#
Periodic CO_{2} Dosing Strategy for Dunaliella salina Batch Culture

^{1}

^{2}

^{*}

^{†}

## Abstract

**:**

_{2}dosing strategy for D. salina 19/30 batch culture is proposed. A model of periodic CO

_{2}dosing including dosing time calculation, dosing interval estimation and final chlorophyll yield prediction was established. In experiments, 5% CO

_{2}/95% N

_{2}gas was periodically dosed into D. salina culture. Two different gas dosing flow rates were tested. The corresponding dosing time for each flow rate was estimated via the model (10 min·d

^{−1}for 0.7 L·min

^{−1}and 36 min·d

^{−1}for 0.3 L·min

^{−1}). Daily pH measurements showed that the pH of these cultures dosed periodically was always kept between 7.5 and 9.5, which highlights that periodic gas supply can maintain a suitable range of pH for microalgal growth without expensive buffers. Notably the culture dosed for set daily intervals was seen to have similar growth to the culture supplied constantly, but with much higher CO

_{2}capture efficiency (11%–18%) compared to continuous dosing (0.25%). It shows great potential for using periodic gas supply to reduce cost, wasted gas and energy use.

## 1. Introduction

_{2}[1], for it may offer a way to reduce the levels of unwanted CO

_{2}whilst also allowing the production of useful by-products from the algae such as bio-oils, chemicals, fatty acids and substances, etc. [2]. However, the large-scale cultivation of microalgae still faces many problems and barriers. These include the energy required to continuous mix, dewater and process the algal biomass, and the large areas of land needed for such projects [3]. CO

_{2}dosing is one of the concerns highlighted in this study. The major problem with CO

_{2}being dosed into microalgae cultures is that conventionally the bubbles used are fairly large. As Zimmerman et al. [4] discuss, such bubbles will reduce the interfacial area between bubbles and the liquid, and will reduce the overall mass transfer for both CO

_{2}dissolution and O

_{2}stripping. To counteract the resulting low mass transfer, large flow rates of CO

_{2}enriched gas are often continuously bubbled into the cultures. While the mixing effect on the culture is beneficial for ensuring the even distribution of light and nutrients, the intense turbulence produced by large flow rate can damage the algal cells within the culture and reduce the productivity [5]. A further disadvantage of using high flow rate, especially with low mass transfer, is that most of the gas bubbled into the culture will pass through and be wasted. Carvalho and Malcata [6] agree that when CO

_{2}is bubbled into algal cultures, the mass transfer is not particularly effective and considerable gas is wasted, adding to operational costs. Even with some high mass transfer dosing techniques (e.g., Dissolved Air Flotation systems), hypothetically, continuous dosing is still not a wise option, because when the concentration of dissolved CO

_{2}has reached an equilibrium value, the gas-liquid mass transfer process stops. Any additional input beyond this point would not increase the total amount of dissolved CO

_{2}, but cost energy and waste CO

_{2}.

_{2}microbubble-dosing technology was studied by Ying et al. [7,8,9], which has proved that the microbubble-dosing technology can enable a higher CO

_{2}mass transfer and consequently lead to a higher microalgal growth rate and a greater CO

_{2}capture efficiency. Nonetheless, the operational parameters (e.g., dosing time, dosing interval and flow rate, etc.) for microbubble-dosing technology still need to be engineered. In this study, an optimal periodic CO

_{2}dosing strategy is proposed and a model established based on D. salina (19/30) cultures. The main hypothesis for this study is that by using a microbubble driven airlift bioreactor, high mass transfer can be attained in a batch culture by supplying gas periodically (with little CO

_{2}wasted and less energy cost) and achieve similar algal growth compared to when the gas is supplied continuously.

## 2. Results and Discussion

#### 2.1. Model of Periodical CO_{2} Dosing

_{2}dosing. Secondly, the dosing time should only be long enough for CO

_{2}to reach its equilibrium concentration. Meanwhile, the equilibrium pH (corresponding to CO

_{2}equilibrium concentration) is expected to be the lower limit of the suitable pH range for the microalgal species being utilized. Thirdly, the time period without dosing (dosing interval) should ensure that the microalgae use up the dosed CO

_{2}, whilst ensuring that the pH increase to the upper limit is within the suitable pH range. Following these three principles, a culture with periodic dosing, compared to one with continuous dosing, should have sufficient (but not excess) CO

_{2}and an optimal pH range to support optimal growth (achieving similar growth as with continuous dosing), while with minimal amount of CO

_{2}wasted and less energy input. Based on these three principles, the dosing time, dosing interval and final algal yield can be estimated.

#### 2.1.1. Estimation of Dosing Time

_{2}] can be calculated by Equation (1) [7,8,9,10,11].

_{2}needed to be transferred to the medium can simply be estimated as the difference between dissolved CO

_{2}level at pH=B and pH=A (assuming the CO

_{2}uptake rate is negligible compared with CO

_{2}gas-liquid mass transfer rate). The dosing time is thereby calculated as the amount of CO

_{2}needed to be transferred to the medium divided by the average CO

_{2}mass transfer rate, shown in Equation (2), where ν'

_{MTR}represents CO

_{2}average mass transfer rate (mol·L

^{−1}·min

^{−1}) and can be calculated as Equation (3). The derivation of Equation (3) was explained in Ying et al. [9]. Finally, assuming the pH before dosing was controlled at B which gives the initial dissolved CO

_{2}level as [CO

_{2}]

_{pH=B}, the optimal valid dosing time needed to drop the pH from B to A can then be estimated through Equation (4), which is obtained by combining Equations (2) and (3). As long as the suitable pH range for a particular type of algae is given and the K

_{L}a for a certain dosing condition is known, the optimal dosing time can be estimated.

_{L}a estimation is more accurate based on the changes in [C

_{T}], especially when pH > 8.4. Therefore, Equation (4) can also be written in terms of [C

_{T}], shown as Equation (5).

#### 2.1.2. Estimation of Dosing Interval

_{2}by microalgae, until the pH achieves the upper limit of the suitable range, then dosing needs to be started again. Therefore, the effective estimation of dosing interval is crucial for periodic dosing, either too long or too short could cause the pH to exceed the upper or lower limit of the suitable range and adversely affect the algal growth.

_{2}expected to be absorbed by the CO

_{2}uptake rate. However, the instantaneous growth rate differs with the concentration of the algae [12], which leads to changes in CO

_{2}uptake rate. Instead of instantaneous CO

_{2}uptake rate, the average CO

_{2}uptake rate for the whole active growth period is therefore used to simplify the estimation of dosing interval Equation (6).

_{2}[13], however, when pH was more than 8.4, the changes in dissolved CO

_{2}cannot fairly represent the CO

_{2}uptake by algae, as both HCO

_{3}

^{−}and CO

_{3}

^{2−}would generate dissolved CO

_{2}to compensate for the consumption of CO

_{2}. In other words, the amount of CO

_{2}consumed by algae should be more than the changes in dissolved CO

_{2}, as HCO

_{3}

^{−}and CO

_{3}

^{2−}would also contribute to the amount of CO

_{2}consumption. Therefore, the changes in total carbon [C

_{T}] should be considered instead of the changes in [CO

_{2}]. The [C

_{T}] can be calculated by Equation (7) [9]. Equation (6) should be converted into Equation (8). The dosing interval can then be estimated as long as the average CO

_{2}uptake rate is known.

_{2}uptake rate is assumed to be the same as in the culture with continuous or excessive CO

_{2}dosing. According to the information from previous D. salina cultures [7], the correlations for CO

_{2}uptake rate versus algal biomass concentration (measured as chlorophyll content) was described by Equation (9) [7], and the relation between total chlorophyll content increase and total CO

_{2}uptake was given (based on the cultures with excessive gas dosing) as Equation (10) [7].

_{2}uptake rate can be fairly described as:

_{c}is the selected culture time period (t

_{c2}–t

_{c1}), beginning from t

_{c1}(the start of log growth phase) and ending by t

_{c2}(the end of log growth phase).

^{−1}) is equal to the initial concentration ([Chl]

_{0}, mg·L

^{−1}) plus the amount of its increase (ν'

_{Chl}× t),

_{2}uptake rate can then be obtained by solving Equation (9), Equation (11)–(13), which gives:

^{−1}) of dissolved CO

_{2}only changes the pH by one unit [7], while the CO

_{2}uptake rates for D. salina cultures are in the range of 10

^{−4}to 10

^{−3}mol·L

^{−1}·d

^{−1}, therefore, the pH value may only increase over the upper limit of a target range by no more than 0.1 unit. Plus empirically the target pH range for algal culture can be slightly narrowed down during the dosing interval calculation in order to prevent the pH value exceeding the real upper limit. The second concern is dissolved oxygen accumulation. According to the basic photosynthetic reaction equation, the O

_{2}generation rate equals to CO

_{2}uptake rate. Therefore, 10

^{−3}mol·L

^{−1}·d

^{−1}of CO

_{2}uptake rate could result in around 32 mg·L

^{−1}of daily O

_{2}accumulation (300% over saturation with respect to air), which will diminish the rate of photosynthesis [14]. Thus, the dosing interval has to be limited to no more than 1 day so that Dissolved Oxygen (DO) can be removed by CO

_{2}dosing in time. In terms of O

_{2}stripping, it takes less than 10 min to reduce the dissolved oxygen from 32 to 0.03 mg L

^{−1}by microbubble (d

_{32}= 388 μm) dosing of 5% CO

_{2}/95% N

_{2}under 0.7 L·min

^{−1}, according to the previous mass transfer study [7].

_{0}= 5 mg·L

^{−1}and t

_{c}= 8 days). Conservatively, the dosing interval of 1 day was suggested for best results in practice, so that DO can be removed by CO

_{2}dosing in time.

#### 2.1.3. Prediction of Final Concentration of Chlorophyll Content

_{d}and dosing interval t

_{i}are known, the total dosing time (t

_{Total}) through the culture period t

_{c}can be calculated as: t

_{d}× t

_{c}/(t

_{d}+ t

_{i}). The total amount of CO

_{2}uptake can be predicted by Equation (16):

_{MTR}× t

_{d}≤ [CO

_{2}]*).

#### 2.2. Experimental Results

_{2}can be attained in a culture by “micro-bubbling” gas periodically (with little CO

_{2}wasted and less energy cost) whilst similar algal growth can be achieved compared to when the gas is supplied continuously, but with higher CO

_{2}capture efficiency.

_{2}. As can be seen, the growth of these cultures, indicated by their chlorophyll content, appears to be fairly similar. This strongly supports the idea that D. salina growth is proportional to the total amount of CO

_{2}that has been effectively transferred from gas phase into liquid phase, while extra CO

_{2}dosing beyond the valid range does not improve the productivity. The final chlorophyll contents for the cultures with different dosing conditions were expected to be the same, as the total amount of CO

_{2}mass transfer was kept identical. The contrast between the growth of the cultures fed with 5% CO

_{2}and the control cultures can also be seen from Figure 1. Unsurprisingly the chlorophyll content of the control cultures remained much lower than other cultures, and a similar phenomenon was also observed in the study of Ying et al. [7].

_{2}capture efficiency, the cultures with different dosing conditions were compared (Table 1). As can be seen, by applying a periodic dosing strategy, CO

_{2}capture efficiency achieved is about 10%–20%. It is expected that the capture efficiency could be further enhanced by improving the CO

_{2}mass transfer (e.g., by further reducing the microbubble size). In contrast, with continuous dosing, capture efficiency was only 0.25% of CO

_{2}supplied, which indicates that most of the CO

_{2}was wasted rather than been captured. With a view to using microalgae for CO

_{2}sequestration, this will mean not only wasted energy to dose CO

_{2}, but also any CO

_{2}that was prevented from entering the atmosphere by fixation in the algae culture, will be greatly exceeded by the amount of CO

_{2}passing straight through the culture into the atmosphere. Therefore, this result shows the potential for both economic and energy savings by adopting a periodic dosing strategy, as it appears that similar algal growth to a culture supplied continuously with CO

_{2}can be achieved with periodic dosing, but with minimal CO

_{2}waste and minimal energy consumption on dosing.

**Figure 1.**Daily chlorophyll content of the D. salina cultures with different dosing conditions. The cultures with 0.7 L·min

^{−1}of periodic dosing were conducted in parallel, as were the control cultures. Therefore, the daily chlorophyll content for these two dosing conditions shown in this graph is the average value, with the error bars given separately.

Dosing Type (5% CO _{2}) | Flow Rate (1 atm, 25 °C) | Dosing Time (in Total) | CO_{2} Total | ||
---|---|---|---|---|---|

Absorption | Input | η | |||

Periodic | 0.7 L·min^{−1} | 10 min·d^{−1} × 8 d | 0.89 g | 5.03 g | 18% |

Periodic | 0.3 L·min^{−1} | 36 min·d^{−1} × 8 d | 0.86 g | 7.75 g | 11% |

Continuous | 0.3 L·min^{−1} | 24 h·d^{−1} × 8 d | 0.81 g | 311.1 g | 0.26% |

_{2}input

_{total}= (CO

_{2}% × Q × t

_{d}× t

_{c}× P)/(R × T); CO

_{2}absorption = ∆[CO

_{2}]

_{uptake}Equation (16) × V

_{L}; η = CO

_{2}absorption/CO

_{2}input.

^{−1}of gas (5% CO

_{2}, 95% N

_{2}).

**Figure 2.**Daily pH values for D. salina cultures supplied periodically with 5% CO

_{2}. There are two pH values for each day, a higher one and a lower one, representing the pH values before and after CO

_{2}dosing, respectively.

**Table 2.**Comparisons between estimated final concentrations of chlorophyll and real values for D. salina cultures with different dosing conditions.

Logarithmic Growth Time Period (d) | Dosing Condition | Estimated [Chl] _{t} (mg/L) | Real [Chl]_{t} (mg/L) | Error |
---|---|---|---|---|

8 d | 0.7 L·min^{−1}, 10 min·d^{−1} | 26.90 | 26.09 | 3% |

8 d | 0.3 L·min^{−1}, 36 min·d^{−1} | 26.07 | 26.48 | 2% |

## 3. Experimental Section

#### 3.1. Experimental Setup

_{d}) and dosing interval (t

_{i}) were estimated based on Equations (5) and (15) and applied for the cultures engaged with periodic dosing strategy (No. 2, 4 and 5). pH was measured for each culture daily (for the culture dosed periodically, pH was measured twice per day, before and after dosing). 15 mL of sample for each culture was taken after gas dosing, followed by topping up the culture with 15 mL of fresh medium (For flask cultures, a sterilized glass stick was inserted into the culture for a proper stirring. Samples were taken after that.). The chlorophyll content for each sample was determined by measuring each sample’s optical density for wavelengths of 645 and 663 nm using the same method to that used in Zimmerman et al. [16] and Ying et al. [7].

**Figure 3.**Setup for D. salina cultures. Six bioreactors containing D. salina cultures (50 mL of inocula to 2.5 L culture medium) were used for this experiment. Two of the six (No. 1 and 6) were flask cultures with no CO

_{2}enriched gas being bubbled through the culture. Like the other four cultures, these control cultures were kept in identical ALBs to ensure that the illumination through these cultures was the same as those being supplied with gas. The remaining four reactors were supplied with CO

_{2}enriched gas (5% CO

_{2}, 95% N

_{2}). Among them, No. 3 was dosed continuously with fine-bubbles, while No. 2, 4 and 5 were connected to a fluidic oscillator, dosed with microbubbles using periodic dosing strategy. No. 2 and 5 were conducted under same dosing condition for error analysis. The detailed dosing conditions for each reactor are listed in Table 4. The dosing time selected for each condition was estimated based on Equation (5). The detailed calculation is shown in Section 3.2. The temperature for each culture was maintained at ambient temperature around 24 °C. Two fluorescent lamps (90 μmol·m

^{−2}·s

^{−1}) were situated behind all reactors for illumination. Growth medium for D. salina cultures is shown in Table 3.

Composition of Growth Medium Per Litre |
---|

0.5 M·NaCl; 0.185 mM·H_{3}BO_{3}; 10 mM·KCl; 0.007 mM MnSO_{4}; 20 mM·MgCl_{2}; 0.8 × 10^{−3} mM·ZnCl_{2}; 10 mM CaCl _{2}; 0.2 × 10^{−4} mM CoCl_{2}; 24 mM MgSO_{4}; 0.2 × 10^{−6} mM·CuCl_{2}; 5 mM NaNO_{3}; 24 mM·Na _{2}SO_{4}; 0.0119 mM·NaHCO_{3}; 0.1 mM NaH_{2}PO_{4}; 0.0015 mM·FeEDTA |

**Table 4.**The dosing conditions for each culture. The average size for the microbubbles and fine bubbles were measured by using high speed camera [17].

Reactor | Dosing Conditions | Represent | |||
---|---|---|---|---|---|

Bubbles | Fluidic Oscillator | Dosing Flowrate | Dosing Time Equation (5) | ||

No. 1 | No bubbles | Not Engaged | 0 | 0 | Flask culture |

No. 2 | Microbubble (d_{32}: 388 μm) | Engaged | 0.7 L·min^{−1} | 10 min·d^{−1} | Periodic dosing |

No. 3 | Fine-bubble (d_{32}: 719 μm) | Not Engaged | 0.3 L·min^{−1} | 24 h·d^{−1} | Continuous dosing |

No. 4 | Microbubble (d_{32}: 388 μm) | Engaged | 0.3 L·min^{−1} | 36 min·d^{−1} | Periodic dosing |

No. 5 | Microbubble (d_{32}: 388 μm) | Engaged | 0.7 L·min^{−1} | 10 min·d^{−1} | Duplication of No. 2 |

No. 6 | No bubbles | Not Engaged | 0 | 0 | Duplication of No. 1 |

#### 3.2. Estimation of the Dosing Time and Dosing Interval

^{−1}were calculated based on Equation (5). The detailed calculation was shown as follows.

_{3}in the culture medium was 0.0119 mol·L

^{−1}, [C

_{T}] at pH = 7.5 and pH = 9.5 can be calculated based on Equation (7), which gives: [C

_{T}]

_{pH = 7.5}= 0.0128 mol·L

^{−1}; [C

_{T}]

_{pH = 9.5}= 0.0106 mol·L

^{−1}.

_{L}a for CO

_{2}mass transfer in the real algal culture was previously determined to be 0.044 min

^{−1}for 0.3 L·min

^{−1}of bubbling flow rate and 0.174 min

^{−1}for 0.7 L·min

^{−1}[9]. Finally, t

_{d}was calculated to be about 36 min for 0.3 L·min

^{−1}of bubbling flow rate and 10 min for 0.7 L·min

^{−1}.

_{0}= 5 mg·L

^{−1}and t

_{c}= 8 days, t

_{i}is calculated to be 1.5 days. Conservatively, 1 day of dosing interval was used in the experiment so that DO can be removed by CO

_{2}dosing in time.

## 4. Conclusions

_{2}dosing strategy for D. salina culture is proposed, with a model of periodic CO

_{2}dosing including dosing time calculation, dosing interval estimation and final chlorophyll yield prediction established. The cultures applying periodic CO

_{2}dosing strategy provide a similar productivity to the culture with continuous dosing, but with a greater CO

_{2}sequestration efficiency. Due to the time limitation, only two different gas dosing flow rates were tested in this study. More flow rates can be tested to explore the relation between dosing flow rate and CO

_{2}capture efficiency in the future studies.

## Acknowledgments

## Author Contributions

## Abbreviations

Δ[Chl] | changes in the concentration of chlorophyll content, mg·L ^{−1} |

Δ[CO_{2}]_{dosed} | the difference between the concentration of dissolved CO _{2} before and after dosing, mol·L^{−1} |

Δ[CO_{2}]_{uptake} | changes in the concentration of CO _{2}(aq) due to the CO_{2} consumption by algal growth, mol·L^{−1} |

Δ[Na^{+}] | the concentration of sodium ions in the liquid which comes from NaHCO _{3}, mol·L^{−1} |

CO_{2}% | the concentration of CO _{2} in the gas stream, % (v/v) |

d_{32} | the Sauter mean diameter of the bubbles, μm |

η | the CO _{2} capture efficiency, % |

K_{L}a | volumetric mass transfer coefficient (min ^{−1}), where “K_{L}” is the CO_{2} gas-liquid mass transfer coefficient (m·min^{−1}); “a” means the gas-liquid interfacial area (m^{−1}) |

P | standard atmospheric pressure, 101.325 Kpa |

pH | the pH value of the liquid |

R | universal gas constant, 8.314 J·mol ^{−1}·K^{−1} |

T | the temperature, K |

t_{c} | culture time period, d |

t_{d} | CO _{2} dosing time, min |

t_{i} | CO _{2} dosing interval, min |

ν'_{Chl} | average productivity of chlorophyll content, mg·L ^{−1}·d^{−1} |

ν_{CO2 uptake} | instantaneous algal CO _{2} uptake rate, mol·L^{−1}·min^{−1} |

ν'_{CO2 uptake} | average algal CO _{2} uptake rate, mol·L^{−1}·min^{−1} |

Q | the gas dosing flowrate, L·min ^{−1} |

ν'_{MTR} | CO _{2} average mass transfer rate, mol·L^{−1}·min^{−1} |

V_{L} | the volume of the liquid, L |

[C_{T}] | total concentration of inorganic carbon in the liquid, mol·L ^{−1} |

[C_{T}]_{pH=A} | total concentration of inorganic carbon in the liquid at pH=A, mol·L ^{−1} |

[C_{T}]_{pH=B} | total concentration of inorganic carbon in the liquid at pH=B, mol·L ^{−1} |

[Chl] | concentration of chlorophyll content in the culture, mg·L ^{−1} |

[Chl]_{0} | initial concentration of chlorophyll content at the beginning of log growth phase, mg·L ^{−1} |

[CO_{2}] | dissolved carbon dioxide concentration in the liquid, mol·L ^{−1} |

[CO_{2}]^{*} | dissolved carbon dioxide equilibrium concentration in the liquid, mol·L ^{−1} |

[CO_{2}]_{0} | initial dissolved carbon dioxide concentration in the liquid, mol·L ^{−1} |

[CO_{2}]_{pH=A} | dissolved carbon dioxide concentration in the liquid at pH=A, mol·L ^{−1} |

[CO_{2}]_{pH=B} | dissolved carbon dioxide concentration in the liquid at pH=B, mol·L ^{−1} |

[CO_{3}^{2−}] | the concentration of carbonate, mol·L ^{−1} |

[HCO_{3}^{−}] | the concentration of bicarbonate, mol·L ^{−1} |

## Conflicts of Interest

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**MDPI and ACS Style**

Ying, K.; Gilmour, D.J.; Zimmerman, W.B.
Periodic CO_{2} Dosing Strategy for *Dunaliella salina* Batch Culture. *Int. J. Mol. Sci.* **2015**, *16*, 11509-11521.
https://doi.org/10.3390/ijms160511509

**AMA Style**

Ying K, Gilmour DJ, Zimmerman WB.
Periodic CO_{2} Dosing Strategy for *Dunaliella salina* Batch Culture. *International Journal of Molecular Sciences*. 2015; 16(5):11509-11521.
https://doi.org/10.3390/ijms160511509

**Chicago/Turabian Style**

Ying, Kezhen, D. James Gilmour, and William B. Zimmerman.
2015. "Periodic CO_{2} Dosing Strategy for *Dunaliella salina* Batch Culture" *International Journal of Molecular Sciences* 16, no. 5: 11509-11521.
https://doi.org/10.3390/ijms160511509