Using seawater-based Na₂CO₃ medium for scrubbing the CO₂ released from Bio-CNG plant for enhanced biomass production of Pseudanabaena limnetica

Abstract

The concentration of CO₂, one of the most important greenhouse gases (GHG), has reached to 409.8 ± 0.1 ppm in 2019. Although there are many carbon capture and storage (CCS) methods, they are very costly and their long term use raises concern about environmental safety. Alternatively, bio-sequestration of CO₂ using microalgal cell factories has emerged as a promising way of recycling CO₂ into biomass via photosynthesis. In the present study, Indigenous algal strain Pseudanabaena limnetica was cultivated in pneumatically agitated 60-L flat-panel photobioreactor system. The gas was released from Bio-CNG plant as by-product into Na₂CO₃-rich medium and cultivated in semicontinuous mode of operation. It was observed that when CO₂ was sparged in seawater-based 0.02 M Na₂CO₃ solution, maximum CO₂ was dissolved in the system and was used for algal cultivation. Control system produced 0.64 ± 0.035 g/L of biomass at the end of 15 days, whereas CO₂ sparged Na₂CO₃ medium produced 0.81 ± 0.046 g/L of biomass. When CO₂ from Bio-CNG station was fed, it resulted in biomass production of 1.62 ± 0.070 g/L at the end of 18 days compared to 1.46 ± 0.066 g/L of biomass produced in control system which was not fed with gas released from Bio-CNG plant as by-product. Thus, feeding CO₂ directly into Na₂CO₃ medium and operating the system semicontinuously would be efficient for scrubbing CO₂ from commercial Bio-CNG plant. This study proves that feeding CO₂ gas from Bio-CNG plant into Na₂CO₃-rich alkaline system can be used to feed algae for enhanced biomass production.

1 Introduction

Global warming is being caused due to emission and increase in three greenhouse gases (GHGs), namely CO₂, methane and N₂O. Atmospheric CO₂ concentration in 2018 was 407.3 ppm which increased by 2.5 ± 0.1 ppm in 2019 [1]. The Kyoto Protocol and the Paris Agreement (2015) have asked the participating countries to curb climate change impact by setting up policies out of which crucial are CO₂ emissions by reducing fossil fuel usage and increasing carbon capture and sequestration [2, 3].

Most of chemical or physical means of capture of CO₂ from smoke stack emission involve three major steps which are carbon capture, separation and storage which enormously increase the cost of project. [4]. Microalgae possess inbuilt mechanisms to capture CO₂ from the atmosphere even in smallest concentrations. These mechanisms are referred to as carbon capture mechanisms (CCM) [5]. Also, microalgae can withstand toxic waste gasses such as CO₂ and NO_x, SO_x coming from industrial and sewage wastewater sources. It is a known fact that microalgal cultures show enhanced growth when they are provided with CO₂-enriched air. Hence, microalgae are used as biological scrubbers which utilize CO₂ from the industrial gas released as by-product resulting into enhanced biomass production. Biomass can be utilized for applications such as food, animal feed, fuel and other value-added by products which could be incorporated into cosmetics and nutraceutical industry [4].

Microalgae captures CO₂ in three ways like conversion of bicarbonates into CO₂ which diffuses into cell directly, assimilation of CO₂ directly via plasma membrane and direct uptake of bicarbonates though cell membrane transporters [6]. One of the challenges in providing CO₂ into algal systems is its efficient transport to algal cells. The most common way of feeding CO₂ is through fine-pore spargers but the barrier is the solubility of CO₂ in aqueous medium. Sparging excess CO₂ does not mean that it will not cause CO₂ limitation because most of the sparged CO₂ escapes into the atmosphere. This conventional way of CO₂ feeding does not become economical, especially when large-scale algal cultivation is a target [7]. Alternative way of capturing CO₂ present in gas released from thermal power plant or Bio-CNG plant is to absorb CO₂ using solvents such as NaOH, K₂CO₃, etc., and this CO₂-loaded solvent is then pumped directly into raceway ponds or photobioreactors and used for algal cultivation [8].

Present communication deals with the use of two different solvents NaOH and Na₂CO₃ for absorption of CO₂. The CO₂ released from CNG station was sparged in 0.01 M NaOH solution. After feeding CO₂, the NaOH was converted to NaHCO₃ and then Na₂CO₃. The conversion of NaOH to NaHCO₃ and Na₂CO₃ was checked using pH double indicators. The amount of Na₂CO₃ solution formed was also calculated. This CO₂-enriched Na₂CO₃ solution was used as carbon source while preparing the culture medium for growth of our own algal strain Pseudanabaena limnetica (Lemm.) komàrek isolated from salt pans of eastern suburban regions of Mumbai capable of sustaining elevated concentration of Na₂CO₃ [9]. It will serve dual purpose, sequestration of CO₂ from Bio-CNG station and enhancement of algal biomass which can be utilized for several commercial applications.

2 Material and methods

2.1 Strain selected

Microalgal strain selected was Pseudanabaena limnetica (Lemm.) komàrek, which is an indigenous halophilic strain isolated from salt pans of Mumbai suburban region.

2.2 Culture medium

BG-11 medium used for growth of cyanobacterial strains was prepared as mentioned by Rippka et al. 1979 [10]. Modified seawater BG11 medium (MSWBG11) was formulated from the BG11 medium. MSWBG11 medium was modified BG11 seawater-based medium containing ten times higher carbonate (Na₂CO₃) compared to normal BG11 composition [11].

2.3 Design and culture conditions

To conduct the experiments for CO₂ sequestration, the operationally optimized 60-L photobioreactor was utilized. The photobioreactor was made up of glass having dimensions of 20 cm × 50 cm × 70 cm (breadth × length × height). Construction and operational standardization were carried out in our previous study by Magar and Deodhar 2018 [11]. The culture was supplied with light intensity of 185–222 µmol m⁻² s⁻¹for 12 h every day using white fluorescent light. The agitation was provided using spargers at the rate of 4LPM using acrylic rotameters (Napro Scientific, Pune). Perforated horizontal tubes were placed at the base of the photobioreactor through which air was sparged into the system [11].

2.4 CO₂ feeding

The CO₂ supply was fed from the commercially available 100% compressed CO₂ cylinders (Super Industrial Gases, Thane). The flow rate of CO₂ was controlled using a commercial pressure regulator. Further for accurate flow rate, rotameter of 0.001–10 LPM capacity was procured from Napro Scientific, Pune. The gaseous CO₂ was fed into the medium at a flow rate of 0.001 LPM through the borosilicate sintered glass sparger (Deepali enterprises, Mumbai). For experiment purpose, flow rate of ≤ 0.001LPM was maintained using the same rotameter and sparger system.

2.5 pH, Temperature, dissolved carbon dioxide (DCO₂) detection and biomass estimation

The detection and monitoring of pH, temperature and DCO₂ were carried out using electronic sensors, which were immersed in the working liquid system of the photobioreactor. Each of the sensors, i.e. pH and DCO₂, along with inbuilt temperature sensor in each of them was obtained from Mettler-Toledo India Pvt. Ltd. The dry weight of the biomass (DWB) was estimated gravimetrically.

2.6 Experimental setup for dissolution of CO₂ in different solvent systems

Experiment was performed by adding two different salts like NaOH and Na₂CO₃ having concentration of 0.01 M and 0.02 M, respectively, in tap water as well as seawater. Both alkaline solutions were separately prepared, each one having total volume of 5L. CO₂ gas was sparged at the rate of 0.001 LPM in the system using sintered glass sparger as mentioned above. Sampling from each of the sets was done every 10 min, and molar concentration of NaOH, Na₂CO₃ and NaHCO₃ was estimated and calculated by volumetric titration method using double indicators, namely phenolphthalein and methyl orange [15]. Change in pH was also measured every 10 min using pH sensor. Solution capable of capturing maximum CO₂ was further used to study its effect on growth of algae.

2.7 Effect of CO₂-sparged alkaline solutions on the growth of algae

In control experiment, the microalgae P. limnetica were grown in 60-L photobioreactor in MSWBG11 medium without any artificial CO₂ supplementation. The initial inoculum was added in the reactor which was aerated using spargers at rate of 4 LPM, and the algal growth was continued for 15 days. Culture was exposed to white fluorescent light of 10,000–12,000 lx intensity for 12 h every day. Dissolved CO₂ (henceforth mentioned as DCO₂) concentration was noted and recorded continuously using data logger system. The associated pH change was also noted with pH sensor. The growth of dried algal biomass (g/L) was weighed after every 3-day interval in order to get measureable change in biomass weight [11].

In Test 1 and test 2, the culture of P. limnetica was inoculated in a MSWBG11 medium in the 60-L-capacity flat-panel photobioreactor. For test 1 and test 2, the carbonate content in MSWBG11 medium was replaced by CO₂-enriched NaOH and Na₂CO₃ solutions, respectively. The culture was exposed to the white fluorescent light of 10,000–12,000 lx intensity for 12 h every day. The effect of CO₂ sparged NaOH /Na₂CO₃ solution on algal growth was interpreted in terms of biomass produced which was measured at the intervals of 3 days. The DCO₂ and pH change was also reported at every hour.

2.8 Semicontinuous mode of operation for maximum biomass yield

Algal culture P. limnetica was cultivated in three different 60-L flat-panel photobioreactors set A, set B and set C. The culture conditions were same as described above in Sect. 2.7. Algal culture was initially allowed to grow for 10 days till log phase was achieved and then semicontinuous mode of operation was adopted. From three sets, set A, set B and set C, three different volumes of culture media (1 L, 2 L and 3 L) were removed every day and it was replaced by the same amount of fresh MSWBG11 medium. The experiment was continued for 60 days. Everyday increase in the algal dried biomass (g/L) was measured gravimetrically and noted.

2.9 Effect of CO₂ from Bio-CNG plant on growth of Algae P. limnetica in semicontinuous mode of operation

Experiment was conducted in the premises of Primove Engineering Pvt. Ltd., Pune. Composition of Bio-CNG coming from plant contains 50% CH₄, 49% CO₂ and 1% other gases. CO₂ gas coming from Bio-CNG plant had 99% purity with less than 1% of other trace gases such as H₂S, N₂, etc. CO₂ gas was directly sparged in MSWBG11 medium. This CO₂-sparged MSWBG11 medium was used for replacing the medium in a semicontinuous mode of operation. Experiment was conducted in 60-L photobioreactor that was inoculated with 0.3 g/L of culture, and operating conditions were the same as mentioned above. DCO₂ concentration (mg/L), % CO₂ consumption, pH and dried biomass yield (g/L) were analysed and calculated daily.

2.9.1 Statistical analysis

All the data were statistically analysed using one-way ANOVA and Tukey’s test (α = 0.05 and 0.01) wherever applicable.

3 Results and discussions

Emission of CO₂ is the major contributor to global climate change. About one-third of global CO₂ fixation occurs due to algae. This is because algal cells have special cell organelles pyrenoids to accelerate the process of CO₂ assimilation. In algal cell, HCO^3¯ from medium passes through plasma membrane to chloroplasts and then to thylakoids where it is catalysed to CO₂. There it is fixed by RuBisCO enzyme to produce many sugars by Calvin’s cycle. Amount of HCO^3¯ thus enhances the rate of photosynthesis, biomass formation and growth rate of algae. When CO₂ gas is directly bubbled into microalgal medium, most of it is wasted due to short resident time and low utilization efficiency of raceway pond. There should be a mechanism to improve efficiency of converting CO₂ to HCO^3¯. CO₂ bicarbonate absorbers are generally adapted to efficiently convert CO₂ to NaHCO₃ which dissolves easily in culture medium and promotes algal growth [12].

Numbers of promising materials are used to capture CO₂. Best ones are aqueous amines. But the major disadvantages in its use are amine evaporation, corrosion of equipment and requirement of heat energy to regenerate the solvent [13]. As alternative alkaline sorbents, K₂CO₃, Na₂CO₃ have received a wide attention. Recently, hydrated Na₂CO₃ was used to absorb CO₂ [14]. Also NaOH was used to capture CO₂ from coal-fired power plant [15]. CO₂ reacts with aqueous NaOH to form Na₂CO₃ and NaHCO₃. In bench-scale reactor, the operational conditions were standardized in such a way that 97% NaHCO₃ was produced which has several industrial applications as baking soda.

In the present study, CO₂ released from Bio-CNG plant was sparged in NaOH or Na₂CO₃ solutions and resulting mixture was used for preparation of culture medium for growing algae.

The algal strain Pseudanabaena limnetica (Lemm.) komàrek indigenous algal strain isolated from salt pan capable of withstanding Na₂CO₃-enriched alkaline seawater medium. The normal concentration of Na₂CO₃ in the BG11 medium is 0.02 g/L, whereas the modified seawater BG 11 medium (MSWBG11) contains ten times greater amount of Na₂CO₃ (0.2 g/L). The 60-litre flat-panel photobioreactor was used for cultivation. At the end of 18 days, the algae produces 30% enhanced biomass than normal BG11 medium [11].

3.1 Use of different alkaline sorbents for feeding CO₂

When gaseous CO₂ is fed into the NaOH solution, CO₂ is first dissolved in water to form aqueous CO₂ (Eq. 1). Subsequently, aqueous CO₂ reacts with OH^ˉ to generate HCO^3¯and CO₃^2¯ (Eqs. 2 and 3):

$${\text{CO}}_{2} \left( g \right) \to {\text{CO}}_{2} \left( {{\text{aq}}} \right)$$

(1)

$${\text{CO}}_{2} \left( {{\text{aq}}} \right) + {\text{OH}}^{ - } \left( {{\text{aq}}} \right)\, \rightleftharpoons {\text{HCO}}_{3}^{ - } \left( {{\text{aq}}} \right)$$

(2)

$${\text{HCO}}_{3}^{ - } \left( {{\text{aq}}} \right) + {\text{OH}}^{ - } \left( {{\text{aq}}} \right) \rightleftharpoons {\text{H}}_{2} {\text{O}}\left( {\text{l}} \right) + {\text{CO}}_{3}^{2 - } \left( {{\text{aq}}} \right)$$

(3)

When gaseous CO₂ is fed into the NaOH solution, CO₂ reacts with aqueous NaOH and forms sodium carbonate (Na₂CO₃) and later forms sodium bicarbonate (NaHCO₃) in turn. These CO₂ absorption reactions are expressed by Eqs. (4) and (5)

$$2{\text{NaOH }} + {\text{ CO}}_{2} = {\text{ Na}}_{2} {\text{CO}}_{3} + {\text{ H}}_{2} {\text{O}}$$

(4)

$${\text{Na}}_{2} {\text{CO}}_{3} + {\text{ H}}_{2} {\text{O}} + {\text{ CO}}_{2} = \, 2{\text{NaHCO}}_{3}$$

(5)

The reaction rates and pH for above reactions 4 and 5 are different [16]. The mole fraction of each carbonate species could be expressed as function of pH which is described in Fig. 1 which is called Bjerrum plot. As seen in Fig. 1, carbonates are prominently produced at alkaline pH 14 to 10 [15]. As more CO₂ is sparged, pH becomes less alkaline and bicarbonates are maximally produced at pH 8. At lower pH 4–6, HCO^3¯ is produced.

Fig. 1

pH versus the mole fraction of carbonate species (Bjerrum plot). Extracted from research paper by Shim et al. (2016)

3.1.1 Dissolution of CO₂ in tap water- and seawater-based 0.01 M NaOH solution

In the present study, 0.01 M NaOH solution was used for sparging of CO₂ gas. CO₂ gas was purged through fine-pore sintered glass sparger at flow rates of 0.005 – 0.01 LPM. Initially, when CO₂ was sparged in the NaOH solution, the pH of solvent is very alkaline (pH 11) At this pH, most of the fed CO₂ reacted with NaOH to form Na₂CO₃ which was analysed by acid base titration method using double indicators (phenolphthalein and methyl orange). Every 10-min interval, sample was harvested and titrated against 0.2 M HCl. Change in pH was also measured. Phenolphthalein end point from purple to orange is an indicator of consumption of all NaOH and Na₂CO₃. And only bicarbonate is present in the solution that is further determined by methyl orange indicator. Methyl orange end point gives the measure of Na₂CO₃ that is converted to NaHCO₃ after titrating with HCl. The amount of OH^¯, CO₃^¯ and HCO₃^¯ formed during the course of CO₂ sparging was calculated as described in the literature [17].

In this experiment, capacity of tap water and seawater to capture CO₂ was studied. When NaOH was added to pure water, it dissociates to form Na⁺ and OHˉ almost completely making the solution highly alkaline. NaOH, Na₂CO₃ and NaHCO₃ species are generated as explained in Sect. 3.1.1. At zero time, the amount of NaOH is highest and it is responsible for highest phenolphthalein reading. The amount of NaOH is 0.054 ± 0.004 mol/L. The pH of solution is 11.72 ± 0.012. As more and more CO₂ was sparged over time, amount of NaOH gradually started decreasing and Na₂CO₃ was formed. When the reaction mixture was titrated with HCl, the Na₂CO₃ was converted to NaHCO₃. The amount of NaOH or Na₂CO₃ decreased from 0.054 ± 0.004 to 0.003 ± 0.002 mol/L. The amount of NaHCO₃ increased from 0.00 to 0.062 ± 0.002 mol/L. During CO₂ sparging, pH of reaction mixture decreased from 11.72 ± 0.012 to 9.06 ± 0.038. At this pH, NaOH is converted to NaHCO₃ that added to additional increase in methyl orange reading (Table 1).

Table 1 Amount of Na₂CO₃ and NaHCO₃ formed during the course of CO₂ sparging in tap water-based 0.01 M NaOH solution

When 0.01 M NaOH solution was prepared, seawater was used as the medium for dissolution of CO₂ (Table 2). It was observed that initial pH of the solution was only 10.05 ± 0.017, which might be because of inherent buffering nature of seawater which restricts drastic changes in pH. CO₂ was sparged in the solution, and changes in NaOH, Na₂CO₃ and NaHCO₃ were analysed. pH decreased from 10.05 ± 0.017 to 8.24 ± 0.006 in just 110 min. Concentration of NaOH decreased from 0.026 ± 0.000 to 0.001 ± 0.000 mol/L, and at the same time, amount of NaHCO₃ increased from 0.010 ± 0.001 to 0.034 ± 0.006 mol/L which was approximately three times lower than that of compared with NaHCO₃ concentration measured in tap water.

Table 2 Amount of Na₂CO₃ and NaHCO₃ formed during the course of CO₂ sparging in seawater-based 0.01 M NaOH solution

3.1.2 Dissolution of CO₂ in tap water- and sea water-based 0.02 M Na₂CO₃ solution

Similarly, 0.02 M Na₂CO₃ solution in tap water and seawater was also used to study the dissolution of CO₂ and generation of NaHCO₃ in the system. Initially, when Na₂CO₃ was added in water, it dissociates to form NaOH and H₂CO₃. Thus, total available Na₂CO₃ in the system reduces. When CO₂ sparging was initiated, NaOH previously formed is converted to Na₂CO₃. And all Na₂CO₃ is converted to NaHCO₃ which was detected by methyl orange indicator. NaOH and Na₂CO₃ concentrations were measured as already mentioned in the above experiment. It was observed that when tap water was used, the initial pH of the system was 11.31 ± 0.010 and initial NaOH/Na₂CO₃ concentration was 0.026 ± 0.000 mol/L (Table 3). As CO₂ was sparged in the system, Na₂CO₃ and NaOH started to reduce and became 0 mol/L moles, whereas NaHCO₃ increased from 0.035 ± 0.001 to 0.061 ± 0.003 mol/L at the end of 130 min.

Table 3 Amount of Na₂CO₃ and NaHCO₃ formed during the course of CO₂ sparging in tap water-based 0.02 M Na₂CO₃ solution

Similarly, when seawater was used to sparge CO₂, initial pH of 0.02 M Na₂CO₃ was 10.77 ± 0.031 which reduces to 7.75 ± 0.050 at the end of 100 min (Table 4). Also initial NaOH/Na₂CO₃ concentration of 0.028 ± 0.001 decreased to 0.00 and concentration of NaHCO₃ increased from 0.030 ± 0.001 to 0.058 ± 0.004 mol/L, which is comparable to tap water system.

Table 4 Amount of Na₂CO₃ and NaHCO₃ formed during the course of CO₂ sparging in seawater-based 0.02 M Na₂CO₃ solution

Salmon et al. used NaOH as sorbent for capturing CO₂ [16]. When CO₂ is sparged in NaOH, it leads to formation of intermediate species like OH^¯, CO₃^¯ and HCO₃^¯. The amount of intermediates was determined by titration with HCl using two colour indicators phenolphthalein and methyl orange. The effect of flow rates at which CO₂ is sparged and the initial concentration of CO₂ on gas liquid mass transfer was studied. They concluded that alkaline solutions in either pure form (NaOH or Na₂CO₃) or mixture of NaOH and Na₂CO₃ can be used effectively for CO₂ capture.

Actually, absorption of CO₂ in aqueous medium depends on gas liquid equilibrium. Henry’s law constant H’ quantifies the concentration that maintains this gas liquid equilibrium. Cheng and Chou studied effect of two alkaline systems NaOH and Na₂CO₃ on partition of CO₂ by calculating Henry’s law constant. The study was carried out in three phases. In phase I, H’ value for absorption of CO₂ in neutral tap water was determined. The study was carried out at two different temperatures 27 and 37 degrees. In phase II, alkaline-buffering agents NaOH and Na₂CO₃ were used. And in phase III, alga was used for CO₂ absorption [18]

When CO₂ was sparged in 150-L reactor, the CO₂ concentration varied from 600 to 2000 ppm. Initially, when the pH was more than 8, H’ value was as low as 2.2 atm/M and at this pH much gaseous CO₂ is absorbed (1500 ppm) as the pH value ranged from 8 to 6, H’ varied from 2.2 atm to 65 atm and CO₂ absorbing capacity gradually falls. When the pH decreases to pH range 4–5, the H’ is in the range of 90 to 94 atm/M and it absorbs very little CO₂.

When NaOH was used as an alkaline buffer and CO₂ was sparged for 2 h, the pH value ranged from 6.6 to 8.9. But when Na₂CO₃ was used as buffering system, the pH value ranged between 9.1 and 9.8. Adding Na₂CO₃ yielded H’ value from 16.3 to 21.3 atm/M. The CO₂ concentration was 603 ppm and aqueous concentration of HCO₃^¯ was 4.085 mg/L. In NaOH buffering system, H’ value was between 27 to 59 atm/M and there was much decline in concentration of CO₂. Thus, weak alkali Na₂CO₃ had greater buffering capacity than stronger alkali.

Also the difference in CO₂ dissolution and holdup capacity of tap water, seawater and modified seawater BG11 medium (MSWBG11) was studied. It was observed that MSWBG11 medium that contained ten times higher Na₂CO₃ in the medium enabled maximum CO₂ dissolution with concentration of 18.30 ± 1.17 mg/L after 3 h of CO₂ sparging. Further on stopping the CO₂ sparging, system was capable of holding DCO₂ up to 4 h and was 3.34 ± 0.37 mg/L. Thus, seawater-based BG11 medium with ten times higher Na₂CO₃ in it (MSWBG11) made the best solution for capturing and holding maximum DCO₂ in the system [9].

In our present study, when NaOH was used as alkaline system and 0.01 M NaOH was dissolved in tap water, the initial pH was as high as 11.72 ± 0.012. After feeding CO₂ for 110 min, the pH was decreased slightly to 10.58 ± 0.026 and amount of Na₂CO₃ produced was 0.040 ± 0.004 mol/L. But when seawater was used for dissolving 0.01 M NaOH, the initial pH was comparatively low, it was 10.5 ± 0.017. This might be due to buffering action of seawater. After feeding CO₂ for 110 min, the pH became 8.24 ± 0.006 and amount of NaHCO₃ was comparatively less 0.034 ± 0.006 mol/L.

But when Na₂CO₃ was used as alkaline system, 0.02 M was added to tap water; the pH was 11.31 ± 0.010. When CO₂ was sparged for 110 min, it became 9.04 ± 0.032 and amount of NaHCO₃ present in system was 0.053 ± 0.0031 mol/L. When seawater was used after addition of 0.02 M Na₂CO₃, the pH was 10.77 ± 0.031. After feeding CO₂ for 100 minutes, it decreased to 7.75 ± 0.050 and amount of NaHCO₃ was 0.058 ± 0.004 mol/L. This concentration of NaHCO₃ using 0.02 M Na₂CO₃ was statistically proven to be best solution for CO₂ sparging and was significantly different with other solutions using one-way ANOVA and Tukey’s test at level of significance of 0.05 (α = 0.05 and 0.01) (Fig. 2).

Fig. 2

Effect of different alkaline solutions of CO₂ dissolution. (n = 3, mean ± SD). * signifies statistically significant difference between four different alkaline solutions for optimum CO₂ dissolution (ANOVA and Tukey’s post hoc test: * p < 0.05, ** p < 0.01)

Thus, seawater along with Na₂CO₃ was found to be efficient system for capturing CO₂ and converting it into NaHCO₃. Hence, for further experiments of algal cultivation, Na₂CO₃ was used for CO₂ sparging.

Our further objective was to utilize these CO₂-sparged NaOH and Na₂CO₃ solutions for algal growth.

3.2 Use of CO₂-enriched NaOH and Na₂CO₃ medium for algal growth

3.2.1 Use of CO₂-sparged NaOH and Na₂CO₃ solutions for preparing MSWBG11 medium

CO₂-sparged 0.01 M NaOH tap water solution and 0.02 M Na₂CO₃ seawater-based solution were used in the preparation of MSWBG11 medium. These media were used for the growth of algae Pseudanabaena limnetica. MSGBG11 medium contains 0.0018 mol/L of Na₂CO₃ which is far less than Na₂CO₃-enriched stock solutions obtained after CO₂ sparging. Hence, the stock solution was diluted accordingly to obtain required moles of Na₂CO₃ for the preparation of medium.

However seawater-based Na₂CO₃ solution contained 0.058 ± 0.004 mol/L of NaHCO₃. The stock was 1:5 times diluted and added to MSWBG11 medium. Both these media were used to study the growth of algae Pseudanabaena limnetica.

3.2.2 Effect of CO₂-sparged alkaline solutions on the growth of algae

3.2.2.1 Effect of CO₂-sparged NaOH medium on growth of P. limnetica

In case of control tank, DCO₂ concentration and pH at day 0 were 2.07 ± 0.031 mg/L and 8.53 ± 0.097, respectively (Table 5). Initial inoculum added was 0.19 ± 0.021 g/L dried biomass weight. As days passed, algal biomass grew gradually to give dried biomass yield of 0.29 ± 0.031 g/L on day 6. Also DCO₂ was decreased to1.50 ± 0.030 mg/L and pH became 10.16 ± 0.060. Maximum biomass yield of algae was obtained on day 15, i.e. 0.64 ± 0.035 g/L, which was confirmed by consumption of CO₂, and DCO₂ concentration of the system decreased from 2.07 ± 0.031 to 1.15 ± 0.050 mg/L on day 15. Also pH became alkaline from 8.53 ± 0.097 to 9.86 ± 0.051 on day 15.

Table 5 Control tank—No CO₂ sparged

In test 1 tank, where CO₂-sparged NaOH solution was used for preparation of MSWBG11 medium, the initial DCO₂ concentration was 2.47 ± 0.070 mg/L (Table 6) which was little higher than control tank (2.07 ± 0.031 mg/L). In Test tank, initial inoculum added was 0.19 ± 0.012 g/L and pH 8.63 ± 0.064, respectively. The highest biomass of algae was observed on 12th day and increase in the biomass was from 0.19 ± 0.012 to 0.58 ± 0.015 g/L which was slightly more than control tank but the increase was not significant. Growth of the algae was supported by consumption of CO₂ and increase in the pH. On day 15, the DCO₂ decreased from 2.47 ± 0.070 mg/L to 1.39 ± 0.105 mg/L, pH got alkaline from 8.63 ± 0.064 to 9.01 ± 0.067 which resulted in significant decrease in biomass yields. These results indicate that CO₂-sparged NaOH solution can be used for algal growth.

Table 6 Test 1—CO₂-sparged NaOH solution using for algal growth

3.2.2.2 Effect of CO₂-sparged Na₂CO₃ medium on growth of P. limnetica

Further third set (test 2) was tested where CO₂-sparged Na₂CO₃ solution was used as the source of carbonate for preparation of MSWBG11 medium. Growth conditions provided were same as given to above two tanks. In this experiment, the initial DCO₂ of the system was 3.92 ± 0.046 mg/L which was substantially more than the other two tanks and pH was 7.82 ± 0.095. Algal inoculum added in the system on day 0 was 0.23 ± 0.046 g/L. As days passed, DCO₂ reduced and pH started getting more alkaline indicating growth of algae in the system. As seen in Table 7 and Fig. 3, right from day 3 up to day 15, there is significant increase in biomass produced in CO₂-sparged Na₂CO₃ medium over control. On day 15, algal biomass was maximum, i.e. 0.81 ± 0.046 g/L, where as in control tank the biomass produced is significantly less, 0.64 ± 0.035 g/L. At the end of 15 days, DCO₂ was substantially decreased from 3.92 ± 0.046 mg/L to 1.23 ± 0.036 mg/L and pH was increased from 7.82 ± 0.095 to 9.54 ± 0.075 (Table 7).

Table 7 Test 2—CO₂-sparged Na₂CO₃ solution used for algal growth

Fig. 3

Effect of CO₂-sparged NaOH and Na₂CO₃ solutions on algal growth and biomass yield. (n = 3, mean ± SD). * signifies statistically significant difference between Control, Test 1 and Test 2 (ANOVA and Tukey’s post hoc test: * p < 0.05, ** p < 0.01)

Thus, the above experiments conclude that when CO₂-sparged Na₂CO₃ solution is added to MSWBG11, it is sufficiently capable of holding maximum DCO₂ as indicated by high DCO₂ in third set, i.e. 3.92 ± 0.046 mg/L on day 0. Also it supports well the algal growth. In control tank and Tank 1 provided with NaOH medium, the biomass yields at the end of 15 days were 0.64 ± 0.035 g/L and 0.58 ± 0.015 g/L, respectively, which were very less compared to test 2 which yielded 0.81 ± 0.046 g/L of yield, thus proving that Na₂CO₃ solution when used to sparge CO₂ suits best as a medium for maximum CO₂ holding capacity and maximum algal yield. Hence, it was continued for further work.

Statistical analysis by one-way ANOVA proved there was significant difference in three sets Control, Test 1 and Test 2 at level of significance of 0.05. Further post hoc Tukey’s test proved there was significant difference in biomass yields of test 2 over Control and Test 1. (α = 0.05 and 0.01) (Fig. 3).

3.3 Semicontinuous mode of operation for high biomass yield of algae

Cultivation of algae can be done in various ways such as batch mode, fed-batch mode or continuous mode. Previous experiments were conducted in batch mode of operation where experiments were run for about 15 days to complete the growth cycle of algae. After 15 days, algal biomass started decreasing indicating stationary phase of algae.

To further increase the biomass production of algae, it was cultivated in semicontinuous mode of operation. Initially, the culture was allowed grow for 6 to 7 days till the log phase is achieved. The semicontinuous operation is initiated by removing some amount of algal culture and replacing it with fresh medium. By doing so, the algal culture remains in log phase continuously producing biomass. In the present work, it was necessary to standardise the amount of culture to be removed in semicontinuous operation in which 1 L (set A), 3 L (set B), 5 L (set C) of culture were harvested and the same volume of fresh media was added into each of the sets every day. Increase in the biomass yield in each set was evaluated gravimetrically on a daily basis.

In set A, initial inoculum was 0.30 ± 0.058 g/L which was allowed to grow for 6 days till the biomass increased to 0.71 ± 0.023 g/L which was considered as day 0 for starting semicontinuous mode of operation. Daily 1 L of culture was harvested and analysed for biomass yield. Same amount of fresh medium was added into the tank to compensate for the lost harvested culture. It was found that in initial 10 days biomass of 0.71 ± 0.023 g/L increased to 1.18 ± 0.049 g/L and further it went on increasing from 10th till 60th day till which the experiment continued. The final biomass that was obtained at the end of 60 days was 2.62 ± 0.032 g/L.

However, in set B, the initial biomass of 0.82 ± 0.021 g/L was gradually increased to 1.43 ± 0.36 g/L at the end of 30 days. But the biomass produced at the end of 30 days in set B was significantly lesser than biomass in set A (Fig. 4). The same trend was found to be continued, on 60th day the biomass produced in set A (2.61 ± 0.032 g/L) which was significantly higher than set B (1.60 ± 0.025 g/L).

Fig. 4

Effect of different volumes in semicontinuous mode of cultivation on biomass yield of algae. (n = 3, mean ± SD). * signifies statistically significant difference in biomass yields of set A (1 L), set B (3 L) and set C (5 L) (ANOVA and Tukey’s post hoc test: * p < 0.05, ** p < 0.01)

Similarly, in set C where 5 L of media was removed every day, the initial biomass was 0.81 ± 0.040 g/L, and at the end of 30 days, it negligibly increased to 1.08 ± 0.015 g/L which was significantly less than biomass produced in set A. The condition was still worst on day 60, and the biomass produced was still reduced to 0.76 ± 0.012 g/L (Table 8, Fig. 4).

Table 8 Effect of amount of daily removal of culture medium in semicontinuous mode on biomass formation

These results indicated that replacing 1 L of culture with fresh media is the suitable way to replenish the lost nutrient into media and also the biomass harvested in 1 L culture can be overcome by exponentially growing culture. On the other hand in set B and C where 3 L and 5 L of culture were harvested, biomass was not able to grow at that fast rate to cope up the loss of culture. (Table 8). This was statistically proven by performing one-way ANOVA and Tukey’s test at α = 0.05 and 0.01 (Fig. 4).

In one of the studies, it was reported that A. braunii when cultivated in batch cultures, the maximum biomass concentration (Xm) was 1588 ± 11 mg L⁻¹ using 20 mM of NaNO₃. A fed-batch process with the addition of 20 mM NaNO₃ each 48 h from the first to the sixth cultivation day reached Xm = 2753 ± 7 mg L⁻¹. The semicontinuous process was effective in eradicating the lag phase of algae allowing it to grow to maximum biomass of Xm = 2399 ± 5 mg L⁻¹. As seen in our experiment semicontinuous mode of operation increased the biomass gradually beyond 18 days till 60th day and reached maximum biomass of 2.62 ± 0.032 g/L [19]. It could be concluded from the data that semicontinuous mode of operation is not only optimum for mass scale production of algae but can also be used to feed CO₂-rich medium in system every day to compensate for the loss of CO₂ in the environment due to high temperatures in tropical environments.

3.4 Using Na₂CO₃-sparged MSWBG11 medium in semicontinuous mode of operation

In this experiment, CO₂ gas coming from Bio-CNG plant was directly sparged in MSWBG11 (Test1). This CO₂-sparged MSWBG11 medium was used for replacing the medium in a semicontinuous mode of operation. Experiment was conducted in 60-L photobioreactor that was inoculated with approximately 0.35 g/L of culture and agitated with 4LPM of ambient air. Set-up was kept in partial shade near Bio-CNG vicinity of CNG station plant in the premises of Primove Engineering Pvt. Ltd., Pune. DCO₂ concentration (mg/L), % CO₂ consumption, pH and dried biomass yield (g/L) were analysed and calculated daily.

In one set, only MSWBG11 medium with no sparged CO₂ was used as the source of medium for semicontinuous mode and this was considered as control tank. In both sets, i.e. test and control, algal biomass was initially cultivated for 10 days till biomass attains log phase. In control set, initial inoculum added was 0.35 ± 0.072 g/L which increased to 0.77 ± 0.052 g/L at the end of 10 days. DCO₂ at day 0 was 1.82 ± 0.012 mg/L which reduced to 1.68 ± 0.070 mg/L at the end of 10 days. Initial pH of 8.75 ± 0.050 increased to 8.91 ± 0.036 after 10 days. However, in Test tank, initial inoculum of 0.34 ± 0.096 g/L increased to 0.77 ± 0.066 g/L at the end of 10 days. Initial DCO₂ and pH of the system were 2.08 ± 0.060 mg/L which reduced to 1.28 ± 0.085 mg/L after 10 days indicating algal culture grew consuming DCO₂ of the system.

After 10 days, the semicontinuous operation was initiated in both control and test tanks, i.e. every day, 1L of culture from both tanks was replaced with same volume of media. In control tank (Table 9), 1L of culture was replaced with same volume of fresh MSWBG11 medium (with no CO₂ sparged in it), whereas in test tank 1L of culture was replaced with same volume of CO₂-sparged fresh MSWBG11 medium containing DCO₂ concentration of 7–8 mg/L.

Table 9 Control: MSWBG11 medium was used in semicontinuous mode of operation

In control tank, it was observed that as semicontinuous mode of operation started, initial DCO₂ of 1.68 ± 0.070 mg/L observed at 7:00 h increased to 1.70 ± 0.015 mg/L at 11:00 h after addition of fresh medium. As culture grew throughout the day, DCO₂ was consumed and its concentration reduced to 1.61 ± 0.012 mg/L till evening at 19:00 h. Growth of the culture was confirmed by increase in dried weight biomass from 0.77 ± 0.052 g/L to 0.85 ± 0.036 g/L at end of day 1. As DCO₂ was consumed by algae, there was decrease in the pH from 8.91 ± 0.036 on day 0 to 8.95 ± 0.068 on day 1. Slowly as days passed and fresh medium was added, DCO₂ gradually increased from 1.74 ± 0.130 mg/L on day 1 to 2.02 ± 0.042 mg/L at the end of 8 days and pH of the system became more alkaline from 8.95 ± 0.068 to 9.42 ± 0.081 after 8 days. Biomass yield gradually increased from 0.85 ± 0.036 g/L on day 1 to maximum on day 8, i.e., 1.46 ± 0.066 g/L.

In test tank (Table 10), initial DCO₂ of 1.28 ± 0.085 mg/L increased to 1.42 ± 0.140 mg/L on addition of CO₂-enriched fresh MSWBG11 medium to the system after 1 day. On addition to DCO₂, pH got alkaline from 9.14 ± 0.040 on day 0 at 7:00am to 9.58 ± 0.075 at 7:00 pm on day 1 and culture grew from 0.77 ± 0.066 g/L to 0.80 ± 0.070 g/L in a day. As days passed, DCO₂ concentration gradually increased from day 1 to day 8, DCO₂ on addition of media at day 1 was 1.42 ± 0.140 mg/L which slightly increased to 1.55 ± 0.120 mg/L on day 8, DCO₂ was continuously available for the growth of algae and algal biomass increased from 0.80 ± 0.070 g/L on day 1 to 1.62 ± 0.070 g/L after 8 days of semicontinuous mode of operation. Significant difference in the biomass yields between Control and Test was proven by using one-way ANOVA (α = 0.05) (Fig. 5).

Table 10 Test: MSWBG11 sparged with CO₂ from CNG station was used for semicontinuous operation

Fig. 5

Effect of CO₂ from Bio-CNG plant on growth and biomass yield of algae. (n = 3, mean ± SD). * signifies statistically significant difference in Biomass yields of Control and Test (ANOVA: * p < 0.05)

Also Yadav et al. set up a small-scale pilot reactor at Oil and Natural Gas Corporation, Hazira, Surat, India, to capture CO₂ from vent gas. It consisted of a cylindrical CO₂ absorber of volume 0.25 m³. The pH of fresh medium was adjusted to 11 by using 0.1 g/L of NaOH and was stored in storage tank. The medium was transferred to column using peristaltic pump. CO₂ containing vent gas was tapped from exhaust stack at Hazira plant and was purged into absorption column at a flow rate 5.1 L/min. The gas was bubbled from bottom to ensure a long contact time between alkaline solution and gas. The pilot carbonation column was able to bring down the initial CO₂ concentration of 30 vol% to 15 vol%, and the pH of medium was decreased from 11 to 6.8 or 7. This CO₂-sparged medium was then allowed to flow into algal raceway pond of capacity 0.2m³. The growth of microalgae consortium was measured as increase in OD from 0.001 to 0.402. Due to algal growth, the pH of pond rose from 8 to 10. This high pH medium was reused for absorbing CO₂ in absorption column [20].

4 Conclusions

In the current research, indigenous halophilic strain Pseudanabaena limnetica (Lemm.) Komárek was used for CO₂ sequestration studies along with mass cultivation. CO₂ sequestration studies were carried out in previously designed, constructed and operationally optimized 60-L flat-panel photobioreactor system for growth of P. limnetica. MSWBG11 medium was the nutrient medium for the growth of the algae.

To enhance maximum CO₂ dissolution and holdup, different alkaline systems were studied in seawater and tap water out of which 0.02 M Na₂CO₃ in seawater was the best solutions which could hold maximum CO₂. To improve the algal biomass productivity, these solutions were used as source of carbonate. To further enhance the biomass, the system was operated in a semicontinuous mode where 1 L of culture replaced with 1 L of fresh medium everyday showed maximum algal biomass yield even after 60 days operation. Further to sequester CO₂ continuously so that it could be used for industrial application, semicontinuous system was set up at premises of Bio-CNG plant. Here, the gas released from Bio-CNG plant as by-product containing CO₂ was sparged in Na₂CO₃-containing medium and 1 L of old culture was replaced with this CO₂-enriched medium every day and proved to be helpful in enhancing the biomass production.