Evaluation of potent cyanobacteria species for UV-protecting compound synthesis using bicarbonate-based culture system

Abstract

The present investigation evaluates the potential of three cyanobacteria species Anabaena cylindrica, Nostoc commune and Synechococcus BDUSM-13 for photo-protecting mycosporine-like amino acids (MAAs) synthesis using bicarbonate-based culture system. Current investigations witnessed noteworthy bicarbonate tolerance of all species (NaHCO₃; 0.5, 1 and 2 g L^{− 1}) in terms of their growth rate, chlorophyll content, biomass productivity and carbon fixation ability. Among all strains, Synechococcus BDUSM-13 showed maximum surge in specific growth rate (i.e. 0.72 day⁻¹) at 1 g L⁻¹, productivity (i.e. 0.92 ± 0.06 g day⁻¹ L⁻¹) and chlorophyll content (i.e. 0.09 g L⁻¹) at 2 g day⁻¹ L⁻¹. Synechococcus cells were also has the 0.48 g dw⁻¹ carbon content with highest CO₂ fixation rate (i.e. 0.653 g.CO₂ mL⁻¹ day⁻¹) at 2 g L⁻¹. Though, they were not able to produce MAAs after long UV-B exposure (i.e. 24 and 48 h). A. cylindrica strain was the most competent species for the bicarbonate-based approach, produced UV-protecting iminomycosporine compound (i.e. shinorine, λ_max at 334 ± 2 nm) along with carbon fixation (i.e. 0.49 g CO₂ mL⁻¹ day⁻¹) at 2 g L⁻¹ NaHCO₃. This suggests the bicarbonate supplementation during cultivation is a promising strategy to increase cellular abundance, biomass productivity and carbon fixation in cyanobacteria. However, UV-B irradiation may cause species-specific differences in the MAAs synthesis to produce UV-protecting compounds.

Introduction

Carbon dioxide (CO₂), a key greenhouse gas that drives global climate change, can be captured by tiny photosynthetic microorganisms to produce diverse range of compounds (e.g. biofuel, bio-fertilizers, cosmetics, nutraceuticals, pharmaceuticals, bioactive substances etc.) (Smith et al. 2008; Singh et al. 2013; Singh et al. 2016a, b; Singh and Dhar 2019; Maroneze et al. 2019). This scheme seems to be an ideal route to reduce the atmospheric CO₂ level, but this is challenged by exceedingly large energy demands for CO₂ gas compression and transportation, as well as significant loss of gaseous CO₂ from outgas if the algae are cultured in an open system. For commercial cultivation purposes at industrial scale, CO₂ is supplied directly to the culture in gaseous form mixed with air. However, direct CO₂ feeding, due to poor solubility of CO₂ and outgassing from culture medium are still major challenges faced at large scale algal cultivation. More than 70% of supplied CO₂ could be outgassed from the culture medium, thus a maximum of only 25% is efficiently captured by the current algal cultivation system. This low carbon utilization efficiency of conventional CO₂-based cultivation is not acceptable to mitigate CO₂ rich flue gasses, emitted from the industries by combustion of fossil fuels (Campos et al. 2016; Blunden and Arndt 2020). An alternative strategy could be to use bicarbonate salts such as sodium bicarbonate (NaHCO₃) as a carbon source for algal biomass, instead of gaseous CO₂. Recently, Lee et al. (2019) evaluated the techno-economic and environmental feasibility of NaHCO₃ manufacturing process that uses sodium carbonate solution with CO₂ gas, obtained from the flue gas of a coal-fired power plant. Results showed that the process efficiently produces 30,000 tons of NaHCO₃ with a purity of > 99% by utilizing approximately 10,000 tons of CO₂ gas per year. It indicates the strong techno-economical potential of the NaHCO₃ production process. This could be further integrated with algal cultivation to develop a sustainable onsite CO₂ mitigation system for industries. This proposed scheme offers many technical and physiological advantages, making the overall mitigation system sustainable, feasible and desirable. Bicarbonate salts are not only more soluble (> 90 g L⁻¹) than CO₂ gas (1.45 g L⁻¹) in water at 25 °C but also preferred inorganic carbon form to utilize by algal cells. Carbon concentration mechanisms (CCM) of microalgae and cyanobacteria have also revealed that they can import both CO₂ and HCO₃^– (Singh et al. 2014a, b, c). However, once imported into the cell, both forms accumulate in the cell mainly as HCO₃^– to prevent CO₂ leakage due to higher permeability through lipid membrane. Bicarbonate affects both the electron donor and acceptor sides of Photosystem II (PSII), known to be important for proper photosynthetic activity (Carrieri et al. 2007). Additionally, Kaplan et al. (1984) showed the essential role of sodium for the maintenance of intracellular pH during HCO₃^– uptake in the bicarbonate transporting system of the cyanobacterium Anabaena variabilis. Thus, use of sodium bicarbonate for algal cultivation would not only result in lower capturing costs than CO₂, which requires intensive energy for compression but also many physiological benefits during photosynthesis. Although it appears promising, the challenge for development of such a mitigation system is to screen potential strains of microalgae or cyanobacteria that can grow in a high bicarbonate concentration. Strains must have capability to overcome the high pH and high ion strength during the cultivation period. Cyanobacteria, a group of Gram-negative photosynthetic prokaryotes are being sought as alluring biofactories for CO₂ sequestration, renewable biofuel production and commercially valuable products. Being phototrophs, cyanobacteria, require CO₂ for photosynthetic activity but only a few strains could utilize gaseous CO₂ efficiently. They must convert gaseous CO₂ into bicarbonate (HCO₃^–) and then utilize it for photosynthesis. An efficient CCM of cyanobacteria works for acquiring dissolved inorganic carbon species (C_i abbreviation for CO_2, HCO₃^– and CO₃^2–) available in the environment for carbon fixation with the RuBisCO enzyme (Badger and Price 1992; Singh et al. 2014a, b, c). Five different C_i uptake systems have been identified, three for the uptake of HCO₃^– and two for the conversion of CO₂, that diffuses into the cell, to bicarbonate. Eukaryotic algae can also employ a CCM, but it works differently from the CCM of cyanobacteria.

In addition to excellent carbon fixation ability, cyanobacteria are also synthesized ultraviolet (UV) protecting compounds to provide a natural protection against damaging ultraviolet (UV-B) and intense sunlight (Stoyneva-Gartner et al. 2020). The continued release of chlorofluorocarbons, chlorocarbons, organobromides and reactive nitrogen species induces rapid depletion of ozone layer thereby enhancing solar UV-B radiation on the earth surface (Sinha 2015). UV-B radiations are known for DNA damage (Buma et al. 2003). However, cyanobacteria could overcome damaging effects of UV exposure with several defence strategies. Many researchers have shown that cyanobacteria possess cellular mechanisms that make them adaptable to survive in extremely harsh conditions, like growth in high pH ranges from 8.4 to 10.8 of soda lakes (Zavarzin et al. 1999) and mitigate the damaging effects of UV exposure (Sinha 2015). Solar radiation exposed cyanobacterial cells produce natural photo-protectants like mycosporine-like amino acids (MAAs) and scytonemin (Sinha et al. 2001). Mycosporines and mycosporine-like amino acids (MAAs) are a family of small intracellular compounds (MW < 400 Da; e.g. hexose-bound porphyra-334, palythine, shinorine, catenelline, etc.) with absorption maxima in between 268 and 362 nm. They are colourless and highly polar (water soluble) due to cyclohexenone or cyclohexenimine chromophore, conjugated with the nitrogen substituent of an amino acid or its imino alcohol (e.g. shinorine). They biosynthesized upon exposure to solar ultraviolet radiation via shikimic acid pathway in several organisms such as lichens, fungi, algae and cyanobacteria to protect against solar radiation (Singh et al. 2012; Corinaldesi et al. 2017). The inherent abilities of cyanobacteria such as excellent carbon fixation ability and synthesis of ultraviolet (UV) protecting compounds must be utilized to develop sustainable and economically viable CO₂ mitigation technology to overcome global climate change. This research examined the possibility that a bicarbonate-based mitigation system may operate in cyanobacteria species (i.e. Anabaena cylindrica, Nostoc commune and Synechococcus BDUSM-13). The tolerance level of each species against NaHCO₃ gradient (i.e. 0.5, 1 and 2 g L^{− 1}) were evaluated by comparing their growth rate, chlorophyll content, biomass productivity and carbon fixation capacity. Subsequently, species were further explored for their potential to produce UV-protecting compounds in optimized NaHCO₃ level. The overall aim of the present study is to identify a suitable route for a successful bicarbonate-based algal cultivation system which could be further commercialized at industrial scale.

Materials and methods

Sources, growth media and culture conditions of cyanobacteria

Three cyanobacteria species were selected based on their culture conditions and ability to produce MAAs. The freshwater species, A. cylindrica and N. commune were obtained from Centre of Biotechnology, University of Allahabad, India. However, Synechococcus BDUSM-13, a marine cyanobacteria, was a gift from National Facility for Marine Cyanobacteria (NFMC) Tiruchirappalli, India. All cultures were grown in 250 mL Erlenmeyer flasks with 150 mL of suitable culture medium, pH 7.5, under 20 ± 2 °C and light intensity of 72 μmol m⁻² s⁻¹ (measured by Lux meter Lutron LX-101A) with a 16:08 h light and dark cycles using cool-white fluorescent lamps (80 W). The species were cultivated under shaking conditions in their appropriate culture media, BG-11^−ve (without NaNO₃) (Stanier and Cohen-Bazire 1977) and ASN^+ve (Rippka et al., 1979) media for freshwater and marine cyanobacteria, respectively.

Experimental treatment with varying NaHCO₃ concentrations

Batch experiments were performed in 250 mL Erlenmeyer flasks, each containing 150 mL of culture media. The culture media, BG-11^−ve and ASN^+ve for freshwater and marine cyanobacteria, respectively, were supplemented with analytical grade sodium bicarbonate (Sigma–Aldrich, USA). Based on preliminary experiments and literature, three different concentrations of NaHCO₃ (i.e. 0.5, 1 and 2 g L⁻¹) were selected for experiments. Flasks without supplemental NaHCO₃ served as the control group. Inocula were added in the experimental and control Erlenmeyer flasks to obtain a similar cell numbers (i.e. 1 × 10⁵ cells.ml⁻¹) in their culture media (BG-11^−ve or ASN^+ve). Abiotic environmental conditions like temperature, luminosity and photoperiod of experimental flasks were maintained at 20 ± 1 °C, 72 μmol photons m² s⁻¹ of light irradiance and 16:08 h (L:D hour) of light, respectively. The pH of the flasks was monitored by a pH sensor (D.O. Apparecchiature Elettroniche) and maintained in between 7.5 to 8.5 by a concentrated HCl solution (i.e. 2 M) to adjust the pH value. The HCl concentration was chosen to avoid substantial volume changes in the experimental flasks. All the experiments in this study were conducted in triplicate (n = 3) and lasted for 16 days. On the 17th day, biomass of cyanobacteria was harvested at the stationary phase of growth by centrifugation at 3000×g for 5 min, filtered, dried at 40 °C till 48 h for further analysis.

Growth analysis

1 mL of cyanobacteria suspension was collected periodically from experimental flasks and analysed for optical density (OD₇₅₀) and cell number analysis. The filament number of cyanobacteria was counted by the method described by Fogg (1949) and Dextro et al. (2018) under a light microscope (Olympus) using a haemocytometer (Neubauer). Dry cell weight (DCW) of cultures was determined by centrifuging algal suspensions with known optical density and drying at 90 °C till a constant weight. Afterwards, standard curves were plotted to find a direct correlation equation between DCW values (g L⁻¹) against corresponding optical density (OD₇₅₀) readings using a spectrophotometer. Based on this relationship, all the OD values were converted to biomass concentration (gDCW L⁻¹) of the cultures using the Equations shown in Table 1.

Table 1.

Equations for biomass concentration (gDCW L⁻¹)

Name of the strain	Biomass concentration (gDCW L−1)	R2 valuea
Anabaena cylindrica	OD750 × 0.36	0.9932
Nostoc commune	OD750 × 0.38	0.9922
Synechococcus BDUSM-13	OD750 × 0.40	0.9934

^aR², is the square of the correlation, measures the amount of linear association between two variables optical density (OD) and biomass concentration

The overall biomass productivity (P_overall, g L⁻¹ day⁻¹) during the culture period was calculated according to the following equation.

$$P_{\mathrm{overall}}=\frac{(X_{\mathrm{t}}-X_0)}{(t_{\mathrm{t}}-t_0)},$$ 1

where X_t was the biomass concentration (g L⁻¹) at the end of growth phase (t_t) and X₀ the initial biomass concentration (g L⁻¹) at t₀ (day), respectively. Specific growth rate (μ) of the microalgae was calculated (Guillard and Ryther 1962), according to the following formula.

$$\text{Specific growth rate}\left(\mu \right)=\frac{({\text{log}}_{10}{X}_{\mathrm{t}}-{\text{log}}_{10}{X}_{\mathrm{o}})}{\mathrm{\Delta }t},$$ 2

where, X₀ = initial cell number, X_t = cell number at time t, ∆t = time duration.

Chlorophyll estimation

The time-course growth of the cyanobacteria species was also estimated by chlorophyll a content analysis. Cyanobacterial cells were harvested from 1 mL culture suspension by centrifugation at 15,000×g at laboratory temperature for 7 min. Chlorophyll a was extracted by re-suspending the pellet in 1 mL of methanol, precooled to 4 °C. Samples were homogenized by mixing, vortexing (2,000 rpm, 4 s) and by gentle pipetting up and down. After covering the samples with aluminium foil, they were incubated further at 4 °C for 20 min and centrifuged at 15,000×g for 7 min to extract the pigments from the cells. Complete extraction was confirmed by visually checking the color of the pellet ranging between bluish and purple with no green color. The absorbance of the extract was measured at 665 and 720 nm (UV–Vis 2900 double-beam spectrophotometer, Hitachi, Japan) with slit width 1 nm and calculated by Eq. (3); Ritchie's formula (Ritchie, 2006).

$$\text{Total chlorophy ll}\left(\mathrm{Chl},a\right)=12.9447\left(A_{665},-,A_{720}\right)\mathrm{\mu }\mathrm{g}{\mathrm{mL}}^{-1},$$ 3

where A₆₆₅ and A₇₂₀ are the absorbance at 665 and 720 nm wavelengths, respectively.

Carbon fixation rate

The organic carbon in the dried algal biomass was analysed by the titration method of Walkley and Black (1934). The known biomass of algae was mixed with potassium dichromate (1N; 10 mL) and conc. sulfuric acid (20 mL) mixture, further diluted with distilled water (200 mL) followed by H₃PO₄ (10 mL) and diphenylamine (1 mL) addition. The reaction mixture was finally titrated against 4N ferrous ammonium sulfate (FAS) solution till the brilliant green color appeared. The carbon content was quantified using Eq. (4) (Walkley and Black 1934).

$$C_{\text{Carbon}}\left({\text{g g}}^{-1}\right)=\frac{3.951}{\text{g}}\left(1-\frac{{\text{FAS}}_{\text{b}}}{{\text{FAS}}_{\text{s}}}\right),$$ 4

where C_carbon is the carbon content, g is the weight of algal sample (g), FAS_b and FAS_s are the ferrous ammonium sulfate with blank and sample (mL), respectively.

The overall carbon fixation rate $R_{{\mathrm{CO}}_2}$(g ${\mathrm{CO}}_2$ L⁻¹day⁻¹) was calculated according to Eq. (5) (Singh et al. 2016a, b).

$$R_{{\mathrm{CO}}_2}=\frac{P_{\mathrm{overall}}\times C_{\mathrm{Carbon}}\times 44}{12},$$ 5

where P_overall, (g L⁻¹ d⁻¹) is overall biomass productivity; 12 (g/mol) and 44 (g/mol) present the molecular weights molar mass of elemental carbon and ${\mathrm{CO}}_2$, respectively.

Ultraviolet exposures and UV-protecting compounds analysis

The UV-B irradiation experiments with culture suspensions of Synechococcus BDUSM-13, A. cylindrica and N. commune were performed with optimized sodium bicarbonate concentration of 2 g L⁻¹in sterile open lid petri dishes. Each species was freshly grown using exponential phase inocula in 40 mL of fresh culture medium (BG-11^−ve and ASN^+ve). Subsequently, samples were exposed to artificial UV-B radiation supplied by a UV-B lamp (Philips, TL 40 W/12 lamp, Hamburg: 290–315 nm). The cyanobacterial cultures were irradiated continuously for 24 and 48 h under UV-B (290–315 nm), Photosynthetically Active Radiation (PAR; 400–700 nm) and PAR + UV-B radiations along with gently agitation by a magnetic stirrer. The desired irradiance of UV-B approximately 100 to 140 mW m⁻² nm⁻¹reaching the culture suspension was obtained by adjusting the distance of 45 cm and cut-off filters (295 and 395 nm filters) in between the UV-B source and the sample. After irradiation with PAR, UV-B and PAR + UV-B, samples were withdrawn at desired intervals for measuring produced UV-protecting compounds (Sinha et al. 1999). For photosynthetic pigment analysis, cold methanol extraction was performed by adding the PAR, PAR + UV and UV-B treated cyanobacterial cells in methanol and incubated the mixtures overnight at 4 °C (Bermejo et al. 1997). Afterwards, leached pigments in methanolic extract were analysed by wavelength scan in the range of 500–700 nm, spectrophotometrically. To extract the photo-protecting MAA from UV-B exposed cyanobacteria cells, methanolic extracts were initially centrifuged at 10,000 rpm for 10 min and then placed at 40 °C in a vacuum evaporator to evaporate supernatant. This dried samples were further dissolved in 2 mL distilled water and filtered through 0.22 μm pore size and 47 mm diameter filters (MF-Millipore™ Membrane Filter). Filtrates were analysed spectrophotometrically in the absorption spectra of 250–400 nm wavelengths.

Measurements of photosynthetic parameters

To determine the photosynthetic efficiency of cyanobacteria species, under PAR, PAR + UV-B and UV-B samples, the fluorescence and P₇₀₀ parameters of photosystem II (PSII) were examined using a Pulse Amplitude Modulation (PAM) Fluorometer (Super Head Fluorometer FL, 3500/F, Photon System Instruments, Czech Republic) measuring system due to its fast, simple and non-invasive operations. Cultures were first adapted in dark for 20 min prior to original fluorescence (F₀) measurement on PAM. The initial fluorescence, F₀ was measured by applying an analytic modulated flash of light when all PSII reaction centres are open. For the modulated chlorophyll fluorescence kinetics of dark-adapted samples, a flash of saturated actinic light pulse with an intensity of 2000 µmol m⁻² s⁻¹ was applied to obtain maximum fluorescence (F_m), where all reaction centres are closed. The maximum photochemical quantum yield of PSII (F_v/F_m ratio) was obtained (Hofstraat et al. 1994) by Eq. (6):

$$\frac{F_{\mathrm{v}}}{F_{\mathrm{m}}}=\frac{\left(F_{\mathrm{m}},-,F_0\right)}{F_{\mathrm{m}}}.$$ 6

Statistical analysis

All observations were taken in triplicates. The results were statistically analysed by ANOVA (Analysis of Variance) and Tukey test with significance level of 0.05 using Minitab 16 software. All the values in the tables and graphs are expressed as (mean ± SD). The values in the stacked bar diagrams are expressed as means of three measurements. All the graphs have been prepared using Origin pro data analysis software (Origin Lab Corporation) and Microsoft Excel (Office 365).

Results and discussion

Impact of NaHCO₃ gradient on specific growth rate and biomass productivity

Cell growth is the most critical parameter to decide the biomass yield, thus growth in terms of cell number was evaluated. Thus, effects of different bicarbonate concentrations (0.5, 1 and 2 g L⁻¹) on specific growth rate of A. cylindrica, N. commune and Synechococcus BDUSM-13 were analysed. In the NaHCO₃ concentration gradient, SGRs of the three species showed a significantly increasing trend in comparison to control samples (see Fig. 1a), confirmed by ANOVA analysis (P < 0.01). Concentration of 2 g L⁻¹ for A. cylindrica and 1 g L⁻¹ for N. commune and Synechococcus sp. and showed increases of up to 24.55, 32.66 and 26.10% in comparison to the control group (see Fig. 1b), respectively. It may be due to the activity of sodium ions (Na⁺) in supplemented NaHCO₃ salts which plays a major role in the mechanism for HCO₃^– uptake in cyanobacteria. Kaplan et al. (1984) showed that apparent photosynthetic affinity of cyanobacterium A. variabilis for extracellular inorganic carbon (C_i) was strikingly increased by sodium ions (Na⁺). They suggested that high Na⁺ supply decreases the apparent K_m(C_i) of the C_i transporting system and to a lesser extent increases the V_max. Higher concentrations of NaHCO₃ (2 g L⁻¹) exerted an inhibitory effect on growth of N. commune and Synechococcus sp. causing the growth rates of the two species to drop, suggesting species-specific responses to NaHCO₃ addition with respect to cell division. Furthermore, there was no significant change (P > 0.05) in between maximum specific growth rates (μ_max) of all three cyanobacteria within the experimental range of NaHCO₃ gradient. As illustrated in Fig. 1a, The specific growth rate for highest concentration was in following ascending sequence, N. commune 0.64 day⁻¹; A. cylindrica 0.67 day⁻¹; Synechococcus BDUSM-13 0.71 d⁻¹ while for lowest concentration the sequence was Synechococcus BDUSM-13 0.64 day⁻¹; N. commune 0.62 day⁻¹ and A. cylindrica 0.58 day⁻¹. Cyanobacterial species examined here have an optimum and threshold tolerance to NaHCO₃ addition above which reduced cell densities were observed. It suggests that the threshold level for supplied inorganic carbon above, which growth inhibition may occur, will be species dependent.

Fig. 1.

Effect of sodium bicarbonate gradient (0.5, 1 and 2 g L^{− 1}) on a maximum specific growth rate (μ_max), b μ_max differences (in %) between the control and treatments

Biomass productivity is another crucial factor to determine the applicability of an algal strain on commercial scale. Study showed that cyanobacterial productivity could be significantly affected by sodium bicarbonate in comparison to control. As illustrated in Fig. 2, initial transitional phase was between 1st and 5th day of experiment in which cells grew up to plateau stage, productivity increased steeply. Afterwards, in between 6 to 8th day, the log phase began which commences growth of algal cells due to available nutrients and conditions, peaked afterwards and then biomass productivity started lagging. However, in all control samples, end of log phase was observed earlier, till the 4–6th day of experiments. Cyanobacterial productivity showed a significant increasing trend in dose-dependent manner because of a direct impact from the available carbon in the culture and reached a peak value at 2 g L⁻¹ NaHCO₃. Among three cyanobacteria, A. cylindrica showed lowest biomass production in all bicarbonate concentrations showing a minimum rate of biomass productivity of 0.32 ± 0.06 g day⁻¹ L⁻¹ at 0.5 g L⁻¹and maximum 0.86 ± 0.02 g day⁻¹ L⁻¹ at 2 g day⁻¹ L⁻¹. Synechococcus BDUSM-13 strain showed significantly higher productivity of 0.92 ± 0.06 g day⁻¹ L⁻¹ at 2 g day⁻¹ L⁻¹ than the other two cyanobacteria grown with similar bicarbonate concentration. This effect of NaHCO₃ concentration was statistically confirmed using ANOVA analysis (p < 0.001). Present results indicate that the supplementation of solid inorganic bicarbonate could not only enhance the growth rate of cyanobacteria but also biomass productivity. All experimental species were able to tolerate a broad range of NaHCO₃ gradient, demonstrated by the rise in production rate and attained the maximum biomass productivity than the control. Many previous studies have also shown that high biomass production rate can be achieved by addition of NaHCO₃ in the culture medium (Costa et al. 2004; Li et al. 2018; Gupta et al. 2020). Similar findings were also reported by Castro et al. (2015) in their experiments with cyanobacterium Arthrospira platensis grown under different sodium bicarbonate concentrations. They found that, under the experimental conditions studied, biomass production tended to increase with an increase in the NaHCO₃ concentration (9 to 18 g L⁻¹) in the medium. Maximum productivity of 1.53 g day⁻¹ L⁻¹ was achieved with 16.18 g L⁻¹ NaHCO₃ addition and higher light irradiance of 111.67 µmol photons m² s^–1. Chi et al. (2013) showed that cyanobacteria Euhalothece ZM001cultured in 1.0 M solid inorganic bicarbonate (NaHCO₃) enhances the biomass production rate of 1.21 g L⁻¹ day⁻¹. Singh et al. (2014a, b, c) demonstrated that addition of bicarbonate to Leptolyngbya sp. showed high growth compared to the control. Kim et al. (2017) also found that growth of Dunaliella salina in sodium bicarbonate (5 g L⁻¹) was 2.84 times higher in specific growth rate than a bicarbonate-free control. In our study, biomass productivity of Synechococcus BDUSM-13 strain was enhanced by 1.5 times and 0.92 ± 0.06 g day⁻¹ L⁻¹at 2 g day⁻¹ L⁻¹ of sodium bicarbonate. Thus, obtained results suggested that the bicarbonate supplementation during cultivation have beneficial effects on cyanobacteria cell growth and improve the biomass production.

Fig. 2.

Biomass productivity (g day⁻¹ L⁻¹) in a Nostoc commune, b Synechococcus BDUSM-13 and c Anabaena cylindrica, strains supplemented with 0.5, 1 and 2 g L^{− 1} of sodium bicarbonate

Impact of NaHCO₃ gradient on pigment concentration, photosynthetic efficiency and carbon fixation

Chlorophyll measurement is a reliable parameter for measuring algal biomass and its physiological state (Knefelkamp et al. 2007) and hence was used as an indicator to analyse the effect of NaHCO₃ addition. Results showed that like biomass productivity, significant positive effect in a dose-dependent fashion on the levels of chlorophyll a content with an approximate doubling or more with the supplementation of 2 g L⁻¹ NaHCO₃ was noted in all species, compared to treatments with no added NaHCO₃. As depicted in Fig. 3, total chlorophyll content increased with bicarbonate addition and maximum level recorded at 2 g L⁻¹ in each of the strains i.e. 0.063 ± 0.003 g L⁻¹ with A. cylindrica, 0.061 ± 0.001 g L⁻¹ with N. commune and 0.091 ± 0.004 g L⁻¹ with Synechococcus BDUSM-13. It seems that an adequate supply of inorganic carbon is essential for regular photosynthesis and growth in cyanobacteria. Duration of the exponential phase in terms of total chlorophyll content was also longer than normal strains which explains the positive impact of NaHCO₃ on cyanobacterial cells. The fluorescence value of Chl a is another widely used parameter for assessing the physiological state of cyanobacteria using a Dual-PAM Fluorometer (Super Head Fluorometer FL, 3500/F, Photon System Instruments, Czech Republic) measuring system. PSII (F_v/F_m) represents the potential maximum photosynthetic efficiency of thalli. During the culture of the three cyanobacteria species, the photosynthetic efficiency (F_v/F_m) parameters above fluctuated markedly and showed similar trends with their total chlorophyll (see Fig. 4a, b), appeared to increase in a dose-dependent manner with increasing bicarbonate addition (P < 0.05). Specifically, for Synechococcus BDUSM-13 cultures, the PSII (F_v/F_m) increased from 0.478 to 0.653 as the culture NaHCO₃ concentration increased from 0 to 2 g L⁻¹ (P < 0.05). However, lowest PSII (F_v/F_m) of 0.42 was observed in N. commune with 0.5 g L⁻¹ bicarbonate concentration. At 2 g L⁻¹ concentration of NaHCO₃, PSII (F_v/F_m) increases of up to 53.43, 40.26 and 68.73% for A. cylindrica, N. commune and Synechococcus sp., respectively, in comparison to the control group (see Fig. 4b). This demonstrates that availability of preferred inorganic carbon form in the culture may help in an accumulation of the energy to prepare for synthesis of photosynthetic pigments. However, it will also undoubtedly be intrinsically linked with other environmental conditions (e.g. light availability and temperature). Castro et al. (2015) showed that with higher sodium bicarbonate concentrations, even under smaller irradiances, high biomass concentration could be obtained. Carrieri et al. (2007) also reported the in vivo requirement for bicarbonate that is both reversible and selective for this anion for efficient water oxidation activity on PSII in the hypercarbonate-requiring cyanobacterium Arthrospira (Spirulina) maxima. It indicates that NaHCO₃ is a crucial factor with the greatest influence on photosynthetic efficiency.

Fig. 3.

Total chlorophyll content of a Nostoc commune, b Synechococcus BDUSM-13 and c Anabaena cylindrica strains supplemented with 0.5, 1 and 2 g L^{− 1} of sodium bicarbonate

Fig. 4.

a Photosynthetic efficiency measured as the ratio (F_v/F_m). b Percentage increase in photosynthetic efficiency of cyanobacterial strains supplemented with 0.5, 1 and 2 g L^{− 1} of sodium bicarbonate

In addition, Zhou et al. (2016) showed that bicarbonate addition enhanced catalytic activity carbonic anhydrase enzyme, which catalyzes the rapid conversion of bicarbonate (HCO₃⁻) into CO₂, could effectively meet the demand for carbon needed for photosynthesis, thereby accelerating the carbon fixation. Our study also showed that increasing the carbon content in cultures enhances the carbon fixation rate in cyanobacterial species (P < 0.05). There was a significant increasing trend in dose-dependent manner because of ease in availability of inorganic carbon in the culture and reached a peak value at 2 g L⁻¹ NaHCO₃. As depicted in Fig. 5, among three cyanobacteria, A. cylindrica showed lowest carbon fixation of 0.35 ± 0.03 at 0.5 g L⁻¹ which increases to 0.49 ± 0.06 g CO₂ mL⁻¹ day⁻¹ with 2 g L⁻¹ NaHCO₃ addition. Synechococcus BDUSM-13 showed the maximum carbon fixation of 0.65 g CO₂ mL⁻¹ day⁻¹ at 2 g L⁻¹ NaHCO₃ concentration thus also has maximum carbon content (0.48 g dw⁻¹). A. cylindrica and N. commune also achieved their maximum carbon fixation of 0.49 and 0.74 g CO₂ mL⁻¹ day⁻¹, respectively, at 2 g L⁻¹ NaHCO_3. In this study, all cyanobacterial species doubled their carbon fixation ability, compared to control, in a bicarbonate rich environment. It seems addition of bicarbonate allows cellular material production by enhancing the inorganic carbon uptake and thereby achieving maximum biomass productivity.

Fig. 5.

Inorganic carbon fixation rate (gCO₂ mL⁻¹ day⁻¹) in cyanobacterial strains supplemented with 0.5, 1 and 2 g L⁻¹ of sodium bicarbonate

The purpose of this study is to investigate if some widely used cyanobacteria species can grow in high concentration of NaHCO₃. So, they can be further explored for their potential to synthesize UV-protecting compounds in a bicarbonate-based algal cultivation system which could be further commercialized at industrial scale. At the commercial scale, where maintaining both high growth rate and biomass productivity is often crucial for excellent product yield, generally, CO₂ is provided in the medium by bubbling it. The dissolved carbon is available in different forms based on pH: carbonic acid (H₂CO₃), bicarbonate (HCO₃⁻) and carbonate (CO₃²⁻) ions. The maximum dissociation occurs in the pH range from 7 to 9. The poor solubility of CO₂ in water limits the availability of carbon source for growing cyanobacterial cells in culture (Aishvarya et al. 2012). Thus, alternative inorganic carbon sources (e.g. salts of bicarbonate like NaHCO₃), could preferentially be utilized. It is also worth mentioning that our previous study showed that the pH value of algal cultures may drop with atmospheric CO₂ addition, from initial 7.2 to values around 5.5–6 range which are too low for growth (Singh et al. 2016a). In this study, we observed in our preliminary experiments that bicarbonate addition increases in pH values from 7.2 to 8.5–9.5 range which has growth inhibitory effect. Thus, pH values of culture medium were controlled by a concentrated HCl solution (2 M) in between 7.5 and 8.5 for appropriate supply of inorganic carbon. The present study also revealed that an adequate supply of inorganic carbon (in our case 2 g L⁻¹ of sodium bicarbonate) is essential to maintain photosynthetic efficiency, thereby enhancing the carbon fixation rate and biomass yield of cyanobacteria. Photosynthetic pigment analysis showed that total chlorophyll content was quite high in bicarbonate-supplemented medium when compared to control samples. Subsequently, these changes may lead to the modifications in the structure of photosynthetic apparatus, i.e. PSII reaction center proteins in cyanobacteria (Carrieri et al. 2007), indicated by photosynthetic quantum yield of PSII (F_v/F_m) results (see Fig. 4). Availability of preferred bicarbonate (HCO₃⁻) form of carbon in bicarbonate-supplemented medium is also a regulating factor which enhances photosynthetic efficiency, resulting in a higher amount of carbon fixation (Fig. 5). Many researchers have demonstrated similar phenomena in photosynthetic microorganisms; microalgae include Scenedesmus quadricauda (Anusree et al. 2017), Chlorella sorokiniana, Dunaliella sp. (Chi et al. 2014) and cyanobacteria such as Arthrospira maxima (Carrieri et al. 2007), A. variabilis (Kaplan et al. 1984), Synechocystis PCC6803, Cyanothece sp. (Chi et al. 2014). This clearly suggests the feasibility to use bicarbonate form of inorganic carbon as feedstock for cyanobacteria culture and to recirculate the regenerated carbonate for carbon fixation inside the cell. Bicarbonate has greater solubility than CO₂, thus reducing issues associated with low retention times. Moreover, storage and transportation of current carbon source, gaseous CO₂ escalates the cost of algal biomass production whereas solid bicarbonate salts can easily be transported to algal facilities and stored until needed (Chi et al. 2014). Thus, results of the study revealed that due to greater solubility than CO₂, sodium bicarbonate could be an effective inorganic carbon source for cyanobacterial cultivation.

Impact of NaHCO₃ on photosynthetic activity of UV-B exposed cyanobacteria

The synthesis of UV-protecting compounds like mycosporine-like amino acids (MAAs) has always been reported in freshwater, marine and terrestrial cyanobacteria to combat stress due to high levels of UV-B radiation (Karsten and Wiencke 1999; Joshi et al. 2018; Boucar et al. 2021). Previous results of this study have shown that supplementation of high inorganic carbon in the form of NaHCO₃ may aid photosynthetic activity and enhance the photosynthetic efficiency by increasing the concentration of total chlorophyll (Fig. 3). Once it become evident that the best concentration of NaHCO₃ for growth is 2 g L⁻¹, it will be interesting to analyse their effect on the cyanobacterial cells grown under the exposure of UV-B radiation. Thus, we further investigated the continuous UV-B and PAR + UV-B stress strategy to produce UV-protecting compounds in Synechococcus BDUSM-13, A. cylindrica and N. commune by time-course experiments for 24–48 h of UV-B and PAR + UV-B irradiation. Pigment content, photosynthetic efficiency and MAAs synthesis were analysed after UV-B and PAR + UV-B irradiation, shown in Figs. 6, 7 and 8.

Fig. 6.

Effect of PAR, UV-B and PAR + UV-B radiation on photosynthetic pigment content in a Anabaena cylindrica, b Nostoc commune and c Synechococcus BDUSM-13

Fig. 7.

a Photosynthetic efficiency measured as the ratio (F_v/F_m). b Comparative percentage reduction in photosynthetic efficiency of cyanobacteria species after the irradiation with UV-B and PAR + UV-B

Fig. 8.

UV–Vis absorption spectrum of partially purified MAA dissolved in milli-Q water extract of a Anabaena cylindrica, b Nostoc commune and c Synechococcus BDUSM-13 after irradiation with PAR, UV-B and PAR + UV-B

In the control PAR exposures, all cyanobacteria species, Synechococcus BDUSM-13, A. cylindrica and N. commune showed an increasing trend in pigment content (see Fig. 6a–c). However, there was a significant gradual decline in both NaHCO₃ supplemented and control samples after the UV-B and PAR + UV-B radiation exposure. Specifically, after 48 h of UV-B exposure, tremendous decline in pigment was seen in all cyanobacteria species. In addition, interestingly, pigment was severely reduced in Synechococcus BDUSM-13 as compared with N. commune and A. cylindrica. During the 48 h of experiments, no significant change was observed among NaHCO₃ supplemented experimental samples than control. However, obtained results revealed a pronounced significant reduction in photosynthetic pigments of cyanobacteria due to UV-B exposure in comparison to PAR and PAR + UV-B. This agrees with El-Sheekh et al. (2020), who showed remarkable significant reductions in chlorophyll and carotenoids contents of freshwater Cyanobacteria, Planktothrix cryptovaginata, Nostoc carneum and Microcystis aeruginosa, the Chlorophyte Scenedesmus acutus and the marine Cyanobacterium Microcystis cultures irradiated for 1, 3, 5 and 7 h of UV-B in comparison to unirradiated culture. They concluded that reduction in chlorophyll contents under UV-B stress could be caused by the damage of PSII and their influence on structure and composition of light harvesting complex result in disruption of chloroplast structure. Hideg et al. (2013) also reported the detrimental impact of UV-B radiation on photosynthetic pigments could be attributed to photobleaching effects or way of reactive oxygen mediated peroxidation.

Furthermore, many studies suggested that PSII is more sensitive and most important target of UV-B damage in cyanobacteria than PS I. Thus effect of bicarbonate supplementation on photosynthetic efficiency of cyanobacteria in stationary phase was also evaluated by measuring the photosynthetic quantum yield of PSII (F_v/F_m) after 24 and 48 h of PAR + UV-B and UV-B exposure (Fig. 7a). Results showed, compared to PAR, significant decline was observed in optimum quantum yield of PSII (F_v/F_m) of all cyanobacterial samples after PAR + UV-B and UV-B exposures (P < 0.05). As depicted in Fig. 7b, 48 h of UV-B exposed samples were severely affected, compared to others. However, it is worth mentioning that despite significant reduction in photosynthetic pigments, NaHCO₃ supplemented experimental samples showed superior PSII (F_v/F_m) activity than control (without bicarbonate). After 24 and 48 h of UV-B exposure, A cylindrica control samples showed PSII (F_v/F_m) activity of 0.28 and 0.25, while bicarbonate samples exhibited photosynthetic efficiency of 0.57 and 0.44, respectively. Among all the samples, A. cylindrica was least affected after 48 h of UV-B exposure and showed only 27.4% of PSII (F_v/F_m) reduction, compared to PAR control. However, at this point, N. commune reduction was 30.9% like 31.8% reduction of its corresponding control (without bicarbonate). Synechococcus BDUSM-13 have shown a maximum 47.9% reduction in photosynthetic efficiency in 48 h of UV-B exposed samples. It seems that pigments of the photosynthetic apparatus of Synechococcus BDUSM-13 have been destroyed by UV radiation (Fig. 6c), with comparative loss of photosynthetic capacity (Fig. 7a, b). Other workers have also speculated that exposure of intact cells of the Synechococcus 6301 to UV-B radiation may induce a loss in PSII electron transport activity, prior to the alteration in pigment complexes (Rajagopal and Murthy 1996). Six et al. (2007) also showed that after 5 h of UV-B exposure (5 h) on the cyanobacterium Synechococcus sp. WH8102, reduction in the amount of D₁ protein was observed which leads to the rapid photoinactivation of the PSII reaction centres. Pigment and quenching analysis of this study also indicate the decrease in photosynthetic efficiency due to reduce PSII (F_v/F_m) activity, particularly at higher UV-B doses (48 h in our case). It may be due to both, direct effects (e.g. PSII apparatus interaction with certain biomolecules that absorb in the UV range) and indirect effects (e.g. decrease in pigment content with the oxidative stress exerted by reactive oxygen species). Tevini and Pfister (1985) shown UV-B-radiation could also inactivate the PSII α-centres in higher plants like spinach chloroplasts. Both effects, decreases in chlorophyll pigments and rate of photosynthesis, proceed towards lower biomass yield. However, photosynthetic microorganisms have evolved several lines of tolerance, avoidance and repair mechanisms which not only protect them against the damaging effects of UV-B exposure but also help to acclimate to survive in such harsh conditions (Rastogi et al. 2014). Our quenching analysis also showed that although photosynthetic efficiency ratio (F_v/F_m) decreases after the exposure of UV-B and PAR + UV-B irradiation in cyanobacteria cells. But continuous irradiation of UV-B and PAR + UV-B also stimulates the synthesis of photo-protecting compounds such as MAAs, particularly in A. cylindrica and N. commune (Fig. 8a, b).

Induction of MAAs synthesis in NaHCO₃ supplemented cyanobacteria

Enhanced UV-B generally decreases pigment content in cells, whereas it may also increase the biosynthesis of UV-B absorbing compounds in many algae. The occurrence of high concentrations of MAAs in cells exposed to high levels of solar radiation has been described by many authors, provide protection as a UV-absorbing sunscreen (Ehling-Schulz et al. 1997; Sinha et al. 1999, 2001). In this study, synthesis of MAAs was stimulated in NaHCO₃ supplemented in cyanobacterial cultures by irradiation of UV-B and PAR + UV-B on cells. As depicted in Fig. 8, among all of the tested strains, only A. cylindrica and N. commune were only able to produce MAAs, showed by a prominent peak (UV-absorption maximum; λ_max) at 334 ± 2 nm (see Fig. 8a) and 312 ± 2 nm (see Fig. 8b), respectively. There was an increase in the absorbance around 334 nm in the samples irradiated with PAR + UV also, but to a much lesser extent, than the samples that received only UV-B. In contrast, Synechococcus BDUSM-13 did not show any peaks at this range (see Fig. 8c). It seems Synechococcus cells were severely affected by UV-B and PAR + UV-B exposures as they are not able to synthesize MAAs. This is in accordance with our previous findings of the last section, where a maximum 47.9% reduction in photosynthetic efficiency was observed in 48 h of UV-B exposed Synechococcus samples. In contrast, A. cylindrica was least affected after 48 h of UV-B exposure and showed only 27.4% of reduction. Thus, present study showed innate competence of A. cylindrica and N. commune to raise MAAs content in response to UV-B radiation and thus may be able to adapt to day-to-day fluctuations in UV irradiation. Similarly, Sinha et al. (1999) detected an ultraviolet-absorbing MAA, shinorine (a bi-substituted MAA), in Anabaena sp., with an absorption maximum at 334 nm. The MAA content was increased in the cultures exposed to PAR + UV in comparison with the cultures exposed to PAR only. Same group has also studied further, wavelength-dependent induction of MAA N. commune under the UV-B radiation and showed the presence of MAA shinorine, containing both glycine and serine groups with an absorption maximum at 334 nm (Sinha et al. 2003). From our results it is difficult to verify that the presence of MAA in N. commune (see Fig. 8b). However, there is a significant increase in OD scan values of the cultures N. commune, exposed with UV-B and PAR + UV-B radiation, than cultures exposed to PAR only.

This shows that UV stress induces the synthesis of MAA in this cyanobacterium and thus may protect the organism against deleterious UV-B effect. However, synthesis rate or yield may be reduced due to impact of NaHCO₃ supplementation in N. commune cultures. Yet, there is no data available on impact of NaHCO₃ supplementation on UV-protecting compounds synthesis by algae or cyanobacteria. In this regard, further research on NaHCO₃ supplementation as an inorganic carbon source for UV-B exposed algal cells is urgently required. Importance of MAAs is already known which act as antioxidants to prevent cellular damage resulting from UV-induced production of active oxygen species (Dunlap and Yamamoto, 1995; Sinha et al. 1999; Ryan et al. 2002; Krabs et al.00202002; Kageyama and Waditee-Sirisattha, 2019). Garcia-Pichel et al. (1993) showed that cells with high concentrations of MAAs prevented 3 out of 10 photons from hitting cytoplasmic targets. Five MAAs variants have been reported so far in Lyngbya sp. (Asterina-330, M-312, Palythine, Porphyra-334 and Shinorine) which are capable of dissipating absorbed radiation as harmless heat without producing reactive oxygen species (de la Coba et al. 2019; Fuentes-Tristan et al. 2019). However, there is an imperative to screen the potent algal species which can effectively utilise the inorganic carbon source and produce valuable photo-protecting MAAs for commercial sunscreen production. It may include traditional optimization for medium composition, temperature, mixing and specific investigation for optimal pH range and bicarbonate concentration, to obtain the maximum biomass yield in each batch of culture.

Conclusion

The tested cyanobacteria species appeared to be tolerant to the high levels of supplemented bicarbonate (2 g L⁻¹). Among all, carbon content of Synechococcus BDUSM-13 was maximum (0.482 g dw⁻¹) with fixation rate of 0.653 g CO₂ mL⁻¹ day⁻¹ at 2 g L⁻¹ sodium bicarbonate concentration. However, Synechococcus BDUSM-13 was not able to produce photo-protecting MAAs under UV-B exposures. It seems A. cylindrica strain is best species for the bicarbonate-based approach as MAAs production was sustained in sodium bicarbonate-supplemented condition. They were able to produce UV-protecting compounds (λ_max at 334 ± 2 nm in PAR + UV-B) along with significant carbon fixation (0.491 gCO₂ mL⁻¹ day⁻¹ at 2 g L⁻¹) at high levels of bicarbonate (2 g L⁻¹). However, selection of MAA production cyanobacteria species using bicarbonate-supplemented culture system is only a beginning step in developing an economically sustainable carbon capturing and many more studies are to be conducted. Further, these findings also provide a promising way forward to introduce bicarbonate-based algal cultivation in the open pond system for generation of commercial scale biomass even under stress conditions. The study unlocks new avenues like carbon sequestration and photo-protecting compounds production for various industrial applications.

Author contributions

All authors provided critical feedback and helped shape the research, analysis and manuscript. SKS, RK, MAR, MM and SS are the authors of manuscript entitled “An integrated approach to produce UV-protecting compounds in cyanobacterial strains coupled with CO₂ sequestration”. Prof. SS and Dr. SKS conceived of the presented idea and encouraged RK and MM investigated the proposed approach and carried out the experiment. Dr. MAR verified the analytical methods. Dr. SKS wrote the manuscript with support from MAR, and SS. The overall project has been completed under the mentorship of corresponding author Prof. SS.

Funding

This work was supported by the University Grants Commission, New Delhi, India [Grant number: EN/19-20/0020].

Declarations

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.