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ISSN: 1948-5948
Journal of Microbial & Biochemical Technology
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Effects of CO2 and pH on Growth of the Microalga Dunaliella salina

Kezhen Ying1*, D James Gilmour2 and William B Zimmerman1

1Department of Chemical and Biological Engineering, University of Sheffield, UK

2Department of Molecular Biology and Biotechnology, University of Sheffield, UK

*Corresponding Author:
Kezhen Ying
Department of Chemical and Process Engineering
University of Sheffield, Sir Robert Hadfield Building
Mappin Street, Sheffield, S1 3JD, UK
Tel: +44 (0) 114 222 7500
Fax: +44 (0) 114 222 7501
E-mail: [email protected]; [email protected]

Received date: February 14, 2014; Accepted date: March 25, 2014; Published date: March 28, 2014

Citation: Ying K, James Gilmour D, Zimmerman WB (2014) Effects of CO2 and pH on Growth of the Microalga Dunaliella salina. J Microb Biochem Technol 6:167-173. doi: 10.4172/1948-5948.1000138

Copyright: © 2014 Ying K, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

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Abstract

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.

Keywords

Isolated impact; pH; CO2; Dunaliella salina

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 [2-8], while some are still worthwhile to be studied, for instance the effects of pH and CO2 on microalgal growth. At saturating light intensities, the rate of CO2 supply is crucial for algal photosynthesis as CO2 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 CO2 on microalgal growth have been well studied by many researchers [9-14], however, neither pH nor dissolved CO2 were solely controlled during their experiments due to the interactions between pH and dissolved CO2. It seems to be infeasible to keep pH constant while varying the dissolved CO2, or vary the pH while keeping dissolved CO2 constant. Therefore it will be interesting to find out the isolated effect of pH or CO2 on algal growth. In this study, a method was proposed to achieve a constant pH and variable dissolved CO2, or a constant CO2 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 CO2 concentrations were tested.

Methodology

Under a constant bubbling condition, the equilibrium concentration of dissolved CO2 ([CO2]*), according to Henry’s law and Two-film theory, should only depend on the CO2 partial pressure in the gas phase under constant gas/liquid properties and temperature. Therefore, for a fixed CO2 percentage in the bubbling gas, the [CO2]* will not be altered when varying the concentrations of NaHCO3 in the medium (assuming the changes in liquid physical properties by adding NaHCO3 into the water are negligible, as long as the concentrations of NaHCO3 are low). On the other hand, the dissolved CO2 concentration is correlated to pH by Equation 1 [15,16]. Since the concentration of Na+ varies for different concentration of NaHCO3, while the [CO2]* does not change, it is therefore reasonable that the equilibrium pH (pH*) changes for the medium with different NaHCO3 concentration.

image         (1)

In Ying et al. [16], the effects of NaHCO3 concentration on equilibrium concentration of dissolved CO2 and CO2 mass transfer rate in water were studied. The results proved the above hypothesis, indicating the feasibility of using NaHCO3 to control the equilibrium pH of the medium without affecting the [CO2]* and CO2 mass transfer rate. However, only one concentration of CO2 (5%) in the bubbling gas was tested, the relationship between pH* and NaHCO3 established was only suitable for 5% CO2 dosing. Therefore, in this study, experiment a) was designed to find a comprehensive model correlating pH*, NaHCO3 and CO2 %, which would facilitate the experimental designs on b) pH impact and c) CO2 effect on algal growth.

Experiment a): Relationship between pH*, NaHCO3 and CO2%

To study the interaction between pH*, NaHCO3 and CO2%, a gas mixture containing a certain percentage of CO2 balanced with N2 is injected to the airlift bioreactor containing 1.5 L of distilled water and a certain concentration of NaHCO3. 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 NaHCO3 and 5 CO2 stream concentrations tested. The equilibrium concentration of CO2 ([CO2]*) was calculated by Equation 1. The experimental set up is shown in Figure 1.

microbial-biochemical-technology-equilibrium

Figure 1: The setup for equilibrium pH measurement.

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 NaHCO3 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 was added to 1.5 L of fresh culture medium for each culture. CO2 gas mixture was constantly dosed into each reactor with a fixed stream concentration (5% CO2 balanced with N2) under 0.3 L/min. Although the algal growth may consume some dissolved CO2, a new equilibrium would be achieved immediately after that due to the constant CO2 dosing (CO2 mass transfer >> CO2 consumption, the consumed CO2 would be balanced with the CO2 transferred into the medium). In other words, the dissolved CO2 is maintained constant at its equilibrium concentration. However, the equilibrium pH for each reactor differs, due to the different NaHCO3 concentrations in the medium. As regard to the specific NaHCO3 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.

Reactor Culture conditions
Dosing condition Concentration of NaHCO3 (mol/L) Expected pH* Expected [CO2]* (mol/L)
No. 1 5% CO2 constant dosing with fine-bubbles (d32: 719 µm) 5.95×10-4 6 0.002
No. 2 2.03×10-3 6.5 0.002
No. 3 6.97×10-3 7 0.002
No. 4 8.17×10-2 8 0.002
No. 5 0.280 8.5 0.002
No. 6 0.957 9 0.002

Table 1: The culture condition of each reactor in the study of pH impact on D. salina growth.

microbial-biochemical-technology-experimental

Figure 2: The experimental setup for studying the impact of pH on D. salina growth.

Experiment c): The effect of dissolved CO2 on algal growth

To study the impact of dissolved CO2 on D. salina growth dissolved CO2 concentration needs to be varied while the pH for each culture should be maintained constant. To achieve this, three different CO2 stream concentrations (5%, 20% and 50%) were applied to provide three corresponding CO2 equilibrium concentrations. The equilibrium pH for each reactor was expected to be 7 by adding the proper amount of NaHCO3. The concentration of NaHCO3 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.

Reactor Culture conditions
Dosing condition Concentration of NaHCO3 (mol/L) Expected pH* Expected [CO2]* (mol/L)
No. 1 5% CO2 dosing 6.97×10-3 7 0.002
No. 2 5% CO2 dosing 6.97×10-3 7 0.002
No. 3 20% CO2 dosing 3.29×10-2 7 0.008
No. 4 20% CO2 dosing 3.29×10-2 7 0.008
No. 5 50% CO2 dosing 9.19×10-2 7 0.020
No. 6 50% CO2 dosing 9.19×10-2 7 0.020

Table 2: The culture condition of each reactor in the study of CO2 impact on D. salina growth.

microbial-biochemical-technology-studying

Figure 3: The experimental setup for studying the effect of CO2 on D. salina growth.

Results and Discussion

The correlations between pH*, NaHCO3 and CO2%

Figure 4 summarized the relations between [CO2]*, NaHCO3 and CO2%. The equilibrium concentration of dissolved CO2 ([CO2]*) is found to be only dependent on the CO2 stream concentration (CO2 %). [CO2]* was enhanced with the higher CO2% supply. The variation of NaHCO3 concentration did not affect [CO2]* when CO2 % 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.

microbial-biochemical-technology-concentration

Figure 4: Plots of [CO2]* versus NaHCO3 concentration for different CO2 stream concentrations.

In terms of equilibrium pH (pH*), its changes along with the NaHCO3 concentration and CO2 stream concentration (CO2%) were plotted in Figure 5a. As can see, for a fixed CO2% in the gas supply, pH* was altered by varying the NaHCO3 concentration. Higher NaHCO3 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 NaHCO3 and CO2% 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 NaHCO3 and CO2% 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 CO2 mass transfer rate needs to be controlled sufficiently to balance the CO2 consumption by algal growth. Otherwise, pH* would not stay constant but increase.

image         (2)

microbial-biochemical-technology-relationship

Figure 5: 3D-plot of the relationship between pH*, NaHCO3 and CO2%. (a) Plot of pH* versus NaHCO3 and CO2%; (b) plot of pH* versus ln(NaHCO3) and ln(CO2%).

microbial-biochemical-technology-calculated

Figure 6: Comparison between experimental pH* value with the one calculated based on Equation 2. This figure consists of 35 points, covering the pH* values under 7 NaHCO3 and 5 CO2 stream concentrations.

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 CO2 concentration for each culture was maintained the same (about 0.002 mol.L-1) through the constant dosing of 5% CO2. 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*- NaHCO3-CO2% 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.

microbial-biochemical-technology-deviations

Figure 7: Plot of the real pH versus the expected pH for the experiment ‘pH effect on D. salina growth’. For each culture, the real pH value presented in this figure was calculated as the average value of the daily recorded pHs of which the standard deviations are shown as error bars.

microbial-biochemical-technology-chlorophyll

Figure 8: The plot of daily chlorophyll content against culture time for different pH levels. 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.

microbial-biochemical-technology-triangles

Figure 9: The specific growth rate of D. salina culture under each pH condition. The hollow triangles represent the specific growth rates of the first 5 days (light-unlimited) while the solid triangles stand for the specific growth rates between day 5 and day 12 (light-limited).

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 O2 yield rate. An example of the typical photosynthetic O2 concentration versus time was plotted in Figure 10, from which the photosynthetic O2 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.

microbial-biochemical-technology-photosynthetic

Figure 10: Typical graph of photosynthetic O2 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).

microbial-biochemical-technology-conditions

Figure 11: The photosynthetic O2 yield rates under different pH conditions. The O2 yield rates were measured on the samples taken at the 5th day of the culture (light-unlimited).

Since the concentration of dissolved CO2 is maintained the same for each culture, the intracellular CO2 concentration was speculated 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 CO2 concentration ([CO2]*) on D. salina growth

In this experiment, the pH level for each culture was designed to be 7 by using ‘pH*-NaHCO3-CO2% 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 CO2 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% CO2 dosing (0.002 mol L-1 of [CO2]*), while a slight growth inhibition was observed when increasing the CO2 dosing concentration up to 20% (0.008 mol L-1 of [CO2]*), in this case the chlorophyll content increased to less than 60 mg L-1 in 11 days. The 50% CO2 dosing (0.02 mol L-1 of [CO2]*) 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 CO2 concentration on D. salina growth in terms of specific growth rate and the photosynthetic O2 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 d-1 by increasing the [CO2 ]* from 0.002 mol L-1 to 0.008 mol L-1, whilst the photosynthetic O2 yield dropped from approximately 0.40 μmol min-1 mgChl-1 to 0.38 μmol min-1 mgChl-1. Under 0.02 mol L-1 [CO2 ]*, although the decrease in chlorophyll content and the negative value of specific growth rate indicated a strong inhibition in photosynthesis, a photosynthetic activity was still detected, showing the photosynthetic O2 rate to be 0.08 μmol min-1 mgChl-1. Due to the significant weakening of photosynthesis at this high CO2 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 CO2 concentrations on D. salina growth remains the same when increasing [CO2]* from 0.002 mol L-1 to 0.008 mol L-1. For 0.02 mol L-1 of [CO2]*, neither specific growth rate nor O2 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.

microbial-biochemical-technology-chlorophyll

Figure 12: The plot of daily chlorophyll content against culture time for different CO2 stream concentrations.

microbial-biochemical-technology-concentrations

Figure 13: The specific growth rate of D. salina culture for different CO2 stream concentrations. Under the 0.02 mol L-1 of dissolved CO2 concentration, the specific growth rate was shown as negative, representing the decrease in algae concentration.

microbial-biochemical-technology-photosynthetic

Figure 14: The photosynthetic O2 yield rates under different dissolved CO2 concentrations. The O2 yield rates were measured on the samples taken at the 2nd day (light-unlimited) and at the 10th day (light-limited) of the culture.

To sum up, under the same extracellular pH, an increase in dissolved CO2 concentration (i.e. the CO2% in a constant dosing condition) resulted in an inhibition of photosynthesis for D. salina culture at 50% CO2 in the dosing stream (or 0.02 mol L-1 of [CO2]* in the culture), this level of CO2 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 CO2 equilibrium concentration, whilst higher extracellular equilibrium CO2 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 CO2, or a constant CO2 level and variable pH for microalgal culture. An empirical equation correlating pH* to NaHCO3 and CO2% 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 CO2 concentration on Dunaliella salina growth was then studied by using ‘pH*-NaHCO3-CO2% system’. According to either specific growth rate or photosynthetic O2 generation rate, pH around 6-9 was found to support D. salina culture. Both specific growth rate and photosynthetic O2 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 CO2 concentration on D. salina growth, both specific growth rate and photosynthetic O2 generation rate decreased when the CO2 concentration increased. Under 0.02 mol L-1 CO2 concentration, a strong growth inhibition was observed. More than 0.02 mol L-1 of dissolved CO2 (i.e. constant dosing of 50% CO2) was fatal to D. salina growth.

Due to the lab limitations, only 3 different CO2 stream concentrations were studied, an optimal dissolved CO2 concentration was not determined for D. salina culture. More CO2 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 CO2 concentrations, and to find out the relationship between intracellular pH and extracellular pH.

Acknowledgements

We acknowledge support for microbubble dynamics from EPSRC EP/ I019790/1. WZ would like to thank the Royal Society for a Brain Mercer Innovation Award. DJG would like to acknowledge support from Carbon Trust.

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