Journal of Applied Phycology

, Volume 25, Issue 1, pp 153–165

The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures

Authors

    • Plymouth Marine Laboratory
  • A. Pagarette
    • Plymouth Marine Laboratory
    • Department of BiologyUniversity of Bergen
  • P. Rooks
    • Plymouth Marine Laboratory
  • S. T. Ali
    • Plymouth Marine Laboratory
Article

DOI: 10.1007/s10811-012-9849-6

Cite this article as:
White, D.A., Pagarette, A., Rooks, P. et al. J Appl Phycol (2013) 25: 153. doi:10.1007/s10811-012-9849-6

Abstract

The addition of bicarbonate (NaHCO3; 0, 1, or 2 g L−1) to microalgal cultures has been evaluated for two species (Tetraselmis suecica and Nannochloropsis salina) in respect of growth and biochemical composition. In batch cultures, addition of bicarbonate (1 g L−1) resulted in significantly (P < 0.05) higher final mean cell abundances for both species. No differences in specific growth rates (SGRs) were recorded for T. suecica between treatments; however, increasing bicarbonate addition decreased SGR values in N. salina cultures. Bicarbonate addition (1 g L−1) significantly improved nitrate utilisation from the external media and photosynthetic efficiency (Fv/Fm) in both species. For both T. suecica and N. salina, bicarbonate addition significantly increased the cellular concentrations of total pigments (3,432–3,587 and 19–37 fg cell−1, respectively) compared to cultures with no additional bicarbonate (1,727 and 11 fg cell−1, respectively). Moreover, final concentrations of total cellular fatty acids in T. suecica and N. salina cultures supplemented with 2 g L−1 bicarbonate (7.6 ± 1.2 and 1.8 ± 0.1 pg cell−1, respectively) were significantly higher than those cells supplemented with 0 or 1 g L−1 bicarbonate (3.2–3.5 and 0.9–1.0 pg cell−1, respectively). In nitrate-deplete cultures, bicarbonate addition caused species-specific differences in the rate of cellular lipid production, rates of change in fatty acid composition and final lipid levels. In summary, the addition of sodium bicarbonate is a viable strategy to increase cellular abundance and concentrations of pigments and lipids in some microalgae as well as the rate of lipid accumulation in nitrate-deplete cultures.

Keywords

Sodium bicarbonateMicroalgaeFatty acidLipidPigments

Introduction

Microalgae are a vast and diverse group of unicellular aquatic organisms with significant potential to produce valuable natural products (Davidson 1995; Plaza et al. 2009; Plaza et al. 2010). Microalgae pigments, especially carotenoids and phycobiliproteins, have been proposed to have applications that range from food colorants to effective agents in disease prevention in humans (Apt and Behrens 1999; Singh et al. 2005; Spolaore et al. 2006; Vilchez et al. 2011). Moreover, microalgal production of polyunsaturated fatty acids (PUFAs) for their nutritive value in aquaculture systems and for human health applications has been recognised (Berge and Barnathan 2005; Khozin-Goldberg et al. 2011). In recent years, focus has turned to the production of microalgae cellular storage lipids (namely triglycerides or TAGs) for the production of biodiesel as sustainable alternatives to petroleum fossil fuels (Chisti 2007; Mata et al. 2010). Microalgae have several potential advantages over terrestrial crops from which biodiesel is currently generated, including higher growth rates and productivity; reduced competition for arable land and associated water resources and effective nutrient utilisation, etc. (Hu et al. 2008; Li et al. 2008; Ahmad et al. 2011). However, despite obvious potential, there are several limitations to the development of this technology, not least the high-cost requirements and practical difficulties associated with the microalgal production process (Greenwell et al. 2010; Scott et al. 2010; Wijffels and Barbosa 2010; Williams and Laurens 2010; Norsker et al. 2011).

Environmental conditions governing microalgae growth and biochemical composition include the availability of light, nutrients, carbon source, salinity, temperature and pH (Guiheneuf et al. 2008; Pal et al. 2011). One factor that has well established implications on lipid production in some but not all microalgae is nitrogen limitation, which shifts the microalgal metabolism towards the production of cellular storage lipids (Parrish and Wangersky 1987; Suen et al. 1987; Alonso et al. 2000). However, under nutrient-limited conditions, the production of cellular storage lipid comes at the expense of regular growth and cell division, which is arrested owing to a reduction in photosynthetic activity and diverted metabolic demand (Hodgson et al. 1991; Larson and Rees 1996). There is evidence to suggest that a significant proportion of cellular storage lipids originates through new synthesis of cellular lipids from de novo carbon fixation (Shifrin and Chrisholm 1981; Suen et al. 1987) or via recycling of existing cellular lipids or carbon from different metabolite pools (Healey 1979; Sukenik and Livne 1991).

New synthesis of cellular storage lipids necessitates adequate supplies of dissolved inorganic carbon in the surrounding media for carbon fixation. Most studies that have examined the effect of inorganic carbon addition and lipid production or indeed biochemical composition in microalgae cultures have focussed on the addition of gaseous CO2 (Tsuzuki et al. 1990; Chu et al. 1996; Gordillo et al. 1998; Hu and Gao 2003; Muradyan et al. 2004; Carvalho and Malcata 2005; Xia and Gao 2005; Chiu et al. 2009). In seawater (ca. pH 8.1), most (>90 %) of the inorganic form of dissolved carbon is in the form of bicarbonate (HCO3), and the rate of spontaneous conversion of HCO3 to CO2 is low (Skirrow 1975). Many microalgae and cyanobacteria species can actively take up HCO3 from the external environment via transport across the plasma membrane into the cytosol and derive CO2 from HCO3 via the action of carbonic anhydrase maintaining a steady state flux to ribulose-1,5-bisphosphate carboxylase oxygenase for photosynthesis. Alternatively, extracellular carbonic anhydrase can catalyse the inter-conversion of HCO3 and CO2. Both mechanisms of HCO3 utilisation have been reported in phytoplankton (Colman and Gehl 1983; Raven 1991; Merrett et al. 1996; Nimer et al. 1997; Huertas and Lubian 1998; Bozzo et al. 2000; Young et al. 2001). In commercial microalgae production situations where CO2 sources, e.g. flumes from power stations, etc., are not readily available, bicarbonate salts could be added to provide adequate carbon source for intensive microalgal production (Ungsethaphand et al. 2009; Chi et al. 2011). Indeed, 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; Jayasankar and Valsala 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 (Guckert and Cooksey 1990; Gardner et al. 2012). This study builds on findings from these previous works by investigating the effects of different levels of sodium bicarbonate on the biochemical composition of microalgal batch cultures and the rate of lipid production in microalgae under nutrient-deplete conditions.

Despite generalised observations of methods to increase lipid production in microalgae, it is apparent that there are species-specific differences in the rates of lipid production and turnover. Moreover, different species exhibit different potentials to utilise HCO3 from the surrounding media (Nimer et al. 1997; Huertas and Lubian 1998; Dason et al. 2004). In this study, the oleaginous eustigmatophyte Nannochloropsis salina and the marine chlorophyte Tetraselmis suecica were chosen as model organisms on which to examine the effects of sodium bicarbonate addition on cellular pigment and lipid production. These species were chosen as they have been reported to have markedly different biochemical compositions, notably in lipid content and profiles. Furthermore, both species may express different modes of inorganic carbon utilisation and may be affected differently by bicarbonate addition, e.g. other Nannochloropsis species do not exhibit external carbonic anhydrase activity and actively uptake bicarbonate ions (Sukenik et al. 1997; Huertas and Lubian 1998; Huertas et al. 2002a, b) whilst carbonic anhydrase enzyme is externally expressed in other Tetraselmis species (Rigobello-Masini et al. 2003). Two experimental approaches have been taken. The first approach was to assess the effects of bicarbonate addition of regular growth and cell composition (including lipid content) in batch cultures of both organisms, and the second, to specifically examine the effects of bicarbonate addition on the rates of lipid production and lipid composition of both microalgae species in nitrogen deplete conditions.

Materials and methods

Stock cultures of Nannochloropsissalina (CCAP 849/2) and Tetraselmissuecica (CCAP 66/38) were obtained from the Culture Collection of Algae and Protozoa (Scottish Association for Marine Science, Oban, Scotland, UK) and maintained in batch cultures (1 L) in f/2 medium (Guillard 1975) and sub-cultured on a weekly basis. Experimental cultures were maintained under 100 μmol photons m2 s−1 irradiance on a 16:8 h light to dark cycle at 15 °C and swirled twice daily to aid gas exchange (no additional aeration was supplied). Analytical grade sodium bicarbonate was used as the source of bicarbonate in all experiments. Estimates of dissolved inorganic carbon (DIC) species were calculated using the Seacarb program (Lavigne and Gattuso 2011) in experimental treatments at 0 g L−1 bicarbonate addition (DIC = 2,119 μmol kg−1, HCO3 = 1,938 μmol kg−1, CO32− = 167 μmol kg−1, CO2 = 14 μmol kg−1 and total alkalinity 2,350 μEq kg−1), 1 g L−1 bicarbonate addition (DIC = 10,820 μmol kg−1, HCO3 = 10,262 μmol kg−1, CO32− = 375 μmol kg−1, CO2 = 181 μmol kg−1 and total alkalinity 11,050 μEq kg−1) and at 2 g L−1 bicarbonate addition (DIC = 19,520 μmol kg−1, HCO3 = 18,554 μmol kg−1, CO32− = 582 μmol kg−1, CO2 = 383 μmol kg−1 and total alkalinity 19,750 μEq kg−1).

Investigation 1: effect of bicarbonate addition in batch cultures

Batch cultures (100 mL) of each species (f/2 medium; n = 3) were grown under different levels of bicarbonate supplementation (0, 1 and 2 g L−1) into early stationary growth phase (10–13 days), where samples were taken for nutrients, photosynthetic efficiency and pigment and cellular lipids analyses (see below). Culture growth was monitored via cell enumeration to determine growth rates and cell densities (see below).

Investigation 2: effect of bicarbonate addition on nitrate-deplete cultures

Cells from cultures of both N. salina and T. suecica were grown in f/2 media in batch culture (n = 3) to late exponential phase (1.5 × 107 and 1.2 × 106 cells mL−1, respectively) and then pelleted by centrifugation, washed in 3 vol of nitrate-deplete f/2 medium and re-suspended in nitrate-deplete medium supplemented with different levels of bicarbonate (0, 1 or 2 g L−1). Cell numbers, cellular lipid content and composition and medium pH were monitored in cultures (n = 3) over 28 days.

Analytical methods

Cells in culture samples (1 mL) were enumerated by light microscope examination in a haemocytometer following staining with Lugols iodine solution (2 %). Specific growth rates (SGRs) were calculated according to the following equation: K′ = ln (N2/N1) / (t2 − t1) where N2 and N1 are the total cells mL−1 at day 3 (t2) and day 0 (t1), respectively.

For nutrient analyses, 10 mL samples of culture were filtered (0.2 μm), and the filtrate was stored (−20 °C) in an acid-washed bottle to await analysis. After thawing, samples were analysed for nitrate and phosphate concentrations using a nutrient autoanalyser (Branne and Luebbe, AAIII, SPX Flow Technology Ltd., UK), employing standard methods (Brewer and Riley 1965; Zhang and Chi 2002). Samples were taken at the beginning and end of the study in investigation 1.

To assess phytoplankton PSII efficiency (Fv/Fm) in batch cultures in stationary phase, discrete variable-chlorophyll fluorescence measurements were acquired using a fluorescence induction and relaxation (FIRe) fluorometer (Satlantic Inc., Canada). Dark-adapted samples were analysed using excitation wavelengths of 450 and 500 nm using a high luminosity blue and green LED array, respectively. The FIRe was employed with a four-step measurement protocol: (1) single turnover (ST) excitation from a 100 μs pulse, (2) ST relaxation from a weak modulated light over 500 ms, (3) multiple turnover (MT) excitation from a 100-ms pulse and (4) MT relaxation from a weak modulated light over 1 s. Ten sequential acquisitions of each four-step sequence were cumulatively averaged for each fluorescence profile to increase the signal to noise ratio. Fluorescence profiles were fitted with the biophysical (KPF) model of Kolber et al. (1998) using the software FIRePro (v. 1.3; Satlantic Inc., Canada).

For pigment analysis, 50 mL samples from batch cultures grown in investigation 1 were centrifuged (3,000 × g), and the algal pellet was stored at −80 °C. Pigments were extracted from algal biomass into 2 mL 100 % acetone containing the internal standard apo-carotenoate (Sigma, UK) using an ultrasonic probe (35 s, 50 W; Sonics Vibra-cell; Sonics and Materials Inc., USA). Extracts were centrifuged to remove cell debris (5 min at 4,000 × g) and analysed using HPLC with a reversed-phase C8 column and gradient elution (Barlow et al. 1997) on a Thermo Accela Series HPLC system with chilled autosampler and photodiode array detector (Thermo Fisher Scientific, USA). The HPLC was calibrated using a suite of standards (DHI, Denmark), and pigments in samples were identified from retention time and spectral match using photo-diode array spectroscopy (Jeffrey et al. 1997).

Fatty acid concentrations and profiles in microalgal cells were determined post-conversion to fatty acid methyl esters (FAMEs) and analysed by gas chromatography–mass spectrometry (GC-MS) (Agilent 7890A GC and 5975 C inert MSD; Agilent Technologies Ltd., UK). Samples (2 mL) were centrifuged (3,000 × g), and resulting pellets were lyophilised. Nonadecanoic acid (C19:0) was added as an internal standard, and cellular fatty acids were converted directly to FAMEs by adding 1 mL of transesterification mix (95:5 v/v 3 N methanolic HCl; 2,2-dimethoxypropane) followed by incubation at 90 °C for 1 h. After cooling, FAMEs were recovered by addition of 1 % w/v NaCl solution (1 mL) and n-hexane (1 mL) followed by vortexing. The upper hexane layer was injected directly onto the GC-MS system, and FAMEs were separated on a fused silica capillary column (15 × 0.1 mm × 0.1 μm; Omegawax™ 100, Supelco, Sigma-Aldrich, UK) using an oven temperature gradient of 140 to 280 °C at 40 °C min−1 followed by 3 min hold time at 280 °C. Helium was used as a carrier gas (0.4 mL min−1), and the injector and detector inlet temperatures were maintained at 280 and 230 °C, respectively. FAMEs were identified using retention times and qualifier ion response and quantified using respective target ion responses. All parameters were derived from calibration curves generated from a FAME standard mix (Supelco, Sigma-Aldrich, UK).

Results

Effect of bicarbonate on growth nutrient utilisation and pH

The growth rates and final cell numbers for both T. suecica and N. salina in batch cultures with different levels of bicarbonate addition were recorded into stationary phase of growth in the first investigation (10 and 13 days, respectively; Fig. 1). For T. suecica cultures, a pronounced lag phase of growth (2 days) was recorded in those cultures supplemented with 2 g L−1 bicarbonate. Final cell abundances were significantly higher in cultures supplemented with 1 g L−1 bicarbonate (1.22 ± 0.04 × 106 cells mL−1) compared to those with 0 and 2 g L−1 supplementation (1.07 ± 0.02 × 106 and 1.04 ± 0.03 × 106 cells mL−1, respectively), which were not significantly different. However, SGR of the different cultures in linear growth phase was not significantly different and ranged from 0.34–0.52 days−1. For N. salina cultures, there was also a more pronounced lag phase (ca. 2 days) in cultures supplemented with 2 g L−1 bicarbonate. Final cell abundances were significantly different between all experimental treatments and highest at 1 g L−1 bicarbonate addition (2.43 ± 0.10 × 107 cells mL−1), followed by 0 g L−1 bicarbonate (1.66 ± 0.24 × 107 cells mL−1) and 2 g L−1 bicarbonate addition (1.07 ± 0.09 × 107 cells mL−1). Final SGR values were significantly different between all experimental treatments and highest at 0 g L−1 bicarbonate addition (1.12 ± 0.11 days−1), followed by 1 g L−1 bicarbonate addition (0.75 ± 0.15 days−1) and 2 g L−1 bicarbonate addition (0.47 ± 0.09 days−1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9849-6/MediaObjects/10811_2012_9849_Fig1_HTML.gif
Fig. 1

Growth curves for (a) T. suecica and (b) N. salina batch cultures supplemented with no additional bicarbonate (NaHCO3, filled circle); 1 g L−1 bicarbonate (empty circle) or 2 g L−1 bicarbonate (filled triangle) in investigation 1 (n = 3, ±SD)

The levels of primary nutrients (nitrate and phosphate) at the end of investigation 1 when cultures were deemed to be in stationary phase can be seen in Table 1. For both species, irrespective of the level of bicarbonate addition, the final levels of phosphate were <3 μM and can be considered to be depleted. Significant levels of nitrate (>50 μM) were remaining in T. suecica and N. salina cultures with no added bicarbonate and in N. salina cultures with 2 g L−1 bicarbonate addition.
Table 1

pH and medium nutrient concentrations in algal batch cultures in stationary phase supplemented with different levels of bicarbonate (NaHCO3) in investigation 1 (n = 3, ±SD)

 

Bicarbonate (g L−1)

pH

Nitrate (μM)

Phosphate (μM)

T. suecica

0

9.45 ± 0.10a

336.4 ± 7.1a

0.10 ± 0.04a

1

9.73 ± 0.01b

2.2 ± 0.9b

0.04 ± 0.01a

2

9.67 ± 0.12b

1.8 ± 1.1b

0.20 ± 0.01b

N. salina

0

9.61 ± 0.21x

496.5 ± 27.9x

2.70 ± 0.04x

1

9.70 ± 0.05x

4.2 ± 0.5y

0.20 ± 0.10y

2

9.28 ± 0.06y

52.2 ± 19.9z

0.40 ± 0.01y

Superscript notation denotes significant difference between T. suecica and N. salina treatments, respectively. Measured values of nitrate and phosphate in media at the beginning of the investigation were 889 and 29 μM, respectively

The addition of bicarbonate had significant effects on the final pH levels in the cultures. In the first investigation, the final pH of the cultures in stationary phase was compared (Table 1). In the T. suecica cultures, the pH of the cultures at 1 and 2 g L−1 bicarbonate addition (pH 9.73 ± 0.01 and 9.67 ± 0.12, respectively) was significantly higher (P < 0.05) than those cultures with no bicarbonate addition (pH 9.45 ± 0.10). In N. salina cultures, the pH at 0 and 1 g L−1 bicarbonate (pH 9.61 ± 0.21 and 9.70 ± 0.05) was significantly higher than cultures with 2 g L−1 bicarbonate addition (pH 9.28 ± 0.06).

In the second investigation, following re-suspension in nitrate-deplete media, cells of N. salina and T. suecica cultures went through at least one doubling, before remaining constant throughout the experimental period with final mean abundances of 2.9 ± 0.5 × 107 and 2.5 ± 0.3 × 106 cells mL−1, respectively (data not shown). In T. suecica cultures, additional bicarbonate promoted a pH rise in a dose-dependent manner (rate and final pH values), with final pH levels (day 28) of pH 8.9, 9.3 and 9.4 for 0, 1 and 2 g L−1 bicarbonate addition, respectively (Fig. 2). For N. salina cultures, the treatments with 0 and 1 g L−1 bicarbonate rose in pH and peaked at pH 8.8–9.0 after 7 days, but the pH in the cultures with 2 g L−1 bicarbonate peaked at approximately pH 8.8 after 14 days.
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9849-6/MediaObjects/10811_2012_9849_Fig2_HTML.gif
Fig. 2

pH in media of (a) T. suecica and (b) N. salina cultures re-suspended in nitrate-deplete media supplemented with no additional bicarbonate (NaHCO3, filled circle); 1 g L−1 bicarbonate (empty circle) or 2 g L−1 bicarbonate (filled triangle) in investigation 2 (n = 3, ±SD)

Effect of bicarbonate on pigment concentration and composition and photosynthetic efficiency

Cellular pigment levels and relative pigments to Chl-a ratios were only monitored in samples taken at the end of the first investigation (Tables 2 and 3). For both T. suecica and N. salina, the addition of bicarbonate had a significant positive effect on the levels of individual cellular pigments (except for zeaxanthin in N. salina) with an approximate doubling or more in pigment concentrations with the addition of 2 g L−1 bicarbonate, compared to treatments with no added bicarbonate. For T. suecica cultures, there was no significant difference in the cellular concentration of total pigments between cultures supplemented with either 1 or 2 g L−1 bicarbonate. For the individual pigments, only the concentrations of β,β-carotene and Chl-a were significantly different between the 1 g L−1 bicarbonate (86.9 ± 7.2 and 1,563.4 ± 114.9 fg cell−1, respectively) and 2 g L−1 bicarbonate (129.7 ± 26.7 and 1,242.5 ± 51.6 fg cell−1, respectively) treatments. For N. salina cultures, the total levels of pigments increased in a dose-dependent fashion with bicarbonate addition, with maximum levels being recorded in cultures supplemented with 2 g L−1 bicarbonate (36.7 ± 3.5 fg cell−1). Consistent with this, cellular levels of violaxanthin, lutein and Chl-a were maximum in the 2 g L−1 bicarbonate treatments.
Table 2

Photosynthetic efficiency and cellular pigment composition in algal batch cultures in stationary phase supplemented with different levels of bicarbonate (NaHCO3) in investigation 1 (n = 3, ±SD)

 

Pigment (fg cell−1)

 

Bicarbonate (g L−1)

Fv/Fm

Violaxanthin

Zeaxanthin

Lutein

β,β-carotene

Chlorophyll-b

Chlorophyll-a

Total

T. suecica

0

0.60 ± 0.01a

187.7 ± 22.6a

8.6 ± 1.3a

180.7 ± 20.3a

34.7 ± 5.1a

618.1 ± 63.6a

696.8 ± 75.9a

1726.6 ± 187.1a

1

0.67 ± 0.01b

299.7 ± 26.1b

16.7 ± 2.6b

287.2 ± 24.3b

86.9 ± 7.2b

1177.9 ± 88.0b

1563.4 ± 114.9b

3431.8 ± 257.9b

2

0.71 ± 0.01c

365.5 ± 68.8b

16.3 ± 1.9b

303.3 ± 44.9b

129.7 ± 26.7c

1529.9 ± 280.8b

1242.5 ± 51.6c

3587.2 ± 472.6b

N. salina

0

0.54 ± 0.00x

2.3 ± 0.1x

1.5 ± 0.1x

0.7 ± 0.03x

0.6 ± 0.05x

5.8 ± 0.2x

10.9 ± 0.4x

1

0.59 ± 0.01y

3.6 ± 0.8y

1.6 ± 0.3x

1.2 ± 0.3y

1.0 ± 0.2y

11.1 ± 2.4y

18.5 ± 3.9y

2

0.52 ± 0.00z

7.1 ± 0.8z

1.7 ± 0.1x

2.5 ± 0.2z

1.0 ± 0.1y

24.5 ± 2.5z

36.7 ± 3.5z

Table 3

Pigment/Chl-a ratios in algal batch cultures in stationary phase supplemented with different levels of bicarbonate (NaHCO3) in investigation 1 (n = 3, ±SD)

 

Bicarbonate (g L−1)

Violaxanthin

Zeaxanthin

Lutein

β,β-carotene

Chlorophyll-b

T. suecica

0

0.269 ± 0.003a

0.012 ± 0.002a

0.259 ± 0.001a

0.050 ± 0.002a

0.887 ± 0.011a

1

0.192 ± 0.005b

0.011 ± 0.001a

0.184 ± 0.006b

0.056 ± 0.003a

0.753 ± 0.005b

2

0.293 ± 0.045a

0.013 ± 0.001a

0.243 ± 0.028a

0.104 ± 0.018b

1.227 ± 0.180c

N. salina

0

0.392 ± 0.011x

0.254 ± 0.015x

0.120 ± 0.009x

0.099 ± 0.004x

1

0.324 ± 0.007y

0.148 ± 0.020y

0.106 ± 0.003y

0.092 ± 0.009x

2

0.289 ± 0.005z

0.070 ± 0.010z

0.103 ± 0.005y

0.041 ± 0.003y

The effect of bicarbonate addition on the ratios of individual pigments to Chl-a varied significantly between T. suecica and N. salina cultures. In T. suecica cultures, although there were significant differences between treatments, there was no clear relationship between bicarbonate addition and the pigment: Chl-a ratio for individual or total levels of cellular pigment. However, in N. salina cultures, for each individual pigment, increasing levels of bicarbonate addition reduced the respective pigment: Chl-a ratio in a dose-dependent manner. The effect of bicarbonate supplementation on photosynthetic efficiency (Fv/Fm) of microalgae in stationary phase can be seen in Table 2. In T. suecica cultures, the Fv/Fm was maximum (P < 0.05) in the 2 g L−1 bicarbonate addition treatment and appeared to increase in a dose-dependent manner with increasing bicarbonate addition. In N. salina cultures, the Fv/Fm was maximum (P < 0.05) in cultures supplemented with 1 g L−1 bicarbonate and was lowest in cultures supplemented with 2 g L−1 bicarbonate.

Effect of bicarbonate on cellular lipid concentration and composition

In the first investigation, cellular lipid concentration (based on total fatty acid content) and fatty acid profiles were determined in all cultures in stationary growth phase (Table 4). For T. suecica cultures, the total amount of cellular lipid was significantly higher (P < 0.05) in cultures supplemented with 2 g L−1 bicarbonate (7.6 ± 1.2 pg cell−1), compared to cultures with 1 and 0 g L−1 bicarbonate addition (3.5 ± 0.5 and 3.2 ± 0.9 pg cell−1, respectively). Similarly, in N. salina cultures, total lipid was significantly higher in cultures supplemented with 2 g L−1 bicarbonate (1.8 ± 0.1 pg cell−1) compared to cultures with 1 and 0 g L−1 bicarbonate addition (1.0 ± 0.2 and 0.9 ± 0.1 pg cell−1, respectively). There were no significant effects of bicarbonate addition on the contribution (mol%) of saturated, mono- and polyunsaturated fatty acids within cellular lipids for T. suecica cultures. However, the highest addition of bicarbonate promoted a significant increase in the mean level of saturated fatty acids (ca. 40 %) in N. salina, compared to the other treatments (ca. 34–37 %), and 1 g L−1 bicarbonate addition promoted significantly higher mean levels of PUFA (ca. 22 %), compared to the other treatments (ca. 16–17 %). Monounsaturated fatty acids were significantly higher in N. salina cultures with no bicarbonate addition (ca. 46 %), compared to other treatments (ca. 43–44 %).
Table 4

Fatty acid profiles and total cellular lipid in algal batch cultures in stationary phase supplemented with different levels of bicarbonate (NaHCO3) in investigation 1 (n = 3, ±SD)

 

T. suecica

N. salina

Bicarbonate (g L−1)

Bicarbonate (g L−1)

0

1

2

0

1

2

Fatty acid (mol%)

C14:0

5.6 ± 0.2

5.8 ± 0.2

4.7 ± 0.2

C16:0

29.6 ± 0.3

30.1 ± 0.7

30.8 ± 0.4

29.5 ± 0.9

30.9 ± 5.8

33.8 ± 0.9

C16:1

36.8 ± 1.0

34.3 ± 4.8

38.5 ± 0.8

C18:0

1.6 ± 0.0

1.6 ± 0.5

1.6 ± 0.1

C18:1

10.8 ± 0.5

11.9 ± 0.7

11.3 ± 0.9

9.3 ± 0.4

6.5 ± 0.8

5.1 ± 0.3

C18:2 cis

2.8 ± 0.1

2.8 ± 0.1

2.7 ± 0.1

3.0 ± 0.3

2.5 ± 0.1

2.2 ± 0.0

C18:3 (n-3)

34.8 ± 0.3

33.3 ± 0.6

33.6 ± 0.7

C20:4

2.5 ± 0.1

4.1 ± 0.4

2.3 ± 0.1

C20:5

10.6 ± 0.1

10.7 ± 0.3

10.6 ± 0.7

11.7 ± 1.4

14.4 ± 2.0

11.8 ± 0.9

Saturates

31.8 ± 0.2a

32.4 ± 1.3a

33.2 ± 0.5a

36.8 ± 0.6x

34.7 ± 1.8x

40.2 ± 0.5y

Monounsaturates

14.1 ± 0.1a

15.4 ± 0.5a

14.7 ± 0.8a

46.1 ± 0.6x

43.3 ± 0.2y

43.5 ± 0.5y

PUFA

54.1 ± 0.2a

52.2 ± 1.1a

52.0 ± 1.3a

17.2 ± 1.2x

22.0 ± 1.9y

16.3 ± 1.0x

Total lipid (pg cell−1)

3.2 ± 0.9a

3.5 ± 0.5a

7.6 ± 1.2b

0.9 ± 0.0x

1.0 ± 0.2x

1.8 ± 0.1y

Fatty acids, contributing less than 1 % to total fatty acid profile, have been omitted. Total lipid was based on total FAME analysed by GC-MS

In the second investigation, when re-suspended in nitrate-deplete media, both T. suecica and N. salina cultures showed a positive effect (P < 0.05) of bicarbonate supplementation on the production of cellular lipid (Fig. 3). In T. suecica cultures, the rate of lipid synthesis appeared to be significantly enhanced by adding bicarbonate in a dose-dependent manner with rates of 0.5, 0.8 and 1.3 pg cell−1 day−1 for 0, 1 and 2 g L−1 bicarbonate supplementation, respectively. Maximum cellular lipid levels in T. suecica cultures reached after the 28-day experimental period were 15.8 ± 2.4, 31.8 ± 1.9 and 32.5 ± 3.8 pg cell−1 for 0, 1 and 2 g L−1 bicarbonate supplementation, respectively. In N. salina cultures, the time taken to reach the maximum cellular lipid levels differed according to the level of bicarbonate addition with values of 1.3 ± 0.1 pg cell−1 (7 days), 1.7 ± 0.2 pg cell−1 (7 days) and 2.7 ± 0.2 pg cell−1 (14 days) for 0, 1 and 2 g L−1 bicarbonate supplementation, respectively. Following maximum cellular lipid levels being reached, all N. salina cultures showed a general decline in cellular lipid levels.
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9849-6/MediaObjects/10811_2012_9849_Fig3_HTML.gif
Fig. 3

Cellular lipid production in (a) T. suecica and (b) N. salina cultures re-suspended in nitrate-deplete media and supplement with no additional bicarbonate (NaHCO3, filled circle); 1 g L−1 bicarbonate (empty circle) or 2 g L−1 bicarbonate (filled triangle) in investigation 2 (n = 3, ±SD)

Following re-suspension in nitrate-deplete media, there was a significant change in the relative composition (mol%) of different fatty acids for both species (Fig. 4). There was a significant decrease in the levels of polyunsaturated fatty acids from time 0 to 28 days in both T. suecica (from ca. 62 to 37–45 %) and N. salina (from ca. 16 to 4 %) and with a notable increase in the levels of saturated fatty acids (from ca. 33 to 45–53 % and from ca. 35 to 45 %, respectively). Levels of monounsaturated fatty acids rose slightly in T. suecica cultures (from 7 to 10–12 %) and remained relatively consistent in N. salina (range 48–52 %) throughout the 28-day experimental period. Increasing addition of bicarbonate in the nitrate-deplete media had a significant effect on the rate of change and in the final levels of PUFAs and saturated fatty acids in T. suecica cultures (increasing bicarbonate addition increased the rate of PUFA decline and correspondingly increased saturated fatty acid content) but had no significant effect on the rate of change in the relative composition on fatty acids in N. salina.
https://static-content.springer.com/image/art%3A10.1007%2Fs10811-012-9849-6/MediaObjects/10811_2012_9849_Fig4_HTML.gif
Fig. 4

Levels of (a) saturated (b) monounsaturated and (c) polyunsaturated fatty acids in (i) T. suecica or (ii) N. salina cultures re-suspended in nitrate-deplete media supplemented with no additional bicarbonate (NaHCO3, filled circle); 1 g L−1 bicarbonate (empty circle) or 2 g L−1 bicarbonate (filled triangle) in investigation 2 (n = 3, ±SD)

Discussion

An adequate supply of inorganic carbon is essential for regular photosynthesis and growth in photoautotrophic microalgae. This may be achieved through the supply of gaseous CO2 to the media in which the microalgae are growing. However, in commercial situations where the supply of adequate CO2 may be limited, alternative inorganic carbon sources, e.g. bicarbonate salts (NaHCO3), could potentially be utilised. Furthermore, bicarbonate has greater solubility than CO2, thus reducing issues associated with low retention times (Hsueh et al. 2007). However, interspecies differences in utilisation of bicarbonate as a carbon source may result in differences in metabolic efficiency and biochemical composition (Giordano et al. 2005). This study has been carried out to assess the effect of bicarbonate addition on the growth and biochemical composition of model microalgal species with the focus on lipid synthesis which is relevant for biodiesel production. The onset of significant lipid storage production in microalgae is often promoted by stress conditions, not least the depletion of essential nutrients like nitrogen (Suen et al. 1987; Alonso et al. 2000). Different approaches have been proposed for lipid production in commercial growth environments, including growing algae under nutrient-limited (but not deficient) conditions, or a two-stage process whereby algae are grown under nutrient-replete conditions to high densities before transferal into a nutrient-deplete situation to encourage maximum lipid production (Su et al. 2011). In this study, the effects of bicarbonate addition on growth and lipid production were examined in microalgae grown in both batch cultures to stationary phase of growth and following transferal of microalgal cells to nitrate-deplete conditions.

At the end of the first investigation, when microalgae were deemed to be in stationary phase of growth, it was clear that bicarbonate addition had had significant effects on growth and nutrient utilisation of both microalgae species. Cultures of both species exposed to the highest level of bicarbonate addition expressed extended lag periods of growth, and N. salina cultures did not grow to similar cell abundances compared to other treatments. Moreover, SGRs in N. salina cultures were reduced by bicarbonate addition, whereas bicarbonate addition had no significant effect on the growth rates of T. suecica cultures, suggesting species-specific responses to bicarbonate addition with respect to cell division. Indeed, recently, it was shown that bicarbonate addition was able to stop replication in the chlorophyte Scenedesmus sp., but not in the diatom Phaeodactylum tricornutum (Gardner et al. 2012). However, in the present study, some bicarbonate addition (1 g L−1) did result in significantly higher final cell abundances in both species compared to cultures with no added bicarbonate, suggesting the microalgal species examined here have an optimum and threshold tolerance to bicarbonate addition above which reduced cell densities/biomass are recorded. This is in agreement with other authors who have demonstrated that Chlorella vulgaris has an optimum level of sodium bicarbonate addition of 1,000 mg L−1 for biomass production (Yeh et al. 2010). As demonstrated in this study, the threshold level for inorganic carbon above, which growth inhibition may occur, will be species dependent and will undoubtedly be intrinsically linked with light availability and other environmental conditions (Carvalho and Malcata 2005).

When considering final levels of nutrients in cultures in stationary phase of growth, it is interesting to note that phosphate was depleted in all cultures, but nitrate was not, suggesting phosphate may have been the limiting nutrient promoting growth cessation. Indeed, compared to cultures with no added bicarbonate, for both species examined, nitrate utilisation was significantly increased with bicarbonate addition, although this did not necessarily translate into better growth of cultures (based on growth rates and final cell densities). These results suggest that the addition of inorganic carbon may have driven increased metabolic demand for nitrogen for synthesis of cellular components. Indeed, increased synthesis of cellular lipids and pigments (see below) may have in part accounted for this. This is consistent with previous studies that have demonstrated increased nitrogen uptake with increasing CO2 levels in microalgae associated with increased protein (nitrogenous compounds) production (Larsson et al. 1985; Xia and Gao 2005). It is not possible from this investigation to determine whether the biological demand for nitrogen was consistent throughout the growth phases of the batch cultures or more extensive in certain growth phases, e.g. higher demand for nitrogen in stationary phase, due to the increased production of nitrogenous secondary metabolites. Interestingly, the photosynthetic efficiency (Fv/Fm) of T. suecica cultures in stationary phase was significantly improved with the addition of bicarbonate. In N. salina, medium levels of bicarbonate addition (1 g L−1) improved photosynthetic efficiency, but high levels caused a significant decrease. This is consistent with the levels of nitrate utilisation for this species which was highest at medium levels of bicarbonate addition and again suggests threshold tolerance levels for bicarbonate addition. Increases in photosynthetic efficiency may suggest increased carbon fixation, presumably owing in part to increased availability of inorganic carbon and an increased metabolic demand.

The pH of all experimental cultures at the end of the first investigation were >9.2. The removal of CO2 by photosynthetic microalgae leads to an increase in pH and a decrease in CO2 partial pressure (providing CO2 replacement occurs more slowly than utilisation). At these pH, the growth of these species will be limited, owing to a shift in the inorganic form of carbon to carbonate (CO32−) which is not readily utilised by photosynthetic marine algae and results in reduced growth and photosynthesis (Chen and Durbin 1994). It is not possible to say here whether the change in pH had resulted in cessation of growth of cultures and the onset of stationary growth phase. Interestingly, in T. suecica cultures with medium and high levels of bicarbonate addition, the final pH was significantly higher compared to cultures without additional bicarbonate, suggesting increased carbon fixation (consistent with nitrate utilisation and photosynthetic efficiency results). Furthermore, these results are consistent with pH measurements in T. suecica cultures following re-suspension of cells in nitrate-deplete media (investigation 2), where pH values were higher in cultures with bicarbonate supplementation. However, in N. salina batch cultures, high levels of bicarbonate addition resulted in significantly lower pH of the cultures compared to the other treatments, suggesting reduced carbon fixation (supported by the lower levels of nitrate utilisation and photosynthetic efficiency). Moreover, the rate of pH increase in N. salina cultures re-suspended in nitrate-deplete media with high levels of bicarbonate addition appeared slower and peaked later, compared to other treatments, suggesting reduced rates of carbon fixation. It is not clear why the final pH values in the second investigation were consistently lower in N. salina cultures compared to T. suecica cultures, as these may have been influenced by interspecies differences in carbon fixation under nitrate-deplete conditions and/or potentially by the accumulation of excreted growth products, etc.

The types of cellular pigments and fatty acids identified at the end of the first investigation are consistent with other works that have studied the occurrence of these compound types in these two species of microalgae (Volkman et al. 1989; Goes et al. 1994; Reitan et al. 1994; Karlson et al. 1996; Forjan et al. 2007; Pal et al. 2011; Ruivo et al. 2011). Unfortunately, comparative cell size/volume was not measured in the current investigation, and subsequently, caution should be displayed when comparing cellular concentration of compounds measured here, with concentrations measured in other investigations, especially those based on cellular dry weight. Nonetheless, at the end of the first investigation when the cultures were deemed to be in stationary phase, results showed that bicarbonate addition had a significant positive effect on the cellular levels of total lipid and pigments. This is in agreement with other authors that have shown changing levels of inorganic carbon (bicarbonate or CO2) in microalgae cultures can increase total amounts of fatty acids and pigments in microalgal cultures (Tsuzuki et al. 1990; Sergeenko et al. 2000; Hu and Gao 2003; Muradyan et al. 2004; Carvalho and Malcata 2005; Xia and Gao 2005; Chiu et al. 2009; Pimolrat et al. 2010). Although no effects of bicarbonate addition could be detected on the composition of fatty acids in T. suecica cultures, there was a marked effect on N. salina cultures. Interestingly, the highest addition of bicarbonate (2 g L−1) resulted in reduced growth in N. salina cultures and also promoted the highest level of fatty acids and more specifically the highest cellular level of saturated fatty acids. This is in agreement with other authors that have shown that high CO2 levels (10 %) in Dunaliella salina and Spirulina platensis cultures inhibited desaturation and elongation of fatty acids and caused an increase in the relative content of saturated fatty acids (Mouradian et al. 1998; Muradyan et al. 2004). Increases in cellular pigment concentration provide potential for increased photosynthesis (Yang and Gao 2003). Indeed, the increased Fv/Fm values recorded in this investigation support this notion for T. suecica. However, despite the highest levels of pigment in N. salina cells being recorded in the 2 g L−1 bicarbonate treatment, the Fv/Fm value was at its lowest value which was in agreement with the comparatively reduced growth witnessed, suggesting a decoupling of cellular pigment content and photosynthetic efficiency at this level of sodium bicarbonate addition.

In the second investigation, following re-suspension in nitrate-deplete media, there was both a significant increase in cellular lipid and a change in the percentage composition of fatty acids. The recorded shift from PUFA to saturated and monounsaturated fatty acids is consistent with the conversion of polar membrane lipids and de novo synthesis of fatty acids into intracellular lipid droplets mainly composed of triacylglycerol when cells are under nutrient stress (Tornabene et al. 1983; Parrish and Wangersky 1987; Suen et al. 1987; Siron et al. 1989; Lombardi and Wangersky 1991; Yongmanitchai and Ward 1991; Reitan et al. 1994; Siaut et al. 2011). There were clear interspecies differences in the rate of cellular lipid production and time taken to reach maximum cellular lipid levels following re-suspension, with N. salina reaching maximum levels within 7 days (at longest), but T. suecica taking at least 28 days under the same conditions. Interestingly, bicarbonate addition increased both the total amounts of cellular lipids; the rate of cellular total lipid production and the change in the relative percentage of saturated and polyunsaturated fatty acids in T. suecica cells, all of which has implications in terms of lipid productivity for microalgal oil production. Consistent with the photosynthetic efficiency results from the first investigation, this suggests that the bicarbonate addition can promote increased carbon fixation to lipid storage reserves and/or lipid turnover under nitrate-deplete conditions for this species. Although bicarbonate addition had significant positive effects on the maximum cellular total lipid in N. salina, its effects on the rate of lipid production or the relative change in composition of saturated and polyunsaturated fatty acids were not clear. However, this may have been a result of insufficient sampling frequency immediately after re-suspension in nitrate-deplete media and the associated rapid lipid turnover in this species.

Overall, it appeared that T. suecica cultures were tolerant to the high levels of bicarbonate (2 g L−1) used in this investigation unlike N. salina where reduced growth, photosynthetic efficiency and nitrate utilisation (compared to 1 g L−1 bicarbonate addition) were recorded. This implies that cell division and replication in N. salina may be hindered by the high levels of bicarbonate; however, the increased levels of cellular pigments and/or lipids suggest at least a base level of inorganic carbon fixation is occurring. Possibly, these differences may be associated with species specific processes of inorganic carbon utilisation i.e. T. suecica likely utilises external carbonic anhydrase to convert HCO3 to CO2 prior to transport into the cell, unlike N. salina that may require an energy dependent active HCO3 uptake mechanism (Huertas et al. 2002a, b; Rigobello-Masini et al. 2003). However, it is unclear why an excess of HCO3 would limit an active uptake process and may suggest the presence of a complex feedback inhibition mechanism. Alternatively, there may have been species-specific inhibition of cell division in response to increased levels of other inorganic carbon species (namely CO32−) or alkalinity of the media, which are greater with increased bicarbonate addition. Clearly, more research is required to elucidate these findings.

In summary, the findings suggest that bicarbonate addition can significantly affect the nutrient utilisation, photosynthetic efficiency and production of cellular compounds including pigments and lipids in microalgae, although the responses are species specific when compared under similar conditions. Furthermore, the rates and levels of lipid synthesis and/or turnover under nitrate-deplete conditions can be promoted with bicarbonate addition in certain microalgal species. Given that the final cell abundances were not negatively affected by some bicarbonate addition (suggesting biomass productivity is unaffected), these findings indicate that higher yields of valuable lipid and pigment compounds may be promoted by utilising bicarbonate addition in commercial production systems where sources of CO2 or efficient air–water gas exchange is limited, although effects are likely to be species specific and would require independent evaluation.

Acknowledgments

The authors would like to thank Helen Findlay and Victor Martinez-Vincente for their assistance in the calculation of DIC species and pigment analyses, respectively. This work was funded by the UK's Carbon Trust ‘Algal Biofuels Challenge’ programme.

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© Springer Science+Business Media B.V. 2012