The Possible Contribution of Chlorella vulgaris to Uptake High Concentration Carbon Dioxide to Alberta’s Climate Leadership Plan.

Hosseini S^1* and Chu Angus^2
*Correspondence: Hosseini S, Department of Environmental Engineering, Research & Education, University of Calgary, Alberta, Canada, Tel: +14032205821, Email:

Keywords

Wastewater treatment; Chlorella vulgaris; Microalgae; GHG mitigation; Water; Nutrients; CO₂

Introduction

An increase in population has led to a rise in the global demand for energy and waste treatment, and this demand has contributed to global warming. Fossil fuels are the primary source of energy all over the world, and their increasing consumption will lead to higher emissions of greenhouse gases (GHG) such as carbon dioxide (CO₂), methane (CH₄), ozone (O₃) and water vapour. GHG reflect incoming radiation from the sun and trap outgoing infrared radiation [1]. In this GHG crisis, CO₂ plays a vital role, which has raised concerns in recent years. The main industrial sources responsible for CO₂ emissions are coal-fired power plants [2]. Coal-fired power plants have historically been Alberta’s inexpensive supply of electricity. Sixteen percent of Alberta GHG emissions come from the electricity sector; this amount is higher compared to other Canadian provinces. According to the latest federal regulations, all coal-fired power plants should phase-out once they become fifty years old [3]. On the other hand, Wastewater is an appropriate, renewable, economical medium, which contains enough nutrients, such as nitrogen and phosphorus, for algae to grow.

As an aquatic plant, algae lack roots, leaves, and stems and have the ability to conduct photosynthesis similar to terrestrial plants. C. vulgaris is a well-known green eukaryotic microalga in the Chlorella species [4]. Microalgae can be found everywhere in the world and grow in either fresh or saline water; they can tolerate the most severe environmental conditions [5]. As a result of the photosynthesis process and presence of carbon substrate and enough nutrients, energy is stored in the produced biomass. In recent decades, microalgae have been used to produce highvalue products, such as pharmaceuticals, cosmetics, and food supplements, biodiesel as engine fuels, and biomass as livestock food [6].

November 20, 2016, Government of Alberta’s Climate Leadership Plan focused on reducing carbon emissions. They implemented a $20/ tonnes carbon price to all GHG emitters on January 1, 2017. Beginning in 2018 this price rose to $30/tonnes carbon and will increase by 2% every year [7]. The new regulations necessitate innovative technologies to mitigate CO₂ to meet Alberta’s aggressive targets while remaining leaders in oil production. This study aims to find the range of optimum optical density that C. vulgaris has higher CO₂ uptake in the domestic wastewater effluent as a medium. Also, growth possibility of C. vulgaris in high CO₂ concentration will be assessed. Finally, assess the contribution of this research result to Alberta CLP.

Material and Methods

Microalgae and medium

Chlorella vulgaris was obtained from the Canadian Phycological Culture Center (CPCC) located at the University of Waterloo, Ontario. The medium used in this experiment was Bold’s Basal Medium (BBM) which consist of KH₂PO₄ 0.12 g l^-1, CaCl₂·H₂O 0.025 g l^-1, MgSO₄·7 H₂O 0.075 g l^-1, NaNO₃ 0.25 g l^-1, K₂HPO₄ 0.075 g l^-1, NaCl 0.025 g l^-1, Na₂EDTA·2 H₂O 0.01 g l^-1, KOH 6.2 g l^-1, FeSO₄·7 H₂O 0.005 g l^-1, H₂SO₄ 1 ml l^-1, trace metal solution 1 ml, H₃BO₃ 0.0081 g l^-1 and F/2 vitamin solution 1 ml. Moreover, since the goal is to use wastewater to grow algae, the BBM was not sterilized afterwards for scaling up to higher volumes nor for use in the follow-up experiments.

Experimental setup

C. vulgaris was cultivated in BBM in three 500 ml Erlenmeyer flasks to have enough stock culture. A 2- litres vacuum flask was chosen as a photobioreactor. The volume of the vacuum flask was checked with a graduated cylinder. 1.5 litres of the flask was occupied by microalgae culture. Using a graduated cylinder, the headspace volume was measured which was 484 mL. According to Nielsen [8], an appropriate CO₂ supply for the saturation of microalgae growth is approximately 5% of the unicellular green algae Chlorella. If there is an excess amount of CO₂, the pH can drop, and the growth of microalgae can be inhibited. Figure 1 illustrates the schematic of the apparatus setup. Also, a closed system is chosen based on its advantages compared to an open system such as reduction of water evaporation and lowering the growth of competitive algal weeds and predators and pathogens that may kill the desired microalgae [9].

In a closed system, the gas in the headspace of the photobioreactor is continuously recirculated throughout the C. vulgaris culture in a loop, passing through the gas sensors. Consequently, gas concentrations changes, due to photosynthesis and respiration of C. vulgaris, so it can be easily monitored in real time. Moreover, using such a configuration is financially beneficial since it avoids the necessity of having more sensors for the gas inlet and outlet simultaneously. To protect the sensors by water trap from the moisture the desiccant beads were bought from Sigma-Aldrich Canada. Also, UV Flux 25% oxygen sensor and COZIRWR high-performance CO₂ infrared gas analyzer (IRGA) and the air pump were purchased from CO₂ Meter Inc. In this experiment, 20% of the headspace was occupied by CO₂. Since we calculated the volume of headspace, we were able to feed the proper amount of CO₂ to the microalgae culture while not inhibiting the growth. Considering the ideal gas law and CO₂ molecular weight and knowing that the concentration of CO₂ in the air which is 400 mg/L so 0.387 liter of air has 0.155 gram of CO₂ therefore, the total CO₂ mass in the headspace is 0.35 gram. Knowing that for 0.35 g of CO₂ is equal to 20% in the CO₂ sensor detector; the initial mass of CO₂ in four experiments can be easily measured. Applying the same method above the mass of oxygen in the headspace was found to be 0.58 g for 15.58% of oxygen detected by the O₂ sensor. In- addition, based on the growth of microalgae, which is similar to other living microorganisms, they have different growth phases, as shown in Figure 2.

In this experiment, the photobioreactor was filled with Chlorella vulgaris, which was in various growth phases, to monitor the ability of microalgae to fix CO₂ in different phases four OD680 were chosen to test the CO₂ mitigation ability of C. vulgaris (Table 1).

The exponential growth phase of microalgae has the most rapid growth possible under the conditions presented in this batch system. Four optical densities were chosen from the exponential phase to conduct this experiment. For the first test, an OD was selected from the beginning of the exponential growth phase; the second test used an average OD from the entire exponential phase. The third test used an OD that was chosen from just before the end of the exponential growth phase, and finally, the fourth OD was selected from the end of the exponential growth phase and the beginning of the stationary phase.

Microalgae growth conditions

The stock culture was cultivated in the pH of 7 and ambient temperature (20- 22°C) the light intensity was 2200 Lux provided by ABI cool light LED light. To provide carbon substrate and proper mixing, 5% of CO₂ in the gas mixture (Air-CO₂) was diffused into culture flask, utilizing stone diffusers. C. vulgaris growth was determined by measuring the OD using HACH (DR/4000 U Spectrophotometer).

Results and Discussion

It is important to know that the efficiency of CO₂ sequestration in a closed system depends on microalgal species, CO₂ concentration, photobioreactor design and physical growth conditions [10]. Also, Figure 3 shows the depletion of CO₂ in the algae photobioreactor. Interestingly, the CO₂ concentration approaches zero for all four experiments. Since the system is airtight and has a fixed solubility of CO₂ in water, the removed CO₂ only can be sequestered by algae cells in the photobioreactor. To prove the photosynthesis process, the potential oxygen production was measured at the same time as CO₂ depleted.

Figure 4 shows the increase of oxygen in the headspace of the photobioreactor. As previously discussed, four algae cultures with varying initial OD-680 were added to the photobioreactor. Figure 4 illustrates that an OD-680 of 0.650, which is at the beginning of the exponential growth phase, had a higher oxygen production rate compared to other tested optical densities. Table 2 presents the results of applying the same method as described before to determine the producer of biomass concentration. Also, the concentration of biomass has converted to the mass of biomass per litre of the bioreactor.

Table 2 illustrates the rate of CO₂ consumption per produced biomass in a period of 35 hours. Razzak et al. and Hu [10,11] reported that most microalgae strains need to uptake 1.7 kg of CO₂ to produce 1 kg of dry weight biomass. However, the reported rate belongs to open pond raceway with constant nutrient feeding. Clearly, in such a closed system such as our experiment, this rate would be way lower because of lack of nutrient and carbon substrate over the time. In the table above, the result shows that in OD of 0.493, both algae production and CO₂ uptake are high values compared to other values. Moreover, in OD of 1.030, although the algae production is the lowest, the CO₂ uptake is the highest value compared to the other OD’s. This is because of the presence of high concentration of biomass which existed in the photobioreactor from the beginning of the experiment. Moreover, Vaičiulyte et al. [12] reported that they could achieve the highest biomass productivity of C. vulgaris at 0.3 g L^-1 d^-1 with a constant 2% CO₂ flow into the culture. This result is because of the presence of an adequate amount of nutrients and carbon substrate. However, the above experiment conducted over two days without adding any nutrients or carbon substrate. Based on the above experiments, the highest productivity of biomass in 24 hours is 0.039 g L^-1 d^-1 for OD of 0.4. Also, it is highly recommended to measure the biomass increase or growth rate to assess the potential of a microalgae culture system for direct CO₂ removal [10]. Table 3 shows the increase in biomass concentration after 35 hours the experiment started which is proof of CO₂ removal.

Table 4 summarizes the existing literature data on the amount of provided CO₂ and produced biomass for C. vulgaris while providing a basis for comparison, differing experimental conditions between this work and the presented literature should be considered Table 5. Specifically, the bioreactors presented in the literature are roughly one third the size of our bioreactor as well as using different mediums and 15 days duration. One of the reasons for low biomass productivity is because the photobioreactor was air-tight. To maintain the system airtight we could not top nutrient and definitely, after a certain period, the nutrient became limiting for algae growth. Also, Bhola et al. [13] stated that in the similar experiment 15% of CO₂ in the photobioreactor led to a three-time lower yield of biomass compared to the presence of 4% CO₂. This is because of the toxic effect of CO₂ by converting CO₂ to carbonic acid. Produced carbonic acid decreases pH and making an acidic environment for algae [14].

Table 6 shows the ratio of produced O₂ mass and consumed CO₂ mass in the period of four hours. The reason for four hours measurement is because the O₂ sensor cannot detect the O₂ concentration above 25% and the O₂ concentration in all four experiments reached the highest point the sensor could read in four hours. Finding corresponding CO₂ uptake in four hours we measured the ratio of produced O₂ and consumed CO₂. Based on the photosynthesis equation stoichiometry, for every CO₂ molecular mass (44 g) which is uptaken by C. vulgaris one O₂ molecular mass (32 g) should be produced (ratio=0.73). In this experiment, the ratio in Table 6 is lower than 0.73 for all four sets of experiments. Comparing the exponential growth phase, the beginning, for its high rate of biomass production and end of the exponential growth phase, for its high accumulate biomass concentration have a greater ratio compared to the middle of the exponential growth phase [15,16].

Conclusion

The closed loop is an easy and direct measurement of CO₂ sequestration with minimal sample preparation and without any pre-measurement. Moreover, an additional benefit of this method is simultaneous oxygen production measurements. As shown in Figures 2 and 3, C. vulgaris has a higher CO₂ sequestration rate and biomass production rate during the beginning of the exponential phase compared to other parts of the exponential phase and stationary phase. During the exponential growth phase, the rate of increase of cells in the culture is correlated to the number of cells present at any time. As a result, if more algae cells are present, then more CO₂ will be fixed. Overall, the efficiency of CO₂ fixation in a closed cultivation system depends on the microalgae species, CO₂ concentration, photobioreactor design, and operating conditions. Klinthong states that C. vulgaris has a maximum CO₂ removal efficiency of 55.3% at 0.15% CO₂ in a membrane photobioreactor while this experiment shows almost 100% of CO₂ removed at 20% of total volume in 36 hours.

The outdoor cultivation is harder to control but usually leads to higher yield and lower cost. On the other hand, indoor cultivation is easier to control but more expensive. According to Alberta’s long cold season outdoor algae cultivation is not possible because of inappropriate growth conditions. Thus this method would not have a great contribution to CLP to reduce GHG emission.

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