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Abstract

Chlorella vulgaris was isolated from sewerage treatment plant and grown in the presence of sodium bicarbonate as carbon source at 0.25, 0.5 and 1.0 g L^− 1 . Highest specific growth rate (0.653 μ d^− 1 ) was obtained with 1 g L^− 1 bicarbonate followed by 0.5 g L^− 1 (0.641 d^− 1 ) on 15th day culturing. Total chlorophyll content of microalgae has increased in a dose dependent fashion with bicarbonate addition and maximum level recorded in 1 g L^− 1 (0.769 ± 0.09 g L^− 1 ). The biomass productivity was in the range of 0.237–0.996 g d^− 1  L^− 1 . Rate of CO₂ fixation and carbon content, in terms of quantity was estimated. Results showed that at 1 g L^− 1 sodium bicarbonate concentration, maximum CO₂ fixation (0.497 g/dry weight) and carbon content (0.69 g mL^− 1  day^− 1 ) was found. Biomass concentration was significantly higher (p < 0.05) in cultures (1.54 g L^− 1 ) supplemented with 1 g L^− 1 bicarbonate whereas there was no much difference in cellular lipid concentration (16 mg mL^− 1 ). GC–MS analysis of fatty acids showed highest amounts of palmitic acid, myristic and stearic acid. In summary, the addition of sodium bicarbonate increases cellular abundance, chlorophyll content and to some extent in the case of lipid content in C. vulgaris integrated with CO₂ sequestration.

Keywords

Microalgae ; Bicarbonate ; Chlorella ; Biomass ; Lipid production

Introduction

Among the atmospheric pollutants, CO₂ contributes significantly to the greenhouse effect. Global warming attributed primarily to the elevated CO₂ level in the atmosphere has led to various CO₂ mitigation strategies. As chemical reaction based CO₂ capturing is relatively costly and energy consuming, it is necessary to develop cost effective and sustainable alternatives. Biological CO₂ mitigation leads to the production of biomass energy and is an alternative strategy of CO₂ fixation through photosynthesis (Kondili and Kaldellis, 2007  ;  de Morais and Costa, 2007 ). Microalgae are more efficient than terrestrial plants in photosynthesis from ambient air and are important for the prevention of increase in atmospheric CO₂ concentration. It converts CO₂ into biomass energy and thus recycles CO₂ (Demirbas, 2004 ). Coupling of CO₂ sequestration with algal cultivation reduces the carbon footprint and sustainable environment.

Microalgae have drawn more attention as a promising source for the production of biodiesel because they possess high growth rate and provide lipid fraction for biofuel production. Microalgal growth and biochemical composition are governed by environmental conditions (Guiheneuf et al ., 2008  ;  Pal et al ., 2011 ). Sodium bicarbonate has been demonstrated to enhance lipid accumulation in both freshwater and marine microalgae (Gardner et al ., 2012  ; Gardner et al ., 2013  ; White et al ., 2013  ;  Peng et al ., 2014 ). Microalgae utilize bicarbonate as the external source of carbon for photosynthesis and derive CO₂ via the action of carbonic anhydrase (Dixon et al ., 1987  ; Nimer et al ., 1997  ;  Bozzo et al ., 2000 ). This work established the growth of Chlorella vulgaris in a medium where sodium bicarbonate serves as a source of inorganic carbon by assessing growth rate, chlorophyll content, biomass and lipid production in batch cultures. Further, rates of inorganic carbon concentration and CO₂ fixation were determined for CO₂ sequestration using bicarbonate.

Materials and Method

Sample Collection and Identification

Algal samples were collected from Bangalore Water Supply and Sewerage Board (BWSSB), Bengaluru (13°04′N, 77°58′E) and poured into a closed 250 mL bottle and exposed in sunlight for 3 weeks. The upper layer of the water was inoculated in agar plates enriched with BG11 medium containing 200 μg mL^− 1 ampicillin to control the growth of bacteria as the sample used was sewage water. Agar plating technique was used to isolate the microalgae and the plates were incubated at 25 ± 2 °C under cool white fluorescent light (40 μmol photons m^− 2  s^− 1 ; 15 h light/9 h dark) until algal growth was detected. The isolates were purified by streak plating and individual colonies were diluted in distilled water. Species of single cells were obtained using capillary pipette under a microscope followed by inoculation into fresh media. After appropriate growth, cells were observed to confirm the single culture and the capillary method was repeated as many times as required to obtain axenic cultures. Identification of Chlorella was using standard protocols as described by Anderson (2005) , Stanier et al. (1971) and the database http://web.biosci.utexas.edu/utex/default .

Growth Under Different Concentrations of Sodium Bicarbonate

Analytical grade sodium bicarbonate was used as the source of bicarbonate in all experiments. Batch cultures (100 mL) of C. vulgaris (BG 11 medium; n = 3) were grown under different levels of bicarbonate supplementation (0.25, 0.5 and 1 g L^− 1 ) into early stationary growth phase (10–15 days), where samples were taken for growth rate, biomass productivity, chlorophyll content and cellular lipid analyses. Media without the addition of bicarbonate were served as control.

Analytical Methods

Biomass Concentration and Productivity

Biomass concentration (g L^− 1 ) of C. vulgaris grown under different bicarbonate concentrations was determined by measuring the optical density (OD₆₈₀ ) using UV–Vis spectrophotometer. The result was converted to biomass concentration using the calibration curve relating OD₆₈₀ (Xia et al., 2014 ) using the following Eq. (1)

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://test.scipedia.com:8081/localhost/v1/":): \mbox{Biomass}\quad \mbox{concentration}=320\times {OD}_{680}.

( 1)

The biomass productivity (mg L^− 1 d^− 1 ) was calculated according to Eq. (2) .

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( 2)

where B₂ and B₁ represent the dry weight biomass densities at the time T (days), at the end and start of the experiment, respectively.

Specific Growth Rate

Specific growth rate (μ) of the microalgae was calculated (Guillard and Ryther, 1962 ) according to the following formula.

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where, N_t and N₀ are the total cells at the end of log phase (T_t ) and start of log phase (T₀ ), respectively.

Chlorophyll Estimation

Chlorophyll a and b of microalgae were estimated according to Mackinney (1941) . Algal suspension was filtered and extracted with methanol in water bath at 60 °C for 30 min. The suspension was cooled, added equal volume of 96% methanol and centrifuged for 6500 g for 10 min. Pigment content of the supernatant was analyzed in a UV–Vis spectrophotometer at 650 nm and 665 nm using 96% methanol as blank and the total chlorophyll was determined using Eq. (3)

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://test.scipedia.com:8081/localhost/v1/":): \mbox{Total}\quad \mbox{chlorophyll}=2.55\times {10}^{-2}.E650+ Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://test.scipedia.com:8081/localhost/v1/":): 0.4\times {10}^{-2}.E665\quad mg\quad {ml}^{-1}

( 3)

where, E650 and E665 are the absorbance at 650 and 665 nm wavelengths respectively.

Estimation of Carbon Content and Carbon Dioxide Fixation Rate

Carbon content of the dried algal biomass was estimated by Walkley and Black (1934) . Dried algal sample was mixed with 1 N potassium dichromate (10 mL) and conc. H₂ SO₄ (20 mL) mixture diluted with distilled water (200 mL) followed by addition of H₃ PO₄ (10 mL) and diphenyl amine (1 mL). The reaction mixture was titrated against 4 N ferrous ammonium sulfate (FAS) until the appearance of brilliant green color and the carbon content was estimated using Eq. (4) .

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( 4)

where, a is the carbon content, g is the weight of algal sample, T and S are the ferrous ammonium sulfate with blank and sample (mL), respectively.

The amount of carbon dioxide fixation rate was estimated using Eq. (5) . (Yun et al., 1997 )

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://test.scipedia.com:8081/localhost/v1/":): R_{{CO}_2}=C_C\times {\mu }_L\left(\frac{M_{{CO}_2}}{M_C}\right)

( 5)

where, R_CO2 and μL are the CO₂ fixation rate (g CO₂  m^− 3  h^− 1 ) and the volumetric growth rate (g dry weight m^− 3  h^− 1 ), respectively, C_C is the average carbon content (algal dry weight [g]). M_CO2 and M_C are the molecular weights of CO₂ and elemental carbon, respectively.

Lipid Extraction

Algal cultures were pelleted and added with 10 mL of ice cold 0.2 N HClO₄ . After 15 min at 4 °C, the sample was centrifuged and the supernatant was added with 10 mL of 0.2 N HClO₄ followed by centrifugation. To the pellet, 10 mL of chloroform–methanol (2:1) solution was added, centrifuged and 0.2 volumes of distilled water were added to the supernatant. The mixture was centrifuged and the lower organic phase was collected, evaporated under a stream of nitrogen to a final volume of 2 mL (Folch et al., 1956 ).

Lipid Estimation

Standard solutions of palmitic acid (5, 10, 20 and 30 mg mL^− 1 ) and algal lipid samples were prepared in chloroform, added with 2 mL of dichromate solution (2.5 g of K₂ Cr₂ O₇ in 1 L of conc. H₂ SO₄ ) and placed in a boiling water bath for 45 min. The mixture was cooled, removed 1 mL from each and diluted to 100 mL with distilled water followed by measuring the absorbance at 350 nm (Marsh and Weinstein, 1966 ).

Fatty Acid Methyl Ester Preparation

The lipid extract was saponified with 5 mL of 0.5 N methanolic solution of potassium hydroxide by boiling for 3–5 min (Hartman and Lago, 1973 ). To this, 15 mL of esterification reagent (2 g of ammonium chloride in 60 mL of methanol and 3 mL of conc. H₂ SO₄ ) was added and refluxed for 3 min. Subsequently, it was transferred to a separating funnel using 25 mL of diethyl ether and 50 mL of saturated sodium chloride solution and the aqueous layer discarded. The diethyl ether layer was washed twice with 25 mL of NaCl solution and the solvent was evaporated under a stream of nitrogen for GC–MS analysis.

GC–MS Analysis

GC–MS analysis of lipid extract was performed using an Agilent GC comprising a Thermo DSQII auto-sampler equipped with a DB 5 MS column (30 m × 0.25 mm ID × 0.25 μm). For GC–MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70 eV. Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1.0 mL min^− 1 and an injection volume of 1 μL was employed in a split mode (1:10). The initial temperature was 40 °C with a hold time of 2 min and the ramp was 310 °C at a rate of 10 °C/min for a hold period of 10 min. Mass spectra were taken at a scan mass range of 30–600 m/z. The solvent delay was 0 to 2 min and the total GC/MS running time was 32 min. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. Quadrupole mass analyzer along with Xcalibur and AMDIS software was adopted to handle mass spectra and chromatograms were compared with NIST 2011.

Statistical Analysis

All the experiments were carried out in triplicates and the results were expressed as mean values and the standard deviation (SD). The statistical differences were obtained through one-way analysis of variance (ANOVA) (p < 0.05).

Results and Discussion

The effect of bicarbonate concentration on specific growth rate (μ) of C. vulgaris is shown in Fig. 1 . Specific growth rate was not significantly different within the concentrations of NaHCO₃ . It showed a similar trend in growth rate with 1 g L^− 1 bicarbonate maintaining the highest specific growth rate (0.653 d^− 1 ) at the end of 15 days culture period followed by 0.5 g L^− 1 (0.641 d^− 1 ).

Fig. 1. Specific growth rate of C. vulgaris culture supplemented with different levels of bicarbonate.

Significant positive effect on the levels of chlorophyll content with an approximate doubling or more with the addition of 1 g L^− 1 bicarbonate when compared to treatments received 0.25 g L^− 1 and 0.5 g L^− 1 (Fig. 2 ) was observed. At the same time, both Chl a and b were significantly varied between lower concentrations of bicarbonate. The total chlorophyll content increased in a dose dependent fashion with bicarbonate addition and maximum level recorded in 1 g L^− 1 (0.769 ± 0.09 g L^− 1 ).

Fig. 2. Chlorophyll content of C. vulgaris culture supplemented with different levels of bicarbonate.

The first equation gives the biomass concentration of the culture. In the 2nd equation, B1 is the early stationary phase and B2 is the end of stationary phase. Stationary phase was considered rather than exponential, as the data is valid at the end of stationary phase in terms of biomass productivity. The addition of bicarbonate had significant effects on the biomass productivity in C. vulgaris ranged from 0.237–0.996 g d^− 1  L^− 1 . As depicted in Fig. 3 , biomass productivity at 1 g L^− 1 (0.996 ± 0.06 g d^− 1  L^− 1 ) was significantly higher (p < 0.05) than those cultures with lower bicarbonate addition. Reduced biomass productivity was observed with lower concentrations of sodium bi carbonate throughout the culturing period.

Fig. 3. Biomass productivity of C. vulgaris culture supplemented with different levels of bicarbonate.

Carbon content of the dried algal biomass was estimated and increasing carbon content enhanced the CO₂ fixation. At 1 g L^− 1 sodium bicarbonate concentration, carbon content was maximum (0.497 g dw^− 1 ) and CO₂ fixation was 0.69 g mL^− 1  day^− 1 . The lowest level of CO₂ fixation was observed in 0.25 g L^− 1 bicarbonate addition (Fig. 4 ).

Fig. 4. Carbon content and CO2 fixation rate in C. vulgaris supplemented with different levels of bicarbonate.

The results of biomass concentration and lipid production at the end of lag phase are compared in Fig. 5 . At 1 g L^− 1 of sodium bicarbonate levels, biomass concentration was significantly higher (1.542 g L^− 1 ) which was triple the amount obtained at 0.5 g L^− 1 bicarbonate addition. However, there was no much difference in lipid production at varying bicarbonate concentrations. Cellular lipid concentration was determined and the total cellular lipid was highest in cultures supplemented with 1 g L^− 1 bicarbonate (16.1 ± 0.07 mg mL^− 1 ) compared to cultures with 0.5 and 0.25 g L^− 1 bicarbonate (14.3 ± 0.14 and 12.4 ± 0.21 mg mL^− 1 ). Based on highest total lipid production, fatty acid profiles in C. vulgaris grown under 1.0 g L^− 1 bicarbonate addition were determined post-conversion to fatty acid methyl esters by GC–MS (Fig. 6 ). Sodium bicarbonate has promoted the fatty acid profiles significantly and the key fatty acids were palmitic, myristic and stearic acid alongside several minor fatty acid components with a carbon number of C16 and C18.

Fig. 5. Biomass and cellular lipid production in C. vulgaris supplemented with different levels of bicarbonate.

Fig. 6. GC–MS profile of fatty acid methyl esters from C. vulgaris at 1.0 g L− 1 bicarbonate concentration.

Cultivation conditions can maximize the algal biomass production rates and lipid concentrations. Carbon limitations due to the poor solubility of CO₂ in water can lead to growth inhibition (Smith and Bidwell, 1989 ; Giordano et al ., 2005  ;  Aishvarya et al ., 2012 ). NaHCO₃ has greater solubility than CO₂ and could be an alternative inorganic carbon source (Hsueh et al., 2007 ) for microalgal cultivation. Further, gaseous CO₂ is costly to store and transport, whereas bicarbonate salts can easily be transported to algal facilities and stored until needed. Enhanced inorganic carbon uptake due to the addition of bicarbonate allows cellular material production and thereby achieving maximum productivity. In this study, maximum biomass of 0.996 g L^− 1 d^− 1 was observed in 1 g L^− 1 sodium bicarbonate addition and this is in agreement with Yeh et al. (2010) who have demonstrated that 1 g L^− 1 sodium bicarbonate was found optimum for biomass production in C. vulgaris . In another study, 1 M NaHCO₃ has produced higher biomass concentration (Chi et al., 2013 ).

Conventionally, chlorophyll is considered as a reliable and standard algal biomass measurement (Wiltshire et al ., 1998  ;  Knefelkamp et al ., 2007 ). Physiological state of microalgae is indicated by carbon to chlorophyll ratio and in this study total chlorophyll content was taken as the indicator of physiology of C. vulgaris influenced by inorganic carbon (sodium bicarbonate). Total chlorophyll content of 0.769 g L^− 1 was determined in cultures grown in the highest concentration of bicarbonate and was lower in decreasing concentrations. Higher chlorophyll biosynthesis was observed in the presence of sodium bicarbonate at 1 g L^− 1 in C. vulgaris ( Kong et al., 2011 ) where as it was 0.005 M in Chlorella salina ( Jayasankar and Valsala, 2008 ). Growth rate of microalgal population is another measure of increase in biomass and increase in specific growth rate was observed at 1 g L^− 1 over time. The duration of exponential phase in terms of specific growth rate was longer which explains impact of sodium bicarbonate in the culture medium. It was clear that higher photosynthetic efficiency due to increase in chlorophyll content indicates sodium bicarbonate would promote the growth rate as well as biomass production. Biofixation of CO₂ using microalgae and converting it into biomass by way of photosynthesis is a promising way to sequester CO₂ ( Watanabe and Hall, 1996 ; Ono and Cuello, 2006  ;  Ryu et al ., 2009 ). High CO₂ tolerant Chlorella sp. with good growth rate was reported ( Sung et al ., 1998  ; Sung et al ., 1999  ;  Yue and Chen, 2005 ).The amount of carbon content and CO₂ fixation rates were determined in C. vulgaris and carbon content in the range of 0.334–0.497 g dry weight^− 1 was obtained. Similar carbon content in C. vulgaris was reported in earlier studies ( Yun et al ., 1997  ;  Pandian and Ravindran, 2012 ). It was observed that at higher bicarbonate levels, increased carbon sequestration was found.

Bicarbonate induces carbon storage metabolic activity and triggers lipid accumulation (Gardner et al ., 2012  ; Gardner et al ., 2013  ;  White et al ., 2013 ). Higher neutral lipid production in microalgae with the addition of bicarbonate was reported by Bywaters and Fritsen (2015) . Sodium bicarbonate has been used as a carbon source for the study of growth and biochemical composition in a range of microalgae species (Guiheneuf et al ., 2008  ; Guiheneuf et al ., 2009  ; Sostaric et al ., 2009  ; Pimolrat et al ., 2010  ;  Yeh et al ., 2010 ) and has been shown to stimulate triacylglycerol accumulation in microalgal species. The length of carbon chain and the number of double bonds in fatty acids affect its fuel properties in which C16:1 and C18:1 are the ideal biodiesel feedstock (Knothe, 2008  ;  Stansell et al ., 2012 ). One of the main objectives of this study is to enhance lipid content of microalgae for biodiesel production hence the lipid class which are suitable for biodiesel production was considered. In this study, triglycerides with carbon chain lengths of C14–C17 were obtained revealing the potential of bicarbonate addition in microalgal cultivation for biofuel production. It was noted that although increased bicarbonate concentration has increased lipid productivity, the values were still in the lower range of those reported for selected species.

Conclusion

This study has the potential to aid industrial algal biofuel production with the use of sodium bicarbonate as an alternative inorganic carbon source to trigger biomass and lipid production. From the results, it was clear that bicarbonate addition had significant effects on pigment, biomass and lipid production in C. vulgaris isolated from sewage treatment plant. Microalgae exposed to 1 g L^− 1 concentration of bicarbonate expressed three times higher biomass concentration compared to other treatments. Further, the amount of total chlorophyll content, CO₂ fixation, biomass and lipid productivity were significantly influenced by the addition of sodium bicarbonate.

Acknowledgement

The authors are grateful to the Karnataka State Biofuel Development Board and the Karnataka State Council for Science and Technology with Grant No. (38S_B_MSc_015 ) for the financial support.

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