. 067
0.970 ± 0. 011
249 ± 11
-
Air
1.502 ± 0.011
0.214 ± 0.002
0.712 ± 0.001
196 ± 5
24.66
2.5
1.601 ± 0.025
0.228 ± 0.033
0.762 ± 0.055
208 ± 5
2.1
5
1.915 ±0.12
0.273 ± 0.012
0.974 ± 0.131
250 ± 13
-
7.5
1.8065 ±0.12
0.258 ±0.023
0.865 ± 0.102
236 ± 10
8.122
Pure FG
1.4 ± 0.015
0.191 ±0.11
0.7 ± 0.0133
175 ± 10
112.11
Flue gas-air ratio
a
CO2 in air is assumed as 0.04%.
b
Each data indicates the mean ± SD and were measured from three independent cultures.
c
Carbon fixation rates were calculated for 0.5 L working volume of microalgal culture.
- Not detectable
29
Table 2 Biomass productivity, CO2 sequestration ,CO2 fixation rate and residual nitrate concentration in experiments with different inoculum density and reactor in series for growing Chlorella sp. with actual flue gas Growth Biomass Biomass CO2 Carbon Residual Nitrate conditions concentration Productivity sequestration dioxide content (g L-1)a (g L-1d-1) (g L-1) fixation rate (g (mg L-1) CO2-1day-1) (For 0.5L)b Inoculum Density 1X
1.4 ± 0.014
0.192 ±0.011
0.705 ± 0.054
176± 10
11.2 ± 1.87
2X
1.981 ± 0.02
0.268 ± 0.124
0.990± 0.012
246± 32
-
3X
2.13 ± 0.01
0.282 ± 0.015
1.065 ± 0.044
259 ± 10
-
4X
2.014 ± 0.27
0.259 ±0. 071
1.007 ± 0. 001
237± 8
1.11 ± 0.0012
Reactor 1
2.13 ± 0.02
0.282 ± 0.12
1.065 ± 0.011
259 ± 7
102.88 ± 0.0003
Reactor 2
2.225 ±0.12
0.296 ± 0.044
1.112 ± 0.09
271 ± 11
-
Reactor 3
1.501 ±0.112
0.193 ±0.053
0.75 ± 0.011
170 ± 65
233.55 ± 0.0054
Reactor in series
a
Each data indicates the mean ± SD and were measured from three independent cultures.
b
Carbon fixation rates were calculated for 0.5 L working volume of microalgal culture.
- Not detectable
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Table 3 Effect of flue gas on Lipid, Carbohydrate and Protein production in Chlorella sp. cultures Growth Biomass Protein (%) Carbohydrate Lipids (%) conditions concentration (%) (gL-1)b
Aira
1.5 ± 0.01
51.3 ± 2.13
12.24 ± 1.2
15.84 ± 1.4
5%
2.2 ± 0.12
55.7 ± 1.5
14.02 ± 0.86
12.72 ± 1.7
10%
1.9 ± 0.13
59.38 ± 2.01
20.4 ± 1.7
5.52 ± 0.85
5%
1.92 ±0.12
56.8 ± 3.02
19.88 ± 1.43
8.65 ± 0.97
Pure FG
1.41 ±0.02
58.39 ± 1.3
24.48 ± 1.3
4.45 ± 1.02
Flue Gas
a b
CO2 in air is assumed as 0.04%. Each data indicates the mean ± SD and were measured from three independent cultures.
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Appendix A. Supplementary material
Fig A.1 Schematic diagram of CO2 fixation process in bubble column reactors for batch microalgal cultivation.
32
Fig A.2 Experimental set–up of three-stage photobioreactors-in-series utilizing actual flue gas. Reactors were continuously supplied with flue gas or pure CO2 during light cycle and air during dark cycles. External illumination of light was provided with 106.6 µmolm−2 s−2 in a 12:12 light: dark photoperiod
33
Highlights: 1. CO2 fixation from industrial flue gas using Chlorella sp. 2. Chlorella sp. tolerates gas stream with 5% of CO2 v/v. 3. Different strategies to minimize toxic effect of flue gas compounds were evaluated. 4. Biofixation efficiency was improved by 54%.