Microalgal strain and culture medium
The microalgal strain N. oleoabundans UTEX1185 was purchased from the Culture Collection of Texas University (USA). It was cultivated in medium that contained NaHCO3, 25.2 g L−1; NaNO3, 0.5 g L−1; NaCl, 0.025 g L−1; MgSO4, 0.05 g L−1; KH2PO4, 0.322 g L−1; CaCl2, 0.02 g L−1; FeCl3·6H2O, 0.005 g L−1; and A5 trace elemental solution 1 mL L−1 [17]. However, at first, the algae could not grow well in culture medium containing 0.3 mol L−1 bicarbonate, although it was reported that this algae can tolerate 0.3 mol L−1 bicarbonate [17]. Thus, the algae were first cultured with 0.08 mol L−1 bicarbonate, and then, the concentration was increased by 0.02 mol L−1 every 4 days until it reached 0.3 mol L−1. This culture was conducted in a 1-L Erlenmeyer flask with a work volume of 0.4 L, and it was bubbled with air at a rate of 100 mL min−1. Every 4 days, 50% of the medium was replaced with fresh medium containing 0.08 mol L−1 bicarbonate. This process lasted for 6 months, and the obtained algal strain adapted to a NaHCO3 concentration of 25.2 g L−1, or 0.3 M.
Algal cultivation and biomass concentration measurement
To test the effect of bicarbonate concentration on growth of the adapted N. oleoabundans strain, batch cultures for 5 days were conducted with bicarbonate at concentrations of 0.1, 0.3, 0.5, and 0.7 mol L−1. Another culture was set up with a zero bicarbonate concentration that contained 0.3 mol L−1 sodium chloride. Both of these were conducted in a 1-L Erlenmeyer flask with a work volume of 0.4 L. To test the recyclability of spent medium, cultures grown in 3.0-L Erlenmeyer flasks were conducted in semi-continuous mode (Fig. 2) with an initial culture volume of 0.8 L. After 2 days, 50% of the culture was removed, and the same volume of fresh medium was added to re-start the culture. The removed cell suspension was then centrifuged, followed by bubbling with pure CO2 for pH adjustment and carbon replenishment, which usually takes approximately 20 min. When the pH of this medium decreased to 8.50, which is the same pH as fresh medium, the bubbling was stopped, and the supernatant was filtered through 0.22-μm hydrophilic membranes, and stored at 4 °C in the refrigerator. After 48 h, when the culture was finished, this CO2-replenished medium was used to replace 50% of the culture in the flask. The removed culture was then treated as such again. To avoid nutrient depletion, the same amounts of nutrients as in fresh medium were replenished in the recycled medium on the 8th and 14th day. To make a comparison, a group that always used fresh medium to replace 50% of the culture in the flask every 2 days was used. The cultures were continuously illuminated at 141.5 μmol m−2 s−1. The flasks were orbital shaken at 140 rpm at a temperature of 25 °C with no air bubbling. Biomass productivity (PBiomass) and specific growth rate (μ) were calculated as
$${P_{\text{Biomass}}}({\text{g}}\;{{\text{L}}^{ - 1}}\;{\text{day}}^{ - 1}) = \frac{{{\text{DC}}{{\text{W}}_2} - {\text{DC}}{{\text{W}}_1}}}{{{t_2} - {t_1}}},$$
(1)
$$\mu ({\text{day}}^{ - 1} ) = \frac{{\ln ({\text{DCW}}_{2} /{\text{DCW}}_{1} )}}{{t_{2} - t_{1} }},$$
(2)
where PBiomass is biomass productivity (g L−1 day−1), μ is the specific growth rate (day−1), and DCW2 and DCW1 are the dry cell weight (DCW) at time t2 and t1, respectively.
DCW was determined according to Zhu et al. [21]. Briefly, triplicates of 40 mL samples were acidified with hydrochloric acid and then centrifuged at 10,000 rpm for 5 min at 25 °C. The algal pellets were then washed twice with a 40 mL ammonium bicarbonate solution. Finally, the pellets were re-suspended in 2 mL of ammonium bicarbonate, and this suspension was dried overnight at 105 °C until a constant weight. DCW was calculated by subtracting the empty dish weight from total weight.
Calculation of apparent carbon utilization efficiency (%)
As the consumed carbon could be from both of initial medium or air, the carbon utilization efficiency in this study is defined as “apparent carbon utilization efficiency”, and it was calculated as follows:
$${\text{Efficiency}}(\% ) = \frac{{\Delta {\text{DCW}} \times C_{\text{C}} }}{{\Delta {\text{Tic}}}} \times \% ,$$
(3)
where the ∆Tic is the variation of Tic during the cultivation process and ∆DCW represents the variation in biomass concentration. CC represents the carbon content in the dry cell weight of the algal biomass, which was measured with a combustion CHNS-analyzer (Element, Germany). In this study, the CC of 45.7 ± 0.18, 51.6 ± 0.17, 54.7 ± 0.14 and 53.1 ± 0.01% was achieved in algal biomasses cultured with bicarbonate concentrations of 0.1, 0.3, 0.5, and 0.7 mol L−1, respectively. To simplify the calculation process, it was assumed that the CC did not change during the whole cultivation process or in different culture modes when using a constant bicarbonate concentration.
Since the alkalinity of the culture media does not change with CO2 consumption or supply [22], total inorganic carbon (Tic) was calculated with the measured pH according to the following equation [23].
$${\text{Tic}} = \frac{\text{Alc}}{{\alpha_{1} + 2\alpha_{2} }},$$
(4)
where Alc is the medium alkalinity and α1 and α2 are the ionization fractions of HCO
−13
and CO32−, respectively, which are obtained as a function of pH and the equilibrium constants k1 and k2 as shown in the following equations.
$$\alpha_{1} = \frac{1}{{\left( {1 + \frac{{H^{ + } }}{{k_{1} }} + \frac{{k_{2} }}{{H^{ + } }}} \right)}},$$
(5)
$$\alpha_{2} = \frac{1}{{\left( {1 + \frac{{H^{ + } }}{{k_{2} }} + \frac{{\left( {H^{ + } } \right)^{2} }}{{k_{1} k_{2} }}} \right)}}.$$
(6)
The equilibrium constants k1 and k2 were theoretically calculated from salinity and temperature according to Millero et al. [24].
In addition, the concentration of dissolved CO2 (CO2D) was calculated from the measured pH value and Tic as follows:
$${\text{CO}}_{{2{\text{D}}}} = {\text{Tic}} \times \frac{1}{{\left( {1 + \frac{{k_{1} }}{{H^{ + } }} + \frac{{k_{1} k_{2} }}{{\left( {H^{ + } } \right)^{2} }}} \right)}}.$$
(7)
In this study, the CO2D* is the liquid-phase CO2D concentration, which is in equilibrium with the air CO2, and the corresponding culture pH was defined as pH*. This equation was solved using Matlab 14.0.
The CO2* can be calculated as follows:
$${\text{CO}}_{{_{2} }}^{*} = H_{{{\text{CO}}_{{_{2} }} }} P_{{{\text{CO}}_{{_{2} }} }} ,$$
(8)
where PCO2 is the partial pressure of saturated CO2 in the atmosphere and HCO2 is the Henry’s constant for CO2.
Alkaline flocculation and measurements of recovery efficiency (RE)
Algal sedimentation was first investigated with fresh medium re-suspended algal cells, as this can well control culture conditions, such as pH and biomass concentration. To test this, algal cells were harvested by centrifugation and then re-suspended in fresh medium. This pretreatment has no significant influence on flocculation [25, 26]. To study the influence of calcium concentration on flocculation, the re-suspended algal suspension was added with different concentrations of calcium and stirred intensively at 1000 rpm for 10 min, followed by a gentle mixing of 250 rpm for another 20 min. The prepared algal suspension was subsequently allowed to settle for 60 min. In addition, the effect of biomass concentration on auto-flocculation was tested, where the re-suspended cell suspension had no pretreatment and was allowed to settle from 0.5 to 24 h. These experiments were carried out in triplicates in 50-mL graduated cylinders with a work volume of 40 mL. The biomass concentration and pH were 0.5 g L−1 and 10.0, respectively.
To test the repeatability of auto-flocculation in spent medium, the recovery efficiency of culture in spent medium was tested with the medium–microalgal cell mixture drawn from bath and semi-continuous mode. Neither of these cultures had any treatment. For batch culture, the cell suspension was drawn after 5 days of batch cultivation, while the recycling semi-continuous culture was drawn per 2 days. To avoid the culture loss of recycling culture, the spent medium in the semi-continuous culture was collected after the flocculation test. However, in our practical experiment, 2 mL of medium was used for sampling, and the rest was recovered by centrifugation, to avoid volume loss. The recovery efficiency was measured by calculating the absorbance difference between the initial (C0) and the final (Ct) optical density at 750 nm.
The recovery efficiency (RE) was calculated as:
$${\text{Recovery}}\;{\text{efficiency}}(\% ) = \frac{{C_{0} - C_{t} }}{{ \, C_{0} }} \times 100,$$
(9)
where C0 is the initial optical density and Ct is the final optical density after settlement.
The concentrating factor (CF) was determined as
$${\text{CF}} = \frac{{H_{1} }}{{H_{2} }},$$
(10)
where H1 is the height of algal suspension in the cylinder at the beginning and H2 is the height of algal sludge at the end of sedimentation.
Statistical analysis
Differences between groups were performed by one-way analysis of variance (ANOVA) tests. The significant differences were considered at p < 0.05, as determined by SigmaStat (version 3.1) software (SPSS).