Bioresource Technology 152 (2014) 225–233

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Carbon dioxide sequestration from industrial flue gas by Chlorella sorokiniana Kanhaiya Kumar, Debopam Banerjee, Debabrata Das ⇑ Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India

h i g h l i g h t s  CO2 sequestration from flue gas of oil producing company using Chlorella sorokiniana.  Techniques for minimizing inhibitory effect of high concentration of CO2 and H2S.  Comparison of fatty acids composition of pure CO2 and flue gas sequestered biomass.  Flue gas caused increase in fatty acids chain length and degree of unsaturation.  Flue gas caused synthesis of some additional pigments and metabolites.

a r t i c l e

i n f o

Article history: Received 1 August 2013 Received in revised form 24 October 2013 Accepted 28 October 2013 Available online 9 November 2013 Keywords: Chlorella sorokiniana Airlift reactor Flue gas Fatty acids Pigments

a b s t r a c t The present study investigated the feasibility of using Chlorella sorokiniana for CO2 sequestration from industrial flue gas. The flue gas emitted from the oil producing industry contains mostly CO2 and H2S (15.6% (v/v) and 120 mg L1, respectively) along with nitrogen, methane, and other hydrocarbons. The high concentration of CO2 and H2S had an inhibitory effect on the growth of C. sorokiniana. Some efforts were made for the maximization of the algal biomass production using different techniques such as diluted flue gas, flue gas after passing through the scrubber, flue gas passing through serially connected photobioreactors and two different reactors. The highest reduction in the CO2 content of inlet flue gas was 4.1% (v/v). Some new pigments were observed in the flue gas sequestered biomass. Fatty acid composition in the total lipid was determined to evaluate its suitability for food, feed, and biofuel. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The threat of global warming is becoming severe because of increasing CO2 concentration in the atmosphere. In 2011, the rate of increase in atmospheric CO2 was 1.94 ppm/year; more than twice the estimated value in 1959. Presently, CO2 is contributing nearly 52% in total global warming (Velea et al., 2009). Biological CO2 sequestration from flue gas is gaining attention because of its eco-friendly and cost effective nature (Kumar et al., 2013, 2011). Use of algae for CO2 sequestration has multiple advantages. Photosynthetic efficiency of microalgae is nearly 10 times greater than that of terrestrial plants (Skjanes et al., 2007). In addition, they are the source of renewable energy and their biomass can be utilized for the production of high value products. Algae can grow in open pond as well as in closed photobioreactors. The advantages of closed photobioreactors are high productivity, easy

⇑ Corresponding author. Tel.: +91 03222 278053; fax: +91 3222 255303. E-mail addresses: [email protected], [email protected] (D. Das). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.098

operation, better control over physiochemical parameters, and sterile operation. Airlift and bubble column photobioreactors have wide acceptance for algal cultivation because of their simplicity in gas–liquid contacting application (Kastanek et al., 2010). Airlift bioreactor is of special interest because of higher mass transfer, regular light and dark cycle, low and homogeneous shear stress to the cells (Kumar and Das, 2012; Vunjak-Novakovic et al., 2005). CO2 sequestration by biological means is limited only to laboratory-scale using air–CO2 gas mixture. Till today, very scarce in situ research is available on algal cultivation using flue gas. Component and composition of flue gas vary with the source of its generation. High temperature, presence of toxic gases like NOx, SOx, H2S, and particulate matters of flue gas are also creating problems in operation for CO2 sequestration using algae. Flue gas components cause acidification to the medium and poses environmental stress to algae. Some of the sources of flue gas used for cultivation of algae are incineration units (Kastanek et al., 2010), coal fired thermal power plant (Maeda et al., 1995), and fossil fuel fired plant (Zeiler et al., 1995). The algae such as C. vulgaris BEIJ 1890 (Kastanek et al., 2010), Chlorella sp. T1 (Maeda et al., 1995), M. minutum

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K. Kumar et al. / Bioresource Technology 152 (2014) 225–233

(Zeiler et al., 1995), Tetraselmis sp. (Matsumoto et al., 1995) are mostly used for this purpose. Previously, continuous supply of flue gas into the culture was found to have an inhibitory effect on the growth of the algal cells (He et al., 2012). Therefore, isolation of SOx, NOx tolerant algae, the addition of CaCO3 to maintain pH near to 7, controlling the pH drop by addition of NaOH were some of the strategies adopted to overcome the flue gas inhibition on the growth of the microalgae (Jiang et al., 2013; Westerhoff et al., 2010). However, SOx and NOx tolerant algae were effective only at lower concentrations of these acidic gases whereas neutralization with NaOH was costly and caused undesirable effects on algae due to high ionic strengths. Recently, on–off pulse of flue gas was adopted by some of the researchers to overcome the flue gas inhibition on algal cells (He et al., 2012; Jiang et al., 2013; Chiu et al., 2011). He et al. (2012) reported frequency of 10 s on-time and 5–9 min off-time as an effective strategy for overcoming high CO2 stress. However, employing this strategy required continuous stirring of the culture for mixing and sophisticated instrument to control the frequency of the flue gas injection. Chlorella sp. grows much quicker and considered as algal weed because of its ability to survive under harsh and high oxidizing environmental conditions. It is also known as a good source of carbohydrates, lipids, proteins, and vitamins. The algal biomass can be used as raw material as single cell protein for human consumption (Mahasneh, 1997) or as feed for fish in aquaculture systems (Hamasaki et al., 1998). Moreover, the physiological response to high CO2 concentration may give rise to several useful metabolites and pigments. Previously, carotenoids have been shown to protect algal cells from photooxidative damage (Siefermann-Harms, 1985). The carotenoids (such as astaxanthin, canthxanthin, lutein, and bcarotene) have significant commercial importance. It has also been reported that carotenoids may inhibit carcinogenesis due to their antioxidant activity. Milbemycins b are a group of macrocyclic lactones with a highly potent antiparasitic activity (Prichard, 2005) besides having anthelmintic and insecticidal properties. Pregnan20-one is a neuroactive steroid which plays a vital role in stress, pregnancy, and CNS neurotransmission (Khisti et al., 2003). These kinds of compounds can be used to aid neurogenesis and epileptic disorders. Further, fatty acids produced from algae have nutritional, biofuel, and pharmaceutical properties. For example, oleic acid has a positive effect on cardiovascular diseases and lowers cholesterol level (Beyhan et al., 2011) whereas lauric, palmitic, linoleic, oleic, stearic, and myristic acids have antibacterial and antifungal properties (Agoramoorthy et al., 2007). In addition, lauric acid is used for manufacturing of cocoa butter, flavourings, alkyd resins, soaps, shampoos, and other surface active agents, including special lubricants. Eicosenoic acid (C20:1) and erucic acid (C22:1) have usefulness in cosmetic products as they provide a protective layer over the skin. Similarly, tricosanoic acid (C23:0) is used in the food and beverage industry. The main objective of the present research work was to determine in-field feasibility of C. sorokiniana for sequestrating CO2 from flue gas. It also aimed to study the effect of stress conditions induced by flue gas on pigments and fatty acid composition.

2. Methods 2.1. Microalgae and culture medium The culture of C. sorokiniana was obtained from Dr. Kari Skjanes (Bioforsk, Norway). Modified TAP (-acetate) medium was used in all experiments (Kumar and Das, 2012). All the experiments were conducted at field condition where temperature was found to vary from 25 to 40 °C. Airlift and bubble column photobioreactors were

designed and fabricated as described by Kumar and Das (2012). The volume of both airlift and bubble column reactors were 1.4 L. The rate of flow of gas into the reactor was 0.33 vvm. Culture was constantly illuminated with cool white fluorescent tubes. Light intensity falling on the surface of reactor from one side was measured approximately 120 lmol m2 s1 as measured with a quantum meter (LX-101, Lutron, Taiwan). 2.2. Scrubber A slurry of zinc oxide (ZnO) of 6.25 g L1 was used as a scrubber for removing the H2S present in the flue gas. In order to ensure continuous mixing of the slurry, Bubble column reactor was used as a vessel. Zinc oxide reacts with hydrogen sulfide and makes zinc sulfide and water as shown in the following Eq. (1).

ZnOðsÞ þ H2 SðgÞ ZnSðsÞ þ H2 OðgÞ:

ð1Þ

2.3. Gas analysis Flue gas of oil producing industry was used in the present study. Gas composition was analyzed by GC (Agilent) using a thermal conductivity detector (TCD) with N2 as a carrier gas. The gas composition of inlet flue gas was 15.65% (v/v) carbon dioxide, 72.79% (v/v) nitrogen, 0.02% (v/v) hexane, 10.63% (v/v) methane, 0.54% (v/v) ethane, and 0.36% (v/v) other hydrocarbons. H2S was measured separately using a piston type hand pump that was used to draw a fixed volume of the gas through a calibrated glass tube filled with lead acetate. 2.4. Dry weight, biomass productivity, and net specific growth rate determination Optical density (OD) of cells was determined in a spectrophotometer at 682 nm (Chemito). Dry cell weight (Dwt) was calculated using a calibration plot between Dwt and OD. Carbon present in dry cell weight of microalgae was assumed as 50% (w/w) which corresponds to a requirement of 1.83 g of CO2 for the production of 1 g dry cell weight of microalgae (Kumar and Das, 2012). The overall biomass productivity Poverall (g L1 d1) in the batch was calculated using the following Eq. (2).

Poverall ¼

DX Dt

ð2Þ

Net specific growth rate, lnet (d1) was calculated from Eq. (3)

lnet ¼

DlnX Dt

ð3Þ

where DX and DlnX are the total amount of biomass and difference of natural logarithmic biomass of initial and final cultivation time, respectively with total cultivation time of Dt (Kumar and Das, 2012; Jiang et al., 2013). 2.5. Growth kinetics using logistic equation Logistic equation was used to determine the growth kinetics of algae. It can explain the entire growth profile (lag, exponential, and stationary phase) without taking substrate consumption into consideration (Kumar and Das, 2012). The following is the logistic Equation.

dC C ¼ K c C ð1  Þ dt C max

ð4Þ

where C, Cmax, and Kc are the dry cell weight (g L1), maximum dry cell weight (g L1), and apparent specific growth rate (d1). On integration and rearrangement, Eq. (4) takes the form of Eq. (5)

K. Kumar et al. / Bioresource Technology 152 (2014) 225–233

C¼

1þ



C max C max Co

  1 eK c t

ð5Þ

where Co is the initial biomass concentration (g L1). These constants were determined by fitting Eq. (5) in experimental data in MATLAB (ver. 7.9.0.529) using the curve fitting tool. A confidence bound of 95% was taken into consideration to find the fit. 2.6. Lipid extraction, preparation of fatty acid methyl esters (FAME), and determination of fatty acids profile At the end of the CO2 sequestration in the duplicate experimental set ups, algal suspension was centrifuged (4000g, 10 min) and the biomass was lyophilized. Lyophilized biomass was further used for lipid extraction. Lipid from algal samples was extracted using methods described by Bligh and Dyer (1959) and FAME was prepared as described in Dey et al. (2011). FAME sample was analyzed by gas chromatography (PerkinElmer, Clarus 500) fitted with Omegawax-250 capillary column (30 m length, 0.25 mm internal diameter, and 0.25 lm film thickness, Sigma) using a flame ionization detector (FID). Chromatographic peaks were identified by comparing to a FAME standard mixture (Supelco 37-Component FAME Mix, Sigma). 2.7. Extraction of carotenoids and identification using thin layer chromatography (TLC) and gas chromatography–mass spectrometric (GC/MS) analysis Biomass collected at the end of algal cultivation was centrifuged (4000g, 10 min) and lyophilized. For the extraction of carotenoids, 0.5 g of the lyophilized biomass was dissolved in MeOH:CHCl3 (1:1). The mixture was then sonicated and kept at 4 °C overnight. Steps were repeated with same biomass until completely depigmented. Thin layer chromatography of the MeOH:CHCl3 (1:1) extract was done using silica gel GF254 as absorbent and acetone:hexane (1:1) as solvent system and Rf values of spots were noted. The probable carotenoids and other secondary metabolites were identified by GC–MS analysis. Mass spectra of the extracts were detected with a m/z scan range at 500–600 nm and mass fragmentation data was matched with existing library (NIST/Wiley). 3. Results and discussion 3.1. Strategies for overcoming flue gas inhibition 3.1.1. Dilution of flue gas using air Flue gas was diluted with air and used for the cultivation to find the suitable concentration of air–flue gas mixture. This gas mixture was passed through water to remove oil and dust particle present in it before passing into the reactor. Schematic diagram of experimental set-ups are shown in Supplementary data sheet. Direct use of flue gas was not found encouraging. Dry cell weight obtained in it was significantly lower than the result obtained in air at similar experimental conditions (Fig. 1). Further, comparing flue gas and pure 15% (v/v) CO2 in previous reports by Kumar and Das (2012), maximum dry cell weight obtained in case of the former was significantly reduced. Moreover, direct flue gas induced a slow growth of C. sorokiniana probably because of time taken by algae for adapting the stress conditions. This observation was similar to Kastanek et al. (2010) who observed a lag time of 3 days in the presence of flue gas. Mixing the flue gas with air enhanced the biomass productivity. However, the maximum biomass, total biomass productivity, and net specific growth rate in air–flue gas mixture in 3:1 proportions were only slightly higher as compared to air–flue gas mixture (1:1) (Table 1). In both the conditions growth profile

227

was found to be overlapping with each other. The higher amount of biomass production on dilution may be because of decreased concentration of CO2 and toxic gases in the inlet of the reactor. Anyhow, it did not improve the biomass production significantly as compared to air most probably due to the comparatively poor reduction of toxic gases into the reactor. Final pH was reduced to nearly 6.8 ± 0.3 when different composition of diluted flue gas was used whereas in case of air the final pH was alkaline. Flue gas contains acidic gases and therefore have tendency to decrease the pH of the medium. Ammonia present in the standard TAP medium further declines the pH as NH4+ uptake by algae leads to H+ production (Goldman and Brewer, 1980). However, modified TAP (-acetate) medium (ammonia substituted by nitrate) was found controlling the sharp decline in the pH (Kumar and Das, 2012). This may be due to the consumption of nitrate in the medium by algae, which increases the alkalinity and pH via OH production (Hulatt and Thomas, 2011; Goldman and Brewer, 1980). 3.1.2. Reactors in series Three different airlift reactors were connected in series. The strategy was similar to reports of de Morais and Costa (2007) where CO2 fixation was carried out in three serially connected bubble column photobioreactor having working volume 1.8 L each with Spirulina species (de Morais and Costa, 2007). The results are summarized in Table 1 and Fig. 2. There was an increase in the maximum biomass production and CO2 sequestration on sparging serially from one reactor to the next. This may be due to the flue gas passed through one reactor; there was consumption of some CO2 during the process of photosynthesis and dissolution of some toxic gases in the culture medium of the previous reactor. Further, the outlet CO2 concentration in the flue gas of 3rd airlift reactor was reduced by 4.1% (v/v). However, equivalent biomass obtained in air–flue gas mixture (3:1) can be extrapolated for CO2 reduction of 4.9% (v/v). These were in accordance with the report that less than 5% (v/v) of CO2 can be sequestered from the input CO2 rich gas stream having more than 1% (v/v) of CO2 (Milne et al., 2009). However, the rate of CO2 utilization is dependent on the amount of CO2 content in the gas mixture as well as their flow rate. For example, higher CO2 reduction is expected when gas mixture is introduced in the reactor in the on–off pulse fashion. These may be some of the reasons for the higher CO2 reduction reported by some of the researchers (Jiang et al., 2013; Chiu et al., 2011; de Morais and Costa, 2007). Along with the reduction in CO2 major hydrocarbons present in the gas was also decreased (1.9% of total hydrocarbon) probably because of their oxidation in the presence of oxygen in the bioreactor during algal cultivation. This has some resemblance as reported by Mann et al. (2009). Furthermore, there was a slight increase in the final pH was observed on moving serially from one reactor to another. This may be due to the improvement of the growth characteristics of the microorganism in the subsequent photobioreactors. 3.1.3. Effect of scrubber and reactors Bubble column reactor was used as scrubber for the pretreatment of flue gas. This gas was considered for the photobioreactor. Airlift and bubble column reactors were used for the cultivation of C. sorokiniana. H2S content in the flue gas was reduced to 20–25 mg L1 from 120–130 mg L1 after passing through the scrubber. Maximum biomass, total biomass productivity and net specific growth rate obtained in case of airlift bioreactor were 1.23 g L1, 0.17 g L1d1, and 0.46 d1, respectively and that of bubble column bioreactor were 1.12 g L1, 0.15 g L1d1, and 0.44 d1, respectively after pretreating flue gas with scrubber (Fig. 3). Improvement of CO2 sequestration compared to direct flue gas may be due to the decrease in H2S concentration. Effect of pH

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K. Kumar et al. / Bioresource Technology 152 (2014) 225–233

2.0

1.5

10

6 1.0

pH

8

-1

Dry cell weight (g L )

12

Air_Dwt 25% (v/v) flue gas_Dwt 50% (v/v) flue gas_Dwt 100% (v/v) flue gas_Dwt Air_pH 25% (v/v) flue gas_pH 50% (v/v) flue gas_pH 100% (v/v) flue gas_pH

4 0.5 2

0.0

0 0

2

4

6

8

10

Time (d) Fig. 1. Comparative growth and pH profile of C. sorokiniana in different dilution of flue gas.

Table 1 Effect of different strategies on CO2 sequestration using C. sorokiniana. Strategies

Air/flue gas

Reactors in series

Dry cell weight (g L1)

Total biomass productivity (g L1 d1)

Net specific growth rate, lnet (d1)

CO2 sequestration Remarks (g L1)

Flue gas pretreated with 0.85 water do 1.14

0.1

0.36

1.56

0.16

0.45

2.09

do

1.68

0.23

0.5

3.07

With different percent of flue Air 0.95 gas 25% Flue gas 1.1 50% Flue gas 0.98 100% Flue gas 0.68 Effect of scrubber and Flue gas pretreated with 1.23 different reactors scrubber 1.12

0.13 0.15 0.13 0.08 0.17 0.15

0.42 0.44 0.43 0.33 0.46 0.44

1.74 2.01 1.79 1.24 2.25 2.05

may not be the influencing factor as the pH profiles were similar in all the experimental conditions using flue gas. Airlift reactor was found to be better as compared to the bubble column reactor. The increase in cell mass concentration may be due to the better light distribution to the cells in defined axial circular mixing patterns of media in an airlift reactor compared to zig-zag mixing pattern in bubble column due to the presence of multiple dead zones (Kumar and Das, 2012). Similarly in airlift reactor, most of the time cells were found residing in the downcomer (photic zone) compared to that of upcomer (dark zone). 3.1.4. Kinetic constants of logistic model Logistic model was found to give reasonably good fit with all the experimental results. The maximum biomass concentration (Cmax) and apparent specific growth rate (Kc) were significantly higher in all cases compared to direct flue gas (Fig. 4). The logistic kinetic constants Co, Cmax, and Kc with direct flue gas were 0.09 g L1, 0.93 g L1, 0.4 d1 (R2 = 0.98). However, in air–flue gas mixture in 3:1 proportions, logistic constants Co, Cmax, and Kc were 0.1 g L1, 0.98 g L1, and 1.3 d1 (R2 = 0.93), respectively. Similarly, predicted Co, Cmax, and Kc of last airlift reactor connected in series were 0.08 g L1, 1.8 g L1, 0.8 d1 (R2 = 0.97), respectively. The logistic kinetic constants Co, Cmax, and Kc with pretreated flue gas using scrubber in an airlift reactor were 0.1 g L1, 1.2 g L1, 1.07 d L1 (R2 = 0. 99). The predicted inoculum concentration was

1st Airlift reactor 2nd Airlift reactor 3rd Airlift reactor Airlift reactor Airlift reactor Airlift reactor Airlift reactor Airlift reactor Bubble column

nearly similar compared to added inoculum in each of the experiments (0.06 g L1). Though the predicted maximum biomass concentration was higher for 3rd airlift in the series compared to 25% (v/v) flue gas diluted with air, the apparent specific growth rate was higher in case of later. Photobioreactors in the series were found most promising. The CO2 present in flue gas was found better than that of atmospheric air under similar experimental conditions. Dry cell weight in the 3rd airlift reactor placed in series was nearly 84% (w/w) compared to 15% CO2–air gas mixture in lab conditions (Kumar and Das, 2012). It was nearly 2.5–3 times lower than the optimal data of CO2 sequestration using the same strain in 5% (v/v) CO2 gas mixture (Kumar et al., 2013; Kumar and Das, 2012). This result is compared with reported data on CO2 sequestration from industrial flue gas using algae (Table 2). The strategy of using reactors in series yielded nearly similar result as of intermittent mixing of flue gas resulting in achieving 90% (w/w) of the theoretical biomass growth under optimal conditions (He et al., 2012). Jiang et al. (2013) reported maximum biomass of 3.63 g L1 using intermittent sparging of simulated flue gas (15% (v/v) CO2, 400 mg L1 SO2, 300 mg L1 NO, balance N2) to S. dimorphus (Jiang et al., 2013). Relatively lower maximum biomass production in the present study may be because of uncontrolled experimental condition and use of real flue gas having large number of unknown components. Similarly in another report, maximum biomass and growth rate

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K. Kumar et al. / Bioresource Technology 152 (2014) 225–233 12

2.0 1st reactor_Dwt 2nd reactor_Dwt 3rd reactor_Dwt 1st reactor_pH 2nd reactor_pH 3rd reactor_pH

10

8

6

1.0

pH

-1

Dry cell weight (g L )

1.5

4 0.5 2

0.0

0 0

2

4

6

8

10

Time (d) Fig. 2. Comparative growth and pH profile of C. sorokiniana in serially connected airlift reactors.

1.4

12

1.2

10

8 0.8 6

pH

Dry cell weight (g L-1)

1.0

0.6 4 0.4

Untreated flue gas_Airlift_Dwt Pretreated flue gas_Airlift_Dwt Untreated flue gas_Airlift_pH Pretreated flue gas_Airlift_pH Pretreated flue gas_Bubble column_Dwt Pretreated flue gas_Bubble column_pH

0.2

0.0 0

2

4

6

8

2

0

10

Time (d) Fig. 3. Effect of scrubber and reactors on the growth and pH profile of C. sorokiniana.

Dry cell weight (g/L)

2 1.8

Experimental_direct flue gas Predicted_direct flue gas

1.6

Experimental_air-flue gas mixture (3:1) Predicted_air-flue gas mixtur (3:1) Experimental_use of scrubber in airlift

1.4

Predicted_use of scrubber in airlift

1.2

Experimental_3rd airlift reactor in series Predicted_3rd airlift reactor in series

1 0.8 0.6 0.4 0.2 0

0

1

2

3

4

5

6

7

8

Time (d) Fig. 4. Comparative simulated cell growth profile of C. sorokiniana in direct flue gas, air–flue gas mixture (3:1), pretreated flue gas with scrubber and 3rd airlift reactor of serially connected photobioreactor. Experimental data is fitted with logistic equation. Data points are average of experimental runs.

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K. Kumar et al. / Bioresource Technology 152 (2014) 225–233

Table 2 CO2 sequestration using green algae from industrial flue gas. Flue gas producing company

Gas composition

Microorganisms

Photobioreactor

CO2 removal efficiency (%)

Biomass production

References

Co-generation Power Plant at MIT

CO2 (8%), NOx (20 ppm)

Dunaliella parva (UTEXLB1983) Dunaliella tertiolecta (UTEXLB999)

Traingular airlift

–

VunjakNovakovic et al. (2005)

Tedom Ltd., Czech Republic Isogo Thermal Power Plant –

CO2 (8–10.2%), NOx (46 mg m3), SO2 (10 mg m3) CO2 (13%), NOx (150 ppm), SOx (10 ppm) CO2 (42%), CH4 (58%), H2S (438 ppm) CO2 (15%) SO2 (400 ppm) NO (300 ppm) CO2 (23 ± 5%) SO2 (87 ± 9 ppm) NO (78 ± 4 ppm) CO2 (15.65%), CH4 (10.63%), H2S (130 ppm)

Chlorella vulgaris BEIJ 1890

0.26 g L1 d1

Kastanek et al. (2010)

Chlorella sp. T-l

Fat vertical prismshaped closed bubble reactor –

82.3 ± 12.5 (Sunny days) 50.1 ± 6.5 (In cloudy days) –

–

1 g L1

Chlorella vulgaris, SAG 211-11b

–

Scenedesmus dimorphus

Cylindrical intermittent mixing of flue gas Cylindrical intermittent mixing of flue gas

CO2 (1.2%), CH4 (50.1%) H2S (0%) –

3.63 g L1

Maeda et al. (1995) Mann et al. (2009) Jiang et al. (2013) Chiu et al. (2011)

–

Oil producing industry

Chlorella sp. MTF-7

Chlorella sorokiniana

of Chlorella sp in 50 L photobioreactor were 2.87 g L1 and 0.52 g L1d1 in 6 days of cultivation using intermittent flue gas sparging (Chiu et al., 2011). It can be extrapolated that the addition of a number of reactors in series can further enhance the biomass productivity of individual reactor. It can be hypothesized that after diluting the flue gas with air followed by passing through the scrubber and introducing in a serially connected airlift reactor may enhance the biomass productivity. In the present study, all the experiments were conducted in batch. Continuous culture can provide better understanding of effect of flue gas and therefore it has been kept for future scope of study.

3.2. Lipid and pigment content of algal biomass 3.2.1. Fatty acids analysis Lipid content in flue gas grown algal biomass (FGGAB) was 21.1 ± 0.54% (w/w). This was almost similar compared with 5% (v/v) CO2 – air grown algal biomass (CAGAB) mixture having lipid content of 20.93 ± 0.49% (w/w). Previously, total lipid content in pure CO2 and flue gas grown biomass of Chlorella sp. was found nearly equal to 25.2% (w/w) (Chiu et al., 2011). Decrease in the lipid content of algal biomass was reported at higher CO2 content in gas (Yoo et al., 2010). The stresses imposed by flue gas may probably neutralize the negative effect on lipid content of algal biomass. FGGAB was rich in many fatty acids such as (C14:0), (C16:0), (C18:0), (C18:1), (C18:2), and (C20:1). However, (C12:0), (C14:0), (C16:0), and (C18:0) fatty acids were present in higher amounts in CAGAB (Table 3; Fig. 5). The observed fatty acids were similar to those reported in representatives of Chlorophyceae class (Leonardi et al., 2011). Similarly, (C16:0), (C18:1) were found to be major fatty acids in fresh and marine microalgae (Barman et al., 2012). The amount of (C20:1) was higher in FGGAB compared to CAGAB. Some fatty acid though in small amount was present only in either of two samples such as (C15:1) in CAGAB and (C17:0) in FGGAB. While the relative percent of saturated fatty acids (SFA) were reduced to 49.36% (w/w) in FGGAB from 80.39% (w/w) in CAGAB, the concentration of monounsaturated fatty acid (MUFA), and polyunsaturated fatty acids (PUFA) were increased in FGGAB. FGGAB was containing many PUFAs such as (C18:2), (C18:3), and (C20:5) contrary to the presence of (C18:3), lone PUFA in CAGAB. It can be interpreted that the stress imposed by flue gas helps in

Serially connected airlift

–

2.87 g L1, 0.52 g L1 d1

CO2 (4.1%)

1.68 g L1, 0.23 g L1d1

Present study

increasing the degree of unsaturation in the fatty acid carbon chain. CAGAB was containing mostly medium chain fatty acids (C10–C15). However, it was reduced nearly half in FGGAB. Long chain fatty acids (C16–C18) and very long chain fatty acids (PC20) were increased by 31% (w/w) and 64.6% (w/w), respectively in FGGAB compared to CAGAB. Very long fatty acids, commonly of pharmaceutical importance were enhanced greatly in FGGAB probably because of the stress imposed by the flue gas similar to previous reports (Cagliari et al., 2011). So, it can be deduced from the result that the stress imposed by flue gas helps in the biosynthesis of higher chain fatty acids. Quality of biodiesel is defined based on the ignition quality (cetane number), cold flow properties, and oxidative stability. High amount of unsaturated fatty acids improves cold flow properties but causes a poor cetane number and oxidative stability whereas the effect of the high amount of saturated fatty acids has vice versa effect on biodiesel. So, biodiesel formed from the lipid of C. sorokiniana grown in flue gas may be attractive in all three parameters of biodiesel because of the presence of nearly equal amount of saturated and unsaturated fatty acids. In addition, this strain was found to be a good source of essential fatty acids such as palmitoleic acid, oleic acid, linoleic acid, a linolenic acid, and erucic acid which was synthesized only in the stress conditions of flue gas. 3.2.2. TLC and GC–MS analysis of the secondary metabolites The total carotenoid content in FGGAB was 6.35 ± 0.15 lg, a significantly higher value than that of CAGAB having 4.4 ± 0.03 lg mg1 of algal biomass. The carotenoids and other metabolites were identified by TLC and GC–MS. Pigments detected in TLC plate of both the samples were neoxanthin, violaxanthin, astaxanthin, chlorophyll c, and b-carotene (Fig. 6A). However, lutein was also detected in FGGAB in TLC. The data obtained from the GC–MS analysis showed the clear distinction between the carotenoid composition of CAGAB and FGGAB. Different pigments synthesized in CAGAB and FGGAB as detected in mass spectrometry (MS) are shown in Fig. 6B and C. In addition to different pigments, some important metabolites such as pregnan-20 one3,17,21-tris-trimethylsilyl-oxyl and milbemycin-b were also present in FGGAB. The findings can be attributed to the increased tolerance of C. sorokiniana biomass to the elevated levels of CO2 and secondary metabolites production correlating the results to that reported in Dunaliella sp. (Graham and Wilcox, 2000). These

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K. Kumar et al. / Bioresource Technology 152 (2014) 225–233 Table 3 Comparison of fatty acids composition in total lipid of pure CO2 (5%, v/v) and flue gas sequestered biomass of C. sorokiniana. Fatty acids

Algal biomass grown in 5% CO2 (relative percent of fatty acid)

Algal biomass grown in flue gas (relative percent of fatty acid)

Capric acid (C10:0) Lauric (C12:0) Myristic (C14:0) 10-Pentadecenoic (C15:1) Palmitic (C16:0) Palmitoleic acid (C16:1) Margaric (C17:0) Stearic (C18:0) Oleic (C18:1) Linoleic acid (C18:2) a Linolenic acid (C18:3) Arachidic (C20:0) Eicosenoic acid (C20:1) Eicosapentaenoic acid (C20:5) Erucic acid (C22:1) Tricosanoic acid (C23:0) Other unknown fatty acids MUFA PUFA Saturated fatty acids (SFA) Medium chain fatty acids (C10-C15) Long chain fatty acids (C16-C18) Very long chain fatty acids (PC20)

4.6 16.23 19.53 2.21 16.42 3.67 – 16.41 2.12 – 1.82 6.2 1.72 – 3.1 1.0 5.0 12.81 1.82 80.39 42.57 40.44 12.03

2.43 8.7 11.2 – 11.5 2.2 Trace 10.3 15.7 10.0 3.0 4.4 11.3 Trace 2.7 0.8 5.8 31.31 13.6 49.36 22.33 52.70 19.2

(A)

250

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0 2

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150

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Fig. 5. Fatty acids profile of (A) standards, C. sorokiniana grown in (B) pure CO2 (5%, v/v), and (C) flue gas.

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Fig. 6. A: TLC of the MeOH:CHCl3 (1:1) extract from 5% CO2 sequestered (in lane 1) and flue gas sequestered (in lane 2) biomass of C. sorokiniana. The probable compounds predicted by matching with standard Rf values (Lorenz, 1998; Owens et al., 1987) are (A) Neoxanthin (Rf = 0.08), (B) Violaxanthin (Rf = 0.22), (C) Astaxanthin (Rf = 0.33), (D) Lutein (Rf = 0.43), (E) Chlorophyll a (Rf = 0.68), and (F) b-carotene (Rf = 0.95). B: MS spectra of 5% CO2 sequestered biomass of C. sorokiniana (A = b-carotene, B = b-carotene 3,4didehydrol, C = Lutein, D = Astaxanthin). C: MS spectra of flue-gas sequestered biomass of C. sorokiniana (A = b-carotene, B = Canthaxanthin, C = b-carotene 3,4-didehydrol, D = Lutein, E = Pregnan 20-one, F = Ketozeaxanthin, G = Melbemycin b, and H = Astaxanthin).

results suggest that FGGAB of C. sorokiniana can be a potential source of natural products with commercial applications. 4. Conclusion The high concentration of CO2 and H2S present in the flue gas had a negative effect on the CO2 sequestration process. Among the various strategies adopted, result was found most promising when reactors were connected in series. Both the length of the fatty acids and their degree of unsaturation in algae were increased in case of using flue gas. Fatty acid analysis showed its suitability for food, feed, fuel, and other commercially important products. Some of the pigments were enhanced in algal biomass due to stress induced by flue gas along with additional induction of few newly formed pigments.

Acknowledgements The authors gratefully acknowledge Council of Scientific and Industrial Research (CSIR), Govt. of India for senior research fellowship; Royal Norwegian Embassy, New Delhi, India (project BioCO2), Department of Biotechnology (DBT) and Ministry of New and Renewable Energy (MNRE), Govt. of India for the financial support. Authors also acknowledge the valuable suggestions of Prof. S. Ray, Department of Chemical Engineering, IIT Kharagpur.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.10.098.

K. Kumar et al. / Bioresource Technology 152 (2014) 225–233

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