Development of a Process for an Efficient Exploitation of CO2

Article pubs.acs.org/IECR

Development of a Process for an Efficient Exploitation of CO2 Captured from Flue Gases as Liquid Carbonates for Chlorella protothecoides Cultivation Barbara Gris,† Eleonora Sforza,*,† Luca Vecchiato,‡ and Alberto Bertucco† †

Department of Industrial Engineering DII, University of Padova, Via Marzolo 9, 35131 Padova, Italy Eco Management S.r.l., Via Emilia 7, 35043 Monselice (PD), Italy

‡

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S Supporting Information *

ABSTRACT: The use of bicarbonate as carbon supply in a large scale photobioreactor for industrial microalgae production could reduce the compression and transportation costs associated with the direct use of CO2 from flue gas. Some microalgae species are able to directly exploit bicarbonate as a carbon source, but the actual yield in view of large scale production is not clear, in particular for eukaryotic and lipid-rich strains. In this work, the exploitation of bicarbonate as carbon source by Chlorella protothecoides was critically assessed. Both batch and continuous experiments were carried out with the addition of NaHCO3 to the culture medium. The growth of this species was found to be strongly affected by the increased pH, resulting in a reduced yield if compared with that from an air−CO2 bubbling supply. To overcome this issue, a pH control system was applied, so that the pH value was maintained below 8. In these conditions a specific growth rate was measured close to that obtained under CO2 bubbling, probably due to the equilibrium of carbonate, which is shifted to H2CO3 and free CO2. Thus, it appears that C. protothecoides is not able to directly use bicarbonate, but exploits the CO2 made available from the shifting of the carbonate equilibrium. In addition, the presence of bicarbonate also caused a lipid accumulation in C. protothecoides, up to about 40% dry weight, compared to the constitutive value of 10−12% dry weight. On the basis of the results obtained, a process scheme was proposed, integrating microalgae cultivation with CO2 capture by absorption with a recirculating carbonate solution. Process simulation was carried out to show the technical feasibility of this process.

1. INTRODUCTION The global energy crisis has currently promoted the research to develop and optimize technology in biofuels as a sustainable alternative for fuel supply. In this regard, biodiesel from microalgae has shown a great potential that could impact the world’s supply of transport fuel in the medium term. However, moving from laboratory to large scale, microalgal cultivation requires careful design and planning, and a full scale commercial application appears challenging for the time being.1 Significant obstacles still need to be overcome before microalgae-based biofuel production becomes cost-effective.2 In particular, a large scale microalgae cultivation is associated with high demands of nutrients, including carbon, nitrogen, and phosphorus. Almost all pilot scale algal cultures depend on purchased carbon dioxide that contribute substantially (about 50%) to the cost of producing the biomass.3 Algal culture for fuels is not feasible unless carbon dioxide and other nutrients are available for free.4 Therefore, a multipurpose approach must be applied for large scale cultivation, for instance exploiting N and P from wastewaters, and CO2 from flue gas, which turns out to be also a promising carbon capture technique.5−7 However, due to the very large surface area required for extensive microalgae production, CO2 has to be compressed and transported to algae photobioreactors (PBRs) some distance away. More energy is consumed for pumping air to provide sufficient mixing and optimum gas−liquid mass transfer.8 These operations are energy intensive and enhance the unacceptability of production costs in a large scale PBR. © 2014 American Chemical Society

Moreover, the transport of CO2 from gas to liquid is inefficient, requiring very high air flow rate which results in remarkable energy consumption when sufficient carbon source has to be ensured to microalgae during photosynthesis.9 To address these challenges, a process where CO2 is captured as bicarbonate and used as feedstock for algae culture would significantly reduce the costs because the transport of the aqueous bicarbonate solution requires much less energy.10 A number of studies have reported that bicarbonate ion (HCO3−) can serve as an alternative carbon source to grow microalgae. In fact, several unicellular algae possess an inducible CO2-concentrating mechanism (CCM) which is able to elevate CO2 levels around the CO2-fixing enzyme ribulose-1,5-bisphospate carboxylase/ oxygenase (Rubisco). This CCM is highly expressed under low CO2 concentrations (e.g., atmospheric concentration of CO2) compared with cells cultivated under high-CO2 conditions (e.g., 2−5% CO2).11 Generally, HCO−3 does not diffuse across the cell membrane, but the presence at the outer membrane level of carbonic anhydrase (CA) enzyme (which is responsible for the conversion of carbonate to free CO 2 ) facilitates CO 2 assimilation.12 Alternatively, an active HCO−3 transportation on the cell surface allows bicarbonate assimilation by the microalgal cell.2 The direct uptake of bicarbonate by an active Received: Revised: Accepted: Published: 16678

June 10, 2014 October 2, 2014 October 3, 2014 October 3, 2014 dx.doi.org/10.1021/ie502336d | Ind. Eng. Chem. Res. 2014, 53, 16678−16688

Industrial & Engineering Chemistry Research

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transport system has been found in several species.12−14 Compared with cyanobacteria, less information is available on inorganic carbon (Ci) transport systems in eukaryotic algae.10 However, it has been reported that, in addition to Ci transporters in the cell membrane, eukaryotic algae, such as Chlamydomonas reinhartdii, also have chloroplast Ci transporters, because photosynthesis in eukaryote microalgae occurs in the chloroplast.10 Thus, some microalgae can directly fix carbon in the form of soluble carbonates for cell growth.15 On the other hand, according to the equilibrium relationship: 2H+ + CO32 − ↔ H+ + HCO−3 ↔ CO2 + H 2O

gases and natural sources. Process simulation was performed by Aspen Plus following this scheme.

2. MATERIALS AND METHODS 2.1. Algal Species and Culture Media. Chlorella protothecoides 33.80 (from SAG Goettingen, Germany), was maintained in liquid BG11 medium24 for the inoculum. Experiments with bicarbonate were carried out in modified BG11, by adding different concentrations of NaHCO3 or KHCO3 (Sigma-Aldrich), as reported in the corresponding sections of results. 2.2. Experimental Setup and Apparatus. The experiments were performed in both batch and continuous processes. The temperature was controlled at 23 °C in an incubator (Frigomeccanica Andreaus, Padova), and artificial light (white OSRAM neon lamps) was provided continuously at an intensity of 100 μE m−2 s−1 of PAR (photosynthetic active radiation), measured by a photoradiometer (model LI-189, LICOR, USA). Batch experiments were performed in 250 mL-workingvolume glass vertical tubes, with a 5 cm diameter, continuously mixed by a stirring magnet placed at the bottom of the bottle. A first series of runs was carried out without gas supply, by adding bicarbonate as the only carbon source. As a control, batch experiments were carried out in BG11 without inorganic carbon addition. A second series of experiments was carried out with air bubbling with a total gas flow rate of 1 L h−1. All batch experiments were compared to a CO2 air bubbling air (5% v/v of CO2, gas flow rate was 1 L h−1). The preinocula were also grown in this culture conditions. Each batch experiment started with an initial microalgae inoculation of OD750 = 0.5, corresponding to a cell concentration of about 15 × 106 cells mL−1. Continuous flow experiments were carried out in vertical glass tubes of 250 mL working volume with a 5 cm diameter. At the beginning C. protothecoides was inoculated into the reactor at significant concentration (order of 108 cells mL−1) and fresh medium was fed by a peristaltic pump (Sci-Q 400, Watson Marlow,USA). Part of reactor volume was pumped out at the same inlet flow-rate from the top, in order to ensure a constant reactor volume. Thus, the residence time τ results: V τ= R (2) V̇ where VR and V̇ are the volume of the PBR and the flow rate, respectively. It was directly controlled by the peristaltic pump. After a transient period, the outlet biomass concentration was found stable and a steady state was reached. At steady state, the biomass productivity Px was calculated as C Px = x (3) τ where Cx is the average biomass concentration of at least five experimental measures in different days. Continuous experiments were carried out both with CO2 5%air bubbling gas, as a control, and with the supply of bicarbonate solution, in the absence of bubbling. In the latter case, mixing was provided by magnetic bars. In the experiments with pH control, the pH of the culture was controlled continuously by a pH sensor (D.O. Apparecchiature Elettroniche) connected to a chromatographic pump (LC20AT Prominence), which was activated when the pH in the reactor exceeded the set point, by using an HCl solution to

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protons are consumed during the conversion of bicarbonate to CO2. Thus, the use of bicarbonate as the original carbon source for photosynthesis leaves OH− in the cell, which has to be neutralized by H+ uptake from the extracellular environment, unavoidably leading to an increased pH, with subsequent changes of the equilibrium between different Ci species and inhibitions of cell growth. The problem of achieving pH control in microalgal cultures has been addressed, among others, by Scherholz and Curtis,16 who showed how an improved understanding of nutrient utilization can facilitate pH control during growth on excess CO2. Several authors6,10,17,18 recently proposed a new methodology to efficiently use flue gases as CO2 source in the production of photosynthetic microorganisms, where the carbon dioxide is absorbed in an aqueous phase that is then regenerated by microalgae. All of them, however, carried out experiments of microalgal growth with cyanobacteria, which are able to directly utilize bicarbonate19 but cannot accumulate significant amounts of lipids. In this context, literature data reported an increased lipid accumulation due to the addition of sodium bicarbonate in Chlorophytes.20 Gardner et al.21 showed that, by manipulating the inorganic carbon concentration supply, it is possible to induce lipid and starch accumulation during nitrogen depletion in C. reinhardtii. However, to obtain a considerable biomass productivity, higher concentrations of CO2 are needed.21 Thus, if the aim of microalgal cultivation is the production of liquid fuel, the feasibility of using bicarbonate as carbon source for eukaryotic algae has to be assessed, by critically evaluating the productivity actually achievable. Even though several studies reported growth curves of green algae in the presence of bicarbonates, the productivity is generally quite low, in particular if compared to that under CO2-air bubbling.22,23 In addition, to our knowledge, only batch experiments have been carried out with eukaryotic algae and bicarbonate solutions in the open literature so far. In this work, the feasibility of using bicarbonate as carbon source for Chlorella protothecoides cultivation was tested, both in batch and continuous experiments, by comparing the productivity with that obtainable under CO2 bubbling. Algal growth and carbonate ions consumption in the medium were measured, by taking into account the effects of pH, and eventually controlling its value, the cation associated with the bicarbonate anion and the presence of a simultaneous air bubbling. In addition, the effect of bicarbonate on lipid content in C. protothecoides was evaluated. Finally, on the basis of experimental results, a simplified large scale process block flow diagram is proposed which is simultaneously an alternative for microalgae cultivation plant and a way to capture CO2 from flue 16679

dx.doi.org/10.1021/ie502336d | Ind. Eng. Chem. Res. 2014, 53, 16678−16688


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Figure 1. Comparison of growth curves of C. protothecoides obtained using different concentrations of sodium bicarbonate (2, 4, and 10 g L−1, represented respectively as triangles, pentagons, and arrows) and potassium bicarbonate 4.76 g L−1 (circles) with 5% of CO2 and a control curve without air bubbling (rhombus and squares, respectively).

Figure 2. Influence of air bubbling on the growth of C. protothecoides in the presence of different carbon sources. Circles represent growth of C. protothecoides in 4 g L−1 of NaHCO3, compared to 5% CO2 (dotted line) and a control curve (squares) performed without NaHCO3 addition.

concentration of each experiment was measured as dry weight (DW) in terms of g L−1. DW was measured gravimetrically in cells previously harvested with a 0.22 μm filter: the filters were dried for 4 h at 100 °C in a laboratory oven. In continuous experiments, biomass concentration in terms of cell mL−1 and DW were determined daily, and the steady state concentration was averaged on 5 to 10 experimental points, sampled once a day. The carbonate species concentrations were measured daily by using a common titration method, consisting of the use of two different dyes, phenolphthalein, and bromcresol green (Sigma-Aldrich), pH-sensitive, allowing to determinate CO2− 3 and HCO−3 concentrations in the solution.

adjust the pH value. The HCl concentration was chosen to avoid substantial volume changes in the reactor, due to the control. 2.3. Analytical Methods. The growth in both batch and continuous experiments was monitored daily by a spectrophotometric analysis of the optical density (measured at 750 nm by an UV−visible UV 500 double beam spectrophotometer from Spectronic Unicam, UK) correlated to cell concentration, measured by a Bürker Counting Chamber (HBG, Germany). Specific growth rates in batch experiments were measured by a linear regression of 6−10 experimental points of a logarithmic phase of growth, from two or three independent biological replicates. At the end of the growth curve, the final biomass 16680



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Figure 3. Effect of pH control on batch growth of C. protothecoides in sodium bicarbonate added medium. (a) Comparison of growth in CO2 5% and 4 g L−1 of NaHCO3 with and without pH regulation (respectively dotted line, triangles, and dashed line). (b) Time variation of pH value (squares) and species concentration in solution (HCO−3 , CO2− 3 , and total, represented as circles, hexagons, triangles, respectively).

of algal biomass.3 The resulting CO2 flow rate can be absorbed from flue gases by a carbonate solution of suitable concentration, and transferred to the photobioreactor, where it is consumed by the microalgae to grow. The lean carbonate solution is then recirculated back to the absorption column. To simulate this steady-state process, the Aspen Plus software was used. The selected thermodynamic model was elecNRTL (electrolytic nonrandom two liquid), which accounts for all the ionic species of the equilibrium reported in eq 1 and allows the calculation of the pH of the solution. A RADFRAC column model was employed in the absorption section, and a SEP unit was set to simulate the CO2 removal from the liquid phase by the microalgae. A validation of the thermodynamic model was carried out by comparing process simulation data to the experimental results of the continuous experiment at the same operating conditions.

Total lipids of biomass outlet at steady state were extracted overnight from dried cells by using chloroform/methanol (1:2 vol/vol) in accordance with the Bligh&Dyer method,25 in a Soxhlet apparatus. The lipid mass was measured gravimetrically after solvent removal by using a rotary evaporator. 2.4. Statistical Analysis. T-student tests were applied to ascertain significant differences in biomass concentration in terms of g L−1, cellular weights at different pH in the continuous experiments, as average values of steady state measures (5 to 10 experimental points). The level of statistical significance was assumed for P < 0.05. 2.5. Process Simulation. The simulation of a large scale production process was performed with reference to the irradiation impinging on 1 ha. The biomass produced was calculated from the total annual irradiation energy available, which was retrieved by PVGIS Solar Irradiation Data software (http://re.jrc.ec.europa.eu/pvgis/) applied to the location of Padova, Italy (EPD). An effective photosynthetic conversion (η) of 2.5% was assumed, as an average of the data measured in current pilot scale technologies,26 and a lower heating value (LHV) of 24 MJ kg−127 was estimated for the dried microalgae. Thus, the biomass areal production Pm,PD (kg m−2 d−1) was evaluated as

Pm,PD =

E PDη LHV

3. RESULTS AND DISCUSSION 3.1. Batch Experiments. To assess the capability of C. protothecoides to exploit bicarbonate, batch growth curves were measured in the presence of different concentrations of NaHCO3. In Figure 1 the curves obtained by using bicarbonate are compared to the control (no carbon added) and to a curve measured under CO2−air (5% v/v) bubbling. The concentration of 10 g L−1 of NaHCO3 was initially chosen according to the carbon mass balance and yield, by considering the elemental composition of Chlorella.28 Thus, with a C content of 47.5% of DW, 10 g L−1 of NaHCO3 is the amount required to obtain about 3 g L−1 of biomass, which is the biomass

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The CO2 input required by the process was calculated according to the microalgae composition and by taking into account that 1.8 kg of CO2 are needed in order to obtain 1 kg 16681



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Figure 4. Average biomass concentration in terms of g L−1 and total carbonate ions concentration (inlet and outlet, dark and white bars, respectively) obtained at steady-state in continuous flow experiments. The data obtained in continuous experiments with the addition of NaHCO3 5 g L−1 and pH control are reported (named 7, 7.5, and 8 as target values of the pH control) compared to the experiment without pH control (9.87 was the pH measured in these conditions) and to the one with CO2 bubbling as a reference.

growth rate in the presence of bicarbonate was found significantly lower than that under bubbling of 5% of CO2, as well as the final biomass concentration (Supporting Information, Table S1) strongly affects this algae growth, thus suggesting that C. protothecoides is not able to efficiently exploit bicarbonate as the carbon source or, indirectly, the increase of pH, due to the addition of bicarbonate to the culture medium. The pH trends of all the batch experiments (reported as Supporting Information, Figure S1) showed that the addition of bicarbonate caused a rapid increase of pH up to values close to 12 in the first days of growth. While the initial pH is proportional to the bicarbonate concentration in the medium, the final one is similar for all conditions. Interestingly, the increase rate followed the microalgal specific growth rates, suggesting that the consumption rate of carbon is directly related to the pH increase. The inhibition of growth by sodium bicarbonate might also be explained by a specific inhibition due to the sodium ion: in fact, high Na+ concentrations in the soil are usually toxic to most higher plants, where the permeation of sodium through Na+ transporters leads to high cytoplasmatic concentration, causing toxic effects. On the contrary, the K+ ion is commonly used by several plants as a pH regulator.29 To test such a possible explanation, KHCO3 was used as an alternative carbon source instead of NaHCO3. However, as shown in Figure 1, no significant differences were observed between the growth in NaHCO3 and KHCO3. In both cases, the pH increased very rapidly to values higher than 9, causing an

Table 1. Lipid Content of C. protothecoides Grown Using Different Carbon Source, Compared with Total Lipid Content in CO2 5% (*Reference Data from a Previous Work35) experiment CO2 5% NaHCO3 NaHCO3 NaHCO3 CO2 5% NaHCO3 NaHCO3 NaHCO3 NaHCO3

4 g L−1 bubbling 4 g L−1 bubbling pH 8 4 g L−1 pH 8 5 5 5 5

g/L g L−1 pH 8 g L−1 pH 7 g L−1 pH 7.5

batch/ continuous batch batch batch batch continuous continuous continuous continuous continuous

lipid content % (w/w) 11.5 33.4 30.3 38.0 27.3 45.1 45.8 38.7 39.4

± ± ± ± ± ± ± ± ±

3.1* 3.41 3.01 3.92 2.79 4.62 4.66 3.95 4.01

concentration reached in the case of CO2 bubbling (see Supporting Information, Table S1). However, with such a bicarbonate concentration, the pH rapidly increased and the algal growth was strongly inhibited, with a very low specific growth rate (about 0.12 d−1). At lower concentrations (4 and 2 g L−1 of NaHCO3), specific growth rates of 0.241 and 0.334 d−1, respectively, were measured, but no significant biomass concentration was achieved. In particular, the biomass obtained with 2 g L−1 is not significantly different from the control without NaHCO3 addition. Thus, in all cases, the specific 16682



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Figure 5. Scheme process proposal of CO2 capturing from flue gas using carbonates.

Figure 6. Comparison between measured (dark) and simulated (gray) concentration (reported as logarithm of molarity) of CO2 HCO−3 , CO2− 3 and H+ ions at the inlet and outlet of the continuous reactor with NaHCO3 5 g L−1 without pH control. The simulated outlet concentrations were calculated by considering the removal of CO2 in the liquid phase, progressively released by the shifted carbonate equilibrium.

bicarbonate: Yeh et al.22 showed that by adding 1.2 g L−1 of NaHCO3 to culture medium, Chlorella vulgaris was able to utilize 99.6% of the available carbon source (mainly HCO−3 ) with a final biomass concentration of 0.6 g L−1. However, considering the mass balance of carbon (47% of C by elemental composition) and calculating a biomass yield on bicarbonate consumed, it seems very likely that in the experiments of Yeh et al.22 Chlorella utilized the CO2 supplied by the bubbling system, which was probably also responsible for the lower pH data observed, as a consequence of the acidification caused by the CO2 dissolved in water. A possible adaptation of the cells to the presence of bicarbonate and high pH conditions was also

inhibition of growth of C. protothecoides (Supporting Information, Table S1). A general inhibition due to high salinity of the medium can be excluded, considering the wide tolerance of C. protothecoides to salt content up to 35 g L−1 of salts.30 Bozzo et al. and Matsuda and Colman13,14 identified an active transport of bicarbonate ion in two species of the genus Chlorella, activated at low CO2 concentration and a pH higher than 7.5. They also observed an acclimation period of 3 or 5 h to activate the HCO−3 active transport, but no information is available on the efficiency of their process. Other authors reported the capability of Chlorella to directly exploit 16683



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compared with the experiments without any pH regulation. Responsible for this significantly higher growth was probably the part of dissolved HCO−3 , which shifted to CO2 as a result of the equilibrium of carbonates in solution, when the pH increased. In fact, according to the classical equilibrium, three types of inorganic carbon species can be formed depending on pH: the solution is dominated by free CO2 molecules or carbonic acid (H2CO3) below 4.5, by HCO−3 at pH 8.5, and by 31 carbonate (CO2− 3 ) at pH higher than 8.5. Thus, by feeding HCl to the solution, the equilibrium is shifted to the right of eq 1, increasing the concentration of free CO2 molecules, which can be directly exploited by microalgae as the carbon source. The trend of the pH and the concentration of the carbon species in solution as a function of time are reported in Figure 3B. When HCl was added to the culture media, an increase of HCO−3 and a decrease of CO2− 3 concentrations were measured: as an overall result, the total concentration of the ions decreased during the experiments from the initial concentration of 45.8 mM to 6.34 mM, leading to a total consumption of 86.2%. The pH regulation permitted the acquisition of a higher biomass concentration than in experiments without pH control, similarly to those with air− CO2 bubbling. On the other hand, the specific growth rate was found still lower than that under CO2 bubbling. Therefore, in batch cultures, CO2 supply by direct bubbling remains the best method to provide for the carbon needed to support microalgae growth, but pH regulation is a good way to obtain a significant growth in NaHCO3 added medium. 3.4. Continuous Experiments. In the perspective of a large scale production, the growth of C. protothecoides by using sodium carbonate as the carbon source was investigated in a continuous system. Experiments with and without pH control were performed, with a residence time of 1.92 days, according to our previous results.32 Steady-state concentrations maintained for at least 7 days were compared to a continuous experiment under air−CO2 bubbling, which is a reference value of the maximum biomass obtainable (about 0.752 ± 0.076 g L−1 DW) (Figure 4 and Supporting Information, Table S3). It is clear that in continuous experiments the pH control resulted in an effective strategy to obtain significant productivity as well. In fact, without any pH control, with 5 g L−1 of NaHCO3 in the feed, the reactor reached a steady state after a long transient, where cell concentration fluctuated between 3 and 22 million cells/mL. A steady state at about 0.446 ± 0.039 g L−1 was then reached, but maintained only for a few days, suggesting that C. protothecoides suffered the pH change and was not able to acclimate to the presence of bicarbonate. As a next step, the continuous reactor was equipped with a pH regulation system, with a set point at pH 8: in these conditions, biomass concentration reached a steady state concentration of about 0.687 ± 0.071 g L−1, which is higher than the one obtained without pH control. In addition, the steady state was found constant for 10 days, as well as the measured bicarbonate ion concentration in the outlet stream with a higher carbon consumption, suggesting that in these conditions algae can better exploit the carbon source, resulting in a yield of 0.43 g of

Figure 7. Sensitivity analysis of the effect of Na2CO3 concentration in the liquid phase on CO2 (dashed dark) and HCO−3 (dashed gray), + CO2− 3 (solid gray) ions concentration, H (solid dark) reported in logarithmic scale, and carbon captured from flue gas (dotted line) reported as C concentration in g L−1 in the liquid output stream of the absorption column.

tested, by reinoculating a culture grown in the presence of sodium bicarbonate in fresh medium at pH 10.5, but no significant differences were observed (data not shown). Thus, even though the Chlorella genus appears to possess direct transporters of inorganic carbon,13 the C. protothecoides species does not seem capable of exploiting this active transport with the efficiency required for a large scale application, thus suggesting that this species prefers to preferably uptake free CO2. 3.2. Batch Experiments under Air Bubbling. The inhibition of growth, due to the addition of bicarbonate, was probably caused by the rapid increase of the pH value. A possible strategy to overcome this issue is to control the pH in the solution by bubbling air through the culture, in order to maintain the pH in a range more compatible with algal viability. In Figure 2 a growth curve with 4 g L−1 of NaHCO3 in the bubbling system is compared to a control under only air bubbling, without any addition of bicarbonate. The trend of the growth curve under air−CO2 (5% v/v) bubbling is also shown as a reference. In this case, the presence of bicarbonate resulted in a growth rate slower than the control, while the pH rapidly raised to a higher value, suggesting that the concentration of CO2 naturally present in the air is not enough to buffer the system, resulting in a strong inhibition of algal growth. 3.3. Batch Experiment with pH Control. The problem of pH inhibition may be solved by performing a chemical pH control by adding suitable amounts of hydrochloric acid to adjust the pH value at about 8. A first experiment was carried out in a semibatch, where the pH was adjusted daily by a manual addition of a suitable amount of HCl 1 N (with an estimated final concentration of 25 mM). As reported in Figure 3A and Supporting Information, Table S2, a remarkable growth rate and higher cellular concentration were obtained, if

Table 2. Data Assumed for Process Simulation and Calculation. Area and Residence Time Were Chosen as Base of Calculation; Irradiation, Photosynthetic Efficiency and LHW Are Literature Data (see Section 2.5) area [ha]

irradiation [MJ m−2 y−1]

photosynthetic efficiency %

LHW [MJ kg−1]

biomass productivity [ton ha−1 y−1]

biomass production [kg h−1]

CO2 required [kg h−1]

residence time [d]

1

4100

2.5

24

42.7

4.87

8.76

2

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Table 3. Results of Process Simulation tot liquid flow [m3 h−1]

inlet Na2CO3 [M]

total gas flow [m3 h−1]

pHin

pHout

CO2 supply [kg h−1]

biomass Production [kg h−1]

VR [m3]

Cx [kg m3]

10

0.18

10000

8.17

8.63

8.68

4.82

480

0.48

C per gram of biomass. This value is remarkably higher than that without pH control, and is in accordance with the literature data of biomass yield on CO2 (1.8−2 kg of CO2 for 1 kg of biomass, resulting in a carbon yield of about 0.458 g of C per gram of biomass3). On the other hand, a lower biomass concentration was found, with respect to the one obtained under the air−CO2 bubbling experiment, suggesting that, even though the carbon yield is comparable, the microalgae are not able to consume all the carbon present in the medium, which resulted in a partial consumption of the carbon supplied (see Supporting Information, Table S3). This can be explained by considering that part of the carbon is present in the form of carbonate ions which cannot be directly exploited by the algae. Thus, assuming that algae are exploiting the CO2 made available by shifting the equilibrium of carbonates, two other continuous experiments were carried out by regulating the set point of the pH control to 7.5 and 7. As reported in Figure 4, the consumption of the total carbon was inversely related to the pH, while the biomass concentration showed a maximum at pH 7.5, suggesting that in these conditions more CO2 is released and efficiently exploited by the biomass. At a lower pH, CO2 is probably released faster, more than the uptake kinetic rate by microalgae, resulting in a loss of CO2 from the culture medium. However, the statistical analysis performed on biomass concentration data at steady state and different pH suggested that the pH control was necessary to obtain a statistically significant increase in biomass concentration, for all the pH values analyzed. As far as the comparison of pH 7.5 and 8 with 5% CO2 is concerned, the difference in biomass concentration was not significant. By reducing the pH, a significant decrease in biomass concentration was observed, coherently with the previously reported hypothesis of a CO2 release too fast to be efficiently used by microalgae. In fact, looking at the yields of biomass/carbon calculated for each continuous experiment (data reported in Supporting Information, Table S3) it is clear that at pH 7.5 the value is closer to the one of the air−CO2 bubbling experiment, while at pH 7, a lower yield is probably related to the loss of part of carbon as gaseous CO2. Another possible explanation of a lower yield of biomass might be related to the accumulation of exopolysaccharides (EPS),33 which are not detectable by the methods applied in this work, but could affect the calculation of organic carbon fixation yields. However, the effect of pH on EPS formation is not yet clear, the latter being generally triggered by light or nutrient stress in phytoplankton.34 Thus, the hypothesis of carbon loss at lower pH values appears the most likely in this case. 3.5. Effect of Bicarbonate on Lipid Content. Soxhlet extractions were performed by using samples obtained from both batch and continuous experiments (Table 1), with the aim to investigate the effect of sodium bicarbonate addition on the lipid content of C. protothecoides. As reported in White et al.,20 the addition of sodium bicarbonate is an effective strategy to increase lipid accumulation in marine Chlorophytes. In Chlamydomonas reinhardtii high bicarbonate concentrations caused a cessation of cell cycling and accumulation of both TAG and starch.21

A constitutive content of total lipid of 11−16% is reported for C. protothecoides35,36 which, under certain conditions, is able to accumulate larger quantities, up to 55%.36 In Table 1 the results of both batch and continuous experiments are reported, showing a remarkable increase of lipid content when NaHCO3 was added to the culture media. In continuous experiments with bicarbonate addition, the lipid content was found to be about 40%, suggesting that the presence of bicarbonate during growth triggers a higher lipid accumulation: in fact, under CO2 bubbling both in batch and continuous experiments, the lipid content is lower than the one obtained in the presence of bicarbonate. The higher lipid content is also confirmed by looking to the cellular mass measured in the experiments with carbonate addition, which was found higher than those measured in control experiments: A statistical analysis, performed comparing the data of cellular mass obtained in continuous experiments with and without NaHCO3 addition, resulted in a significant difference between such data, suggesting that the cell mass increased in the presence of bicarbonate. It is not clear yet, however, if the presence of the bicarbonate itself was the direct cause of lipid accumulation, which could be also due to a generic stress caused by the increased pH. On the other hand, the effect of pH can be excluded in experiments with pH control, in which a lipid accumulation was observed even though the pH did not increase over 8. Another possible explanation is related to the higher salinity of the medium. It has been demonstrated that a high salt concentration may increase the lipid content of C. protothecoides.37 However, in this case the salt concentration associated with the addition of bicarbonate was not so high to justify such a significant effect. An increase of lipid content of Chlorella vulgaris in the presence of bicarbonate was also reported by Yeh et al.,22 who measured values of about 30−40%, exceptionally high if compared to values reported in the literature for this species (14−22%). Also Gardner et al.21 observed a possible stimulating effect of sodium bicarbonate addition on triacylglycerol accumulation in some microalgal species, even if the bicarbonate addition was carried out at the end of the stationary phase of growth in batch cultures, with most of the biomass amount obtained under CO2 bubbling. In this work, a higher lipid content was found by directly culturing algae with bicarbonate, with a double goal to achieve a large production of biomass which is also rich in lipids. This is particularly interesting in view of industrial bio-oil production. In fact, high lipids accumulation without need of applying any additional step to the cultivation process is very welcome, because it allows a steady lipid production in a single step process, resulting in lower plant costs. 3.6. Integrated Process of CO2 Capture from Flue Gas and C. protothecoides Growth. The experimental results reported above suggest that the algal species herein considered does not consume bicarbonate, but largely CO2 coming from a pH-dependent equilibrium, and that pH control is the predominant problem. To develop a large-scale process based on these findings, the use of chemicals (such as HCl) to regulate the pH should be avoided; therefore, the equilibrium 16685



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h−1 is needed. By selecting the proper liquid flow rate and carbonate concentration, it was possible to provide the carbon needed for growth, with a change of the pH from 8.17 and 8.63. At these conditions, a flow rate of 10 m3 h−1 is required, with a carbonate concentration of 0.18 M, resulting in a liquid stream at pH 8.17. Simulation results are summarized in Table 3. As a final calculation, a PBR volume of about 480 m3 was obtained (eq 1), with a biomass concentration of 0.48 g L−1, which are both realistic values if compared with currently available technology. In addition, with the selected flow rates, an absorption column with a reasonable size can be designed, requiring about 1 m2 of cross section per hectare of PBR. To conclude, even though C. protothecoides is not able to directly use bicarbonate as a carbon source, it is possible to exploit the carbon captured in the liquid phase by manipulating the process variables, in order to capture only the CO2 needed for growth, and consequently avoid the increase of the pH value over the limit of viability of algae. Even though the technical feasibility of the proposed process has been assessed, a deeper investigation into the energy use, the carbon footprint, the water consumption, and ultimately a cost balance analysis would be fundamental in order to test the advantage of using bicarbonate instead of direct CO2 bubbling in large scale microalgae cultivation. 3.7. Conclusions. The exploitation of carbonates as carbon source was investigated for C. protothecoides, both in batch and continuous experiments, in order to verify the actual biomass yield and productivity in view of a possible industrial application. Experiments demonstrated that C. protothecoides is not able to use the bicarbonate ion itself with high efficiency, probably due to the increased pH of the medium, which inhibits the growth. However, a significant growth is achieved by controlling the pH of the culture system, and thus shifting the carbonate equilibrium in favor of free CO2 efficiently exploited by C. protothecoides, resulting in a biomass productivity very close to the one obtained under CO2−air bubbling. In this regard, the selected pH value was crucial to obtain a remarkable productivity: pH 7.5 was found to be the best one to provide the sufficient carbon concentration as CO2, while pH 7 caused a rapid release of CO2 that was not used efficiently by the microalgae, as confirmed by both productivity and yield data. The presence of sodium bicarbonate induced lipid accumulation in C. protothecoides up to about 40% DW. This is particularly interesting in view of large scale application, where the use of bicarbonate could be beneficial to obtain higher values of both biomass productivity and lipids in a single step. Considering the preference of C. protothecoides for free CO2, an integrated process of carbon capture and algal cultivation was proposed, where only the CO2 flow rate needed for the desired algal growth was absorbed. By selecting suitable values of recirculating flow rates and carbonates concentration, the release of this flow rate in the PBR can be achieved, so as to avoid an increase of pH over the viability limit of the algae of interest.

shift of carbonate−bicarbonate systems has been exploited in this respect. The use of carbonate liquid solutions as an alternative carbon source might be a successful way for supplying carbon in a large scale PBR, so as to avoid the high compression/transportation costs and the drawbacks linked to the direct exploitation of CO2 from flue gases.2 From this perspective, a simple process scheme is proposed in Figure 5, similarly to what was suggested by other authors in the case of bicarbonate-exploiting organisms.10,18 Accordingly, CO2 is captured from flue gases by absorption in a solution of sodium carbonate, and fed to the PBR as soluble carbonates, which are exploited by microalgae as the carbon source to produce biomass. The lean carbonate solution is recirculated back to the absorption section and a makeup is needed as part of aqueous solution is lost in the wet algae product. An additional advantage of this approach is the possibility of using the heat released in the absorption step to control the PBR temperature during cold seasons at middle latitudes. A scheme similar to the one in Figure 5, proposed by Fernandez,18 is based on the assumption that algal species are able to directly use bicarbonate as a carbon source. In our case, Chlorella protothecoides preferably exploits the CO2 made available in the liquid phase by the shift of carbonate equilibrium, with a consequent pH increase leading to the inhibition of algal growth at values higher than about 9. To avoid a chemical control of the pH, which would lead to an accumulation of salts in the solution, a carbonate solution is used in the currently proposed process, and its flow rate and concentration are adjusted so that the CO2 uptake by the microalgae is performed within the pH range of viability of algal cells. The effectiveness of the elecNRTL model was initially validated by comparing the simulated profiles to the ion concentrations experimentally measured in the continuous experiments. Results are plotted in Figure 6, which clearly shows that the model correctly calculates the equilibrium composition of the carbonate ions in solution. The absorption was modeled by a column with two ideal stages.38 A sensitivity analysis was carried out showing that, when a typical weight fraction of CO2 (i.e., 10%) is assumed for the flue gas in contact with a carbonate solution, the Na 2 CO 3 concentration is directly responsible for the pH value (Figure 7) and the predominant species is the bicarbonate ion. Thus, even if a carbonate salt is used in the simulation, the resulting liquid solution is comparable to the experimental condition of our work, obtained by the addition of a bicarbonate salt in a closed system. On the other hand, the carbon that can be captured from the flue gas is also proportional to carbonate concentration, an increase of pH corresponding to a higher carbon capture. Nonetheless, if the aim of the absorption step is to only supply an adequate carbon flow rate to the PBR, the carbonate concentration can be set at an optimum value in order to prevent the pH value from increasing too much. In addition, it is possible to set the operating conditions so as to only absorb the CO2 needed for growth, which is then consumed in the PBR, thus changing the pH within a range which turns out to be reasonable for algal viability (i.e., not larger than 9). Calculations for large scale installations were referred to the biomass productivity (Pm,PD calculated according to eq 4) obtainable on 1 ha at middle latitude (see Table 2 for details): based on the algal mass balance, a carbon supply of about 8.7 kg

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ASSOCIATED CONTENT

S Supporting Information *

Tables S1, S2, and S3 reporting data of cell and biomass concentration, as well as their specific growth rate, initial and final pH, total species concentration of all batch experiments, 16686



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Microalgae: How Realistic a Contribution May It Be to Significant CO2 Removal? Appl. Microbiol. Biotechnol. 2012, 96, 577. (19) Su, C. M.; Hsueh, H. T.; Chen, H. H.; Chu, H. Effects of Dissolved Inorganic Carbon and Nutrient Levels on Carbon Fixation and Properties of Thermosynechococcus Sp. in a Continuous System. Chemosphere 2012, 88, 706. (20) White, D. a.; Pagarette, A.; Rooks, P.; Ali, S. T. The Effect of Sodium Bicarbonate Supplementation on Growth and Biochemical Composition of Marine Microalgae Cultures. J. Appl. Phycol. 2013, 25, 153. (21) Gardner, R. D.; Lohman, E.; Gerlach, R.; Cooksey, K. E.; Peyton, B. M. Comparison of CO(2) and Bicarbonate as Inorganic Carbon Sources for Triacylglycerol and Starch Accumulation in Chlamydomonas Reinhardtii. Biotechnol. Bioeng. 2013, 110, 87. (22) Yeh, K.-L.; Chang, J.-S.; Chen, W.-M. Effect of Light Supply and Carbon Source on Cell Growth and Cellular Composition of a Newly Isolated Microalga Chlorella Vulgaris. Eng. Life Sci. 2010, 10, 201. (23) Nayak, M.; Rath, S. S.; Thirunavoukkarasu, M.; Panda, P. K.; Mishra, B. K.; Mohanty, R. C. Maximizing Biomass Productivity and CO2 Biofixation of Microalga, Scenedesmus Sp. by Using Sodium Hydroxide. J. Microbiol. Biotechnol. 2013, 23, 1260. (24) Rippka, R.; Deruelles, J.; Waterbury, J. B.; Herdman, M.; Stainer, R. Y. Generic Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. J. Gen. Microbiol. 1979, 111, 1. (25) Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Physiol. 1959, 37, 911. (26) Norsker, N.-H.; Barbosa, M. J.; Vermuë, M. H.; Wijffels, R. H. Microalgal Productiona Close Look at the Economics. Biotechnol. Adv. 2011, 29, 24. (27) Williams, P. J. L. B.; Laurens, L. M. L. Microalgae as Biodiesel & Biomass Feedstocks: Review & Analysis of the Biochemistry, Energetics & Economics. Energy Environ. Sci. 2010, 3, 554. (28) Phukan, M. M.; Chutia, R. S.; Konwar, B. K.; Kataki, R. Microalgae Chlorella as a Potential Bio-Energy Feedstock. Appl. Energy 2011, 88, 3307. (29) Horie, T.; Schroeder, J. I. Sodium Transporters in Plants. Diverse Genes and Physiological Functions. Plant Physiol. 2004, 136, 2457. (30) Heredia-Arroyo, T.; Wei, W.; Hu, B. Oil Accumulation via Heterotrophic/mixotrophic Chlorella Protothecoides. Appl. Biochem. Biotechnol. 2010, 162, 1978. (31) Dewgoswami, C.; Kalita, M. C.; Talukdar, J.; Bora, R.; Sharma, P. Studies on the Growth Behavior of Chlorella, Haematococcus and Scenedesmus Sp. in Culture Media with Different Concentrations of Sodium Bicarbonate and Carbon Dioxide Gas. African J. Biotechnol. 2011, 10, 13128. (32) Ramos Tercero, E. A.; Sforza, E.; Morandini, M.; Bertucco, A. Cultivation of Chlorella Protothecoides with Urban Wastewater in Continuous Photobioreactor: Biomass Productivity and Nutrient Removal. Appl. Biochem. Biotechnol. 2014, 172, 1470. (33) Clément-Larosière, B.; Lopes, F.; Gonçalves, A.; Taidi, B.; Benedetti, M.; Minier, M.; Pareau, D. Carbon Dioxide Biofixation by Chlorella vulgaris at Different CO2 Concentrations and Light Intensities. Eng. Life Sci. 2014, 00, 1−11. (34) Baines, S. B.; Pace, M. L. The Production of Dissolved Organic Matter by Phytoplankton and Its Importance to Bacteria: Petterns across Marine and Freshwater Systems. Limnol. Oceanogr. 1991, 36, 1078. (35) Sforza, E.; Cipriani, R.; Morosinotto, T.; Bertucco, A.; Giacometti, G. M. Excess CO2 Supply Inhibits Mixotrophic Growth of Chlorella Protothecoides and Nannochloropsis Salina. Bioresour. Technol. 2012, 104, 523. (36) Xu, H.; Miao, X.; Wu, Q. High Quality Biodiesel Production from a Microalga Chlorella Protothecoides by Heterotrophic Growth in Fermenters. J. Biotechnol. 2006, 126, 499. (37) Campennì, L.; Nobre, B. P.; Santos, C. a; Oliveira, a C.; AiresBarros, M. R.; Palavra, a M. F.; Gouveia, L. Carotenoid and Lipid Production by the Autotrophic Microalga Chlorella Protothecoides

steady state cell and biomass concentration, productivity, pH, total carbon concentration in input and output of continuous flow experiments at different conditions, and yield of biomass on carbon consumed; Figure S1 reports the pH trends of batch experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +39-0498275462. Fax: +39-0498275461. Notes

The authors declare no competing financial interest.

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REFERENCES

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