Effects of CO2 and pH on Growth of the Microalga Dunaliella salina

A potentially cost-effective and scalable method to stabilize pH in microalgal batch cultures is proposed in this study. The cultures were supplied with different concentrations of CO2 enriched gas and controlled amounts of bicarbonate were added. An empirical model correlating the equilibrium pH to bicarbonate and CO2 stream concentrations was established experimentally. Finally, the isolated impact of either pH or CO2 concentration on Dunaliella salina growth was studied. Effects of CO2 and pH on Growth of the Microalga Dunaliella salina Kezhen Ying1*, D James Gilmour2 and William B Zimmerman1


Introduction
The cultivation of microalgae has been studied and developed for more than 60 years [1]. Some parameters affecting algal growth have been well studied, e.g. light illumination [2][3][4][5][6][7][8], while some are still worthwhile to be studied, for instance the effects of pH and CO 2 on microalgal growth. At saturating light intensities, the rate of CO 2 supply is crucial for algal photosynthesis as CO 2 is major source for the carboxylation of RuBP. pH is also one of the important factors for algal growth as it can affect the activity of different enzymes. In general, different algal species have various ranges of tolerance to pH.
The effects of pH and CO 2 on microalgal growth have been well studied by many researchers [9][10][11][12][13][14], however, neither pH nor dissolved CO 2 were solely controlled during their experiments due to the interactions between pH and dissolved CO 2 . It seems to be infeasible to keep pH constant while varying the dissolved CO 2 , or vary the pH while keeping dissolved CO 2 constant. Therefore it will be interesting to find out the isolated effect of pH or CO 2 on algal growth. In this study, a method was proposed to achieve a constant pH and variable dissolved CO 2 , or a constant CO 2 level and variable pH. Their effects on microalgal growth were studied based on the culture of the unicellular green alga Dunaliella salina. Six different pH levels and three different dissolved CO 2 concentrations were tested.

Methodology
Under a constant bubbling condition, the equilibrium concentration of dissolved CO 2 ([CO 2 ]*), according to Henry's law and Two-film theory, should only depend on the CO 2 partial pressure in the gas phase under constant gas/liquid properties and temperature. Therefore, for a fixed CO 2 percentage in the bubbling gas, the [CO 2 ]* will not be altered when varying the concentrations of NaHCO 3 in the medium (assuming the changes in liquid physical properties by adding NaHCO 3 into the water are negligible, as long as the concentrations of NaHCO 3 are low). On the other hand, the dissolved CO 2 concentration is correlated to pH by Equation 1 [15,16]. Since the concentration of Na + varies for different concentration of NaHCO 3 , while the [CO 2 ]* does not change, it is therefore reasonable that the equilibrium pH (pH*) changes for the medium with different NaHCO 3 concentration.
In Ying et al. [16], the effects of NaHCO 3 concentration on equilibrium concentration of dissolved CO 2 and CO 2 mass transfer rate in water were studied. The results proved the above hypothesis, indicating the feasibility of using NaHCO 3 to control the equilibrium pH of the medium without affecting the [CO 2 ]* and CO 2 mass transfer rate. However, only one concentration of CO 2 (5%) in the bubbling gas was tested, the relationship between pH* and NaHCO 3 established was only suitable for 5% CO 2 dosing. Therefore, in this study, experiment a) was designed to find a comprehensive model correlating pH*, NaHCO 3 and CO 2 %, which would facilitate the experimental designs on b) pH impact and c) CO 2 effect on algal growth.

Experiment a): Relationship between pH*, NaHCO 3 and CO 2 %
To study the interaction between pH*, NaHCO 3 and CO 2 %, a gas mixture containing a certain percentage of CO 2 balanced with N 2 is injected to the airlift bioreactor containing 1.5 L of distilled water and a certain concentration of NaHCO 3 . The initial temperature is adjusted to 22°C. pH was measured by a SevenGo Duo pro (pH/DO/Ion) meter. When the pH reading stops changing for 10 minutes, this value is recorded and considered as the equilibrium pH. The experimental procedure was repeated 35 times using 7 concentrations of NaHCO 3 and 5 CO 2 stream concentrations tested. The equilibrium concentration of CO 2 ([CO 2 ]*) was calculated by Equation 1. The experimental set up is shown in Figure 1.

Experiment b): The effect of pH on algal growth
Six 1.5 L-airlift bioreactors containing artificial sea water medium designed for Dunaliella species [17,18] but with different NaHCO 3 concentrations were run simultaneously for Dunaliella salina (strain 19/30, Culture Centre of Algae and Protozoa, Oban, UK) culture. (Figure 2) At the beginning, 50 ml of healthy pre-cultured D. salina concentration (5% CO 2 balanced with N 2 ) under 0.3 L/min. Although the algal growth may consume some dissolved CO 2 , a new equilibrium would be achieved immediately after that due to the constant CO 2 dosing (CO 2 mass transfer >> CO 2 consumption, the consumed CO 2 would be balanced with the CO 2 transferred into the medium). In other words, the dissolved CO 2 is maintained constant at its equilibrium concentration. However, the equilibrium pH for each reactor differs, due to the different NaHCO 3 concentrations in the medium. As regard to the specific NaHCO 3 concentration for each culture, it was determined by the empirical model established based on the results from experiment (a). The whole set of cultures were illuminated by a fluorescent cool white lamp providing continuous light of 90 μmol quanta m −2 s −1 . The light intensity was measured by a Hansatech Quantum Sensor. Nontransparent baffles were placed between every two reactors to ensure even illumination for each culture. The temperature for each culture was maintained around 23°C, due to the empirical heat transfer from the fluorescent lamp. pH, OD and chlorophyll content for each culture were measured daily. The photosynthetic oxygen generation rate of each culture was measured at day 5. The method for photosynthetic oxygen generation rate measurement is described in Appendix 1 and 2. The detailed culture condition for each reactor is listed in Table 1. The whole set of experiments were repeated once for error analysis.

Experiment c): The effect of dissolved CO 2 on algal growth
To study the impact of dissolved CO 2 on D. salina growth dissolved CO 2 concentration needs to be varied while the pH for each culture should be maintained constant. To achieve this, three different CO 2 stream concentrations (5%, 20% and 50%) were applied to provide three corresponding CO 2 equilibrium concentrations. The equilibrium pH for each reactor was expected to be 7 by adding the proper amount of NaHCO 3 . The concentration of NaHCO 3 required for each culture is estimated by the empirical equation found from experiment (a). The whole set of cultures was illuminated by a fluorescent lamp providing continuous light of 90 μmol m -2 s -1 . Non-transparent baffles were placed between every two reactors to ensure even illumination for each culture. The temperature for each culture was maintained around 23°C, due to the empirical heat transfer from the fluorescent lamp. pH, OD and chlorophyll content for each culture were measured daily. The photosynthetic activity of each culture was measured at day 5 and day 16. The experimental setup and culture conditions are shown in Figure  3 and Table 2, respectively.

Results and Discussion
The correlations between pH*, NaHCO 3 and CO 2 %    CO 2 %. The equilibrium concentration of dissolved CO 2 ([CO 2 ]*) is found to be only dependent on the CO 2 stream concentration (CO 2 %).
[CO 2 ]* was enhanced with the higher CO 2 % supply. The variation of NaHCO 3 concentration did not affect [CO 2 ]* when CO 2 % was fixed. This phenomenon can be supported by Henry's law that the equilibrium concentration of a gas is in direct proportion to the partial pressure of that gas over the solution.
In terms of equilibrium pH (pH*), its changes along with the NaHCO 3 concentration and CO 2 stream concentration (CO 2 %) were plotted in Figure 5a. As can see, for a fixed CO 2 % in the gas supply, pH* was altered by varying the NaHCO 3 concentration. Higher NaHCO 3 concentration resulted in a higher pH*. Such a trend is also consistent with findings from Ying et al. [16]. An empirical equation correlating pH* to NaHCO 3 and CO 2 % was created in the logarithmic plot ( Figure  5b), shown in Equation 2. The accuracy of Equation 2 was examined by comparing the experimental pH* values with the calculated values, shown in Figure 6. The results showed a less than 5% deviation between the real and the estimated pH* values by using Equation 2. Therefore, under a constant gas bubbling condition, pH can be controlled at a specific level for microalgae culture by choosing the right concentration of NaHCO 3 and CO 2 % in the gas supply, without applying additional 'auto-pH regulating systems' or expensive buffers. For the gas dosing, microbubbles or fine bubbles (e.g. less than 800 -1000 μm in diameter) are recommended as the CO 2 mass transfer rate needs to be controlled sufficiently to balance the CO 2 consumption by algal growth. Otherwise, pH* would not stay constant but increase.

Effect of pH on D. salina growth
To study the pH effect on D. salina growth, six different pH levels were tested (expected pH= 6, 6.5, 7, 8, 8.5 and 9). The dissolved CO 2 concentration for each culture was maintained the same (about 0.002 mol.L -1 ) through the constant dosing of 5% CO 2 . The real pH value for each culture versus the expected value was plotted in Figure  7. The results showed that the pH for each culture was controlled at the expected level, which again proved the feasibility of using pH*-NaHCO 3 -CO 2 % model (Equation 2) for pH control in the real algal culture. The daily algal growth under each pH level was shown in Figure 8. First of all, two different growth phases were observed for each culture. The growth was logarithmic in the first 5 days while 5 days after it became linear-like. The same scenario was discussed by Richmond [8]. For a certain high light intensity, assuming all the photons of a flux density can be captured by the algal culture; cell density will keep  increasing exponentially until all photosynthetically available photons are absorbed. Then, cell density increases linearly until light per cell becomes limiting which leads to growth inhibition. Therefore the cell concentration at 5th day of the culture can be considered as the 'threshold' between light-unlimited growth and light-limited growth, which was about 30-40 mg/L in chlorophyll content. Secondly, no pH level between 6 and 9 was found to completely inhibit to D. salina growth, however, the differences in the growth for different pH conditions were also observed. The specific growth rate for each pH level was compared by plotting Figure 9.
In Figure 9, the differences between the specific growth rates of light-unlimited growth phase and light-limited growth phase were obvious; the former were about 4 times higher than the latter. Therefore, a better geometry design of ALB to extend the light-unlimited growth phase is important and should be mainly considered in future work, for example enhancing the Light/Dark ratio [8]. In terms of the pH effect on D. salina growth, the plot of specific growth rate against each pH condition presented a 'parabola trend' with an optimal value achieved at around pH 7 for either light-unlimited or light-limited growth phase. Besides, D. salina had a wide range of tolerance to pH, and pH between 6 and 9 was found not to completely inhibit growth.
The pH effect on growth was also studied in terms of photosynthetic O 2 yield rate. An example of the typical photosynthetic O 2 concentration versus time was plotted in Figure 10, from which the photosynthetic O 2 generation rate was calculated. An identical 'parabola trend' as in Figure 9 was obtained in Figure 11, again indicating the optimal pH level of around 7.
Since the concentration of dissolved CO 2 is maintained the same for each culture, the intracellular CO 2 concentration was speculated According to the diagram, from day 2 to day 5, the increase in chlorophyll was obviously quicker than the increase between day 5 and day 10. Therefore, for each culture condition, two specific growth rates were calculated on day 2 -day 5 and day 5 -day 10, separately. The method for estimating the specific growth rate is shown in Appendix 2.  to be the same according to the two-film theory, which would suggest that the intracellular equilibrium pH for each culture is identical. In general, the results (Figures 9 and 11) indicated that even for the same intracellular pH, the changes in extracellular pH could still affect the algal growth via an as yet unknown mechanism, possibly related to the pH gradient across the cell membrane. pH around 7 was found to be the optimal pH for D. salina culture.

Effect of dissolved CO 2 concentration ([CO 2 ]*) on D. salina growth
In this experiment, the pH level for each culture was designed to be 7 by using 'pH*-NaHCO 3 -CO 2 % model' (Equation 7.1), while the practical pH value was actually controlled at 6.88 ± 0.08. The daily chlorophyll content change of D. salina under different CO 2 equilibrium concentrations was plotted in Figure 12. The chlorophyll content increased from 10 mg L -1 to 70 mg L -1 within 11 days under constant 5% CO 2 dosing (0.002 mol L -1 of [CO 2 ]*), while a slight growth inhibition was observed when increasing the CO 2 dosing concentration up to 20% (0.008 mol L -1 of [CO 2 ]*), in this case the chlorophyll content increased to less than 60 mg L -1 in 11 days. The 50% CO 2 dosing (0.02 mol L -1 of [CO 2 ]*) strongly inhibited D. salina growth as the chlorophyll content started decreasing from day 2 onwards. Figures 13  and 14 clearly show the effect of dissolved CO 2 concentration on D. salina growth in terms of specific growth rate and the photosynthetic O 2 generation rate, respectively. In the first 4 days, the light was still sufficient for growth due to the low concentration of algae in the culture, the specific growth rate decreased from about 0.39 d -1 to 0.32 Y: 'Range' (%) -Oxygen partial pressure X: Time (min) Figure 10: Typical graph of photosynthetic O 2 concentration (partial pressure) versus time. Each unit in X-axis was set to be 1 min. After several minutes when the recorder system was on, the light was turned on to trigger the algal photosynthetic activity, and the oxygen concentration started to increase. After several minutes, the light was turned off to observe the oxygen consumption (net respiration). The total photosynthetic oxygen generation rate was then calculated assuming that the rate of respiration in the light was the same as the respiration measured in the dark (see Appendix 1). Due to the significant weakening of photosynthesis at this high CO 2 concentration, the photosynthetic activity (assimilation) is highly inhibited and exceeded by the respiration activity (dissimilation), negative growth is therefore observed. When the light become limiting (d4 -d11), the effect of different dissolved CO 2 concentrations on D. salina growth remains the same when increasing [CO 2 ]* from 0.002 mol L -1 to 0.008 mol L -1 . For 0.02 mol L -1 of [CO 2 ]*, neither specific growth rate nor O 2 yield showed any obvious changes, because the growth is inhibited at the beginning of the culture, which did not lead to a light-limited situation.
To sum up, under the same extracellular pH, an increase in dissolved CO 2 concentration (i.e. the CO 2 % in a constant dosing condition) resulted in an inhibition of photosynthesis for D. salina culture at 50% CO 2 in the dosing stream (or 0.02 mol L -1 of [CO 2 ]* in the culture), this level of CO 2 was fatal to D. salina growth. The possible explanation behind the situation is that despite the same extracellular pH, the intracellular pH can be affected by the extracellular CO 2 equilibrium concentration, whilst higher extracellular equilibrium CO 2 concentration leads to a lower intracellular pH which may damage or inhibit the enzymes involved in photosynthesis.

Conclusions and Future work
A methodology was proposed to achieve a constant pH and variable dissolved CO 2 , or a constant CO 2 level and variable pH for microalgal culture. An empirical equation correlating pH* to NaHCO 3 and CO 2 % is obtained. The accuracy of this empirical equation was examined by comparing the experimental pH* values with the calculated values. The results showed a less than 5% deviation between the practical and the estimated pH* values.
The isolated impact of either pH or CO 2 concentration on Dunaliella salina growth was then studied by using 'pH*-NaHCO 3 -CO 2 % system'. According to either specific growth rate or photosynthetic O 2 generation rate, pH around 6-9 was found to support D. salina culture. Both specific growth rate and photosynthetic O 2 generation rate versus different pH levels presented a 'parabola trend' with an optimal value achieved at around pH 7 for either light-unlimited or light-limited growth phase. With regard to the isolated effect of CO 2 concentration on D. salina growth, both specific growth rate and photosynthetic O 2 generation rate decreased when the CO 2 concentration increased. Under 0.02 mol L -1 CO 2 concentration, a strong growth inhibition was observed. More than 0.02 mol L -1 of dissolved CO 2 (i.e. constant dosing of 50% CO 2 ) was fatal to D. salina growth.
Due to the lab limitations, only 3 different CO 2 stream concentrations were studied, an optimal dissolved CO 2 concentration was not determined for D. salina culture. More CO 2 stream concentrations (especially between 5%-20%) are expected to be tested in the future. It will be interesting to measure the intracellular pH under different dissolved CO 2 concentrations, and to find out the relationship between intracellular pH and extracellular pH.