The Effect of NaHCO 3 and Mg 2+ Addition in Haematococcus pluvialis Cultivation by Carbon Injection Method

. The emission of carbon dioxide has been continuously rising year by year. Many efforts that have been used with aim of climate recovery, such as capturing CO 2 with the Carbon Capture Storage (CCS) method, which is the CCS technology is one of the effective tactics for reducing carbon emissions by utilizing energy from biomass of microalgae. This research will discuss about carbon capture using microalgae Haematococcus pluvialis in a lab scale photobioreactor (PBR), and resulting the optimum biomass productivity for Haematococcus pluvialis has occurred in the H 2 variable (50 ppm Mg 2+ ). This happens because of addition of Mg ions above 50 ppm can decrease the yield of biomass productivity, since Haematococcus pluvialis cannot live in high salinity concentrations. Further research should make a calculation of optimal cost incurred at the optimal carbon concentration that can be captured by microalgae, also the results of increasing value of the microalgae biomass produced for the comprehensive use of the microalgae.


Introduction
Global warming is a never end catastrophic climate change for the earth citizens, and the rise of CO2 levels in atmosphere is one of the main causes (Atwoli et al., 2021). The emission of CO2 gas has reached to 412.3 ppm in 2019 (Yoro and Daramola, 2020), and increased to 414.7 ppm in 2021 (Jiang et al., 2022). If the CO2 levels in atmosphere has reached more than the safety level, it will cause the rise of sea level and also threaten the biodiversity and ecosystem functions (Hilmi et al., 2021). The industrial sector has long been the largest contributor to CO2 emissions, because of the high energy consuming can put tremendous pressure on the ecological environment (Wang et al., 2021).
To prevail over the carbon gas emissions, one of the solutive way is capturing the carbon. The methods of carbon capture can be divided into four types, such as chemical method using raw materials of polycarbonates, fuel-based method using raw materials of gasoline, conventional method using a solution, and biological exploitation using microalgae for growth source (Vaz Jr. et al., 2022). As an organic source, microalgae have more better benefits for the environment, such as flexibility of medium growth like sea water and wastewater (Ahmad et al., 2021), also having a potential to preclude the problems of global environment as a fossil replacement, this makes microalgae can reduce the problems of energy stocks and leading the sustainability (Subsamram et al., 2019).
Haematococcus pluvialis is one of unicellular microalgae that lives on fresh water and distributed in many habitats around the world. Haematococcus pluvialis cells are usually spherical in shape with a diameter of 30 μm. Haematococcus pluvialis is considered as the best natural source of astaxanthin and the main producing organism of this commercial product, and most of the applications of Astaxanthin are related to nutrition and human health in the form of food, pharmaceuticals, nutraceuticals, and dietary supplements (Oslan et al., 2021).
Carbon Capture and Storage (CCS) is a process of CO2 separation to a carbon storage, where CCS aims to reduce carbon emissions and help to achieve the sustainable development goals (Mikunda et al., 2021). Using pipelines, the stored CO2 gas will later be compressed and stored to the chemical industries such as methanol production, and other resources (Shaw & Mukherjee, 2022). Microalgae could be able to absorb CO2 as a nutrient and have a carbon concentration mechanism (CCM) that can maximize photosynthetic efficiency under conditions of high and low CO2 (Prasad et al., 2021). Generally, microalgae production requires well-defined conditions, but the maintenance of large-scale @The Author(s). 2022. Published by CBIORE cultures under outdoor conditions of variable irradiance, temperature, rainfall and other influences presents challenges which are not experienced in the controlled conditions of small-scale laboratory cultures (Masojidek et al., 2021). Closed systems can solve the problems that happen in open systems, because open systems are more prone to contaminants (Mustapa et al., 2020). Choosing a development system of microalgae can affect the bio-fixation process and results of microalgae biomass productivity (Merlo et al., 2021). In most cases, microalgae can be cultivated in an open system such as open pond, or using a closed system such as photobioreactor (PBR) (Chua et al., 2022). Closed photobioreactor system has more advantages because of the more controllable of the environment as well as temperature, intensity of light, and nutrient composition (Assunção and Malcata, 2020). Because of the closed system, this can reduce the possibility of contamination during the cultivation process (Lane, 2021). This research aims to knowing the effect of carbon injection against the growth rate of Haematococcus pluvialis and to knowing the effects of adding urea, NaHCO3, and Mg 2+ for the growth rate of microalgae.

Materials
This research use microalgae Haematococcus pluvialis as the main sample. The medium culture contains of RO water, 1 N NaOH, NaHCO3 solution, urea, Mg 2+ , and CO2 gas cylinders. The tools used in this study were a series of photobioreactors 120 L, UV-VIS spectrophotometer, gas analyzer, 250 ml beaker glass, 1000 ml volumetric flask, erlenmeyer, evaporating dish, pH meter, conductivity meter, DO meter, cuvette, syringe, and test tube.

Variables
For the fixed variables, we are using temperature (27-28 °C), nutritional intake for each day of cultivation (10 ml of urea per day and 20 ml of NaHCO3 per 2 days), and 8 hours of lighting duration. The dependent variable used in this research is the cultivation time is 10 days, pH of microalgae and optical density (OD) for Haematococcus pluvialis is 684 nm was measured using a UV-VIS spectrophotometer, both were measured every day. Response variable is pH and OD on each day. For independent variable, we variate the NaHCO3 as a nutrient (0; 100; and 200 ppm) and variations in Mg 2+ as a nutrient (0; 50; 75; 100; 125 and 150 ppm).

Broodstock cultivation and cultivation of Haematococcus pluvialis
Cultivation of broodstock was made in two jars with each capacity of 12 L, and the ratio of microalgae and media is 1 to 3. First, prepare a 9 L media solution that contains a mixture of 500 ppm NaHCO3 and 60 ppm urea. Next, we put 3 L of Haematococcus pluvialis microalgae into the media and perform aeration during cultivation. After that, check the OD value during cultivation until you get an OD value greater than 1. After that, prepare a cultivation medium consisting of 500 ml of RO water and mixed with mixture of 60 ppm urea, NaHCO3 and Mg 2+ (will be added according to the variables), which have been centrifuged previously. Then, add 1 L of microalgae brooders for each variable and adjust the flow rate of CO2 gas according to the variable ( Figure 1). Microalgae was cultivated for 10 days and observing optical density and pH every day during cultivation, and flowing CO2 gas every day at a variable flow rate for 120 minutes or until the pH of the microalgae solution is 7.

Microalgae Harvesting
Microalgae harvesting was carried out after microalgae had been cultivated for 10 days. Harvesting was carried out using 0.05 ppm chitosan flocculant and then allowed to stand for 1 day. After that, the microalgae precipitate was @The Author(s). 2022. Published by CBIORE filtered and centrifuged at a speed of 3500 ppm for 10 minutes and then the precipitate was taken. The centrifuged precipitate was dried in an oven at 60⁰C and checked for 30 minutes once until dry and then weighed.

Growth rate, Biomass Productivity, CO2 Fixation Rate, and Dry Biomass Measurement
Growth rate was measured following equation (1), where µ is growth rate per unit time (d -1 ), Cx is biomass density, and t is time (day). Biomass productivity was measured following equation (2), where Pxvolume is biomass productivity (g/L.day), and µ is growth rate per unit time (d -1 ). CO2 fixation rate was measured following equation (3), where PxCO2 is CO2 fixation rate (g/L.day) and Pxvolume is biomass productivity (g/L.day). While dry biomass was measured by following equation (4), where mDB is dry biomass weight (g), mED is weight of evaporating dish (g), and mWB is wet biomass weight (g).

Optical Density of Haematococcus pluvialis
Optical density measurement has a purpose in the world of microalgae cultivation, besides of obtaining the quantity of microalgae or biomass for each variable, and also used to determine microalgae concentration (Nunes et al., 2021), optical density measurement can re-validate the data obtained in order to determine more precisely growth phase (Rinawati and Prasetyo, 2020). Measurement of optical density using a UV-VIS spectrophotometer with a wavelength for the microalgae Haematococcus pluvialis is 684 nm. From the measurements, the optical density versus time curve obtained as shown in the Figure 2a-c. The main purpose of NaHCO3 and Mg 2+ in a growth medium of microalgae cultivation is for supplying nutrients that used in photosynthesis during microalgae growth. Urea as a nitrogen source, NaHCO3 as a carbon source, and Mg 2+ as a macronutrient which is an absolute essential element (Polat et al., 2020). The OD value will increase concomitantly with the increase of microalgae density, which means that every increase in the absorbance value (OD) will be followed by the number of microalgae, where the OD value also shows the biomass contained in microalgae (Nielsen & Hansen, 2019).

Growth Rate of Haematococcus pluvialis
The growth rate of microalgae was obtained by calculating the ln ratio of the biomass concentration when it reached the exponential phase to the biomass concentration at the start of cultivation divided by the time needed during the exponential phase (Pourjamshidian et al., 2019). From these calculations, the growth rate versus cultivation time curve of microalgae Haematococcus pluvialis obtained as shown in the Figure 3a-c. Based on the graph, microalgae Haematococcus pluvialis entered the exponential phase on day 2 of cultivation. Lag phase occurs the introduction of inoculum into the culture medium, delays the rate of growth because microalgae adapting to the new medium before cell division occurred. Exponential phase occurs the main process of microalgae cultivation, which means there is an increase of biomass. Structure of cells has already in the normal conditions, also the nutrients in the media and the nutrient content in the cells has been balanced. Microalgae will enter the death phase, which is indicated by a decrease in the number of cells. Death phase was indicated by the decreasing of nutrient content in the cultivation media and the decreased metabolic ability of microalgae due to age factor (Yousuf, 2020).

Biomass Productivity of Haematococcus pluvialis
The results of biomass productivity in this study were obtained by calculating the growth rate per unit time (d -1 ) multiplied by the biomass or cell density result (Lucáková et al., 2022). From these calculations, the biomass productivity versus cultivation time curve of microalgae Haematococcus pluvialis obtained as shown in the Figure 4a-c. Based on graph, the optimal biomass productivity of Haematococcus pluvialis for variable NaHCO3 0 ppm is 0.52 g/L.day in day 6 with addition of urea 60 ppm and Mg 2+ 50 ppm, for variable NaHCO3 100 ppm is 0.266 g/L.day in day 4 with addition of urea 60 ppm and Mg 2+ 150 ppm, and for variable NaHCO3 200 ppm is 0.288 g/L.day in day 5 with addition of urea 60 ppm and Mg 2+ 50 ppm. The results of biomass productivity in cultivation with higher CO2 capture are influenced by carbon concentrating mechanism factors, using CA (Carbonic Anhydrase) found in intracellular and extracellular. The function of CA is to support the photosynthetic process of carbonate compounds into biomass. Carbon units that contained in the culture medium will become saturated, and will turn into carbonate compounds when reacted with water. This carbonate compound will be converted into biomass with the help of CA (Wang and Wei, 2020).
The graph on figure above shows a negative values of biomass productivity on the variable H6, H16, and H17. This is because the OD value on day 1 is smaller than the OD value on day 0 because there are impurities in the microalgae. The decrease in OD value on day 1 was also due to the microalgae entering the lag phase where the microalgae conditions were adapting to the given nutrients. The addition of Mg 2+ ions is expected to be a growth promoter which encourages algae photosynthesis by accelerating the conversion of CO2 into bicarbonate substrate. The addition of MgAC (Magnesium aminoclay) to microalgae cultivation with a concentration of 50 ppm had optimal biomass productivity by controlling temperature, light intensity and pH 9 (Kim et al., 2020). The addition of MgAc above 50 ppm caused a decrease in biomass productivity, due to Haematococcus pluvialis could not live in high salinity concentrations. This causes a decrease in the productivity of microalgae biomass at the addition of 150 ppm Mg 2+ in the H12 variable. The nutrient NaHCO3 serves as a carbon source for microalgae during cultivation. However, when CO2 flows through microalgae, microalgae have the potential to become saturated during cultivation because they absorb too much carbon, which causes a decrease in the productivity of microalgae biomass.

CO2 Fixation Rate of Haematococcus pluvialis
The results of CO2 fixation rate in this study were obtained from the yield of biomass productivity multiplied by 1.83 (Pires da Mata Costa et al., 2021). From these calculations, the CO2 fixation rate versus cultivation time curve of microalgae Haematococcus pluvialis obtained as shown in the Figure 5a-c. When we cultivate microalgae with NaHCO3 nutrient and addition of CO2, the carbon source in microalgae biomass comes from HCOand CO2. As for the ideal carbon capture system, microalgae absorb more carbon sources from CO2 than from NaHCO3 medium (Kassim et al., 2020). Therefore, the efficiency of CO2 fixation will increase as the bicarbonate concentration decreases (Zhu et al., 2020) Based on the graph, the optimal CO2 fixation efficiency for microalgae Haematococcus pluvialis occurred in the H2 variable with variations of 60 ppm urea, 50 ppm Mg 2+ , and 0 ppm NaHCO3. While the addition of 100 ppm and 200 ppm NaHCO3 respectively is the H12 variable with variations of 60 ppm urea, 150 ppm Mg 2+ and the H14 variable with variations of 60 ppm urea, 50 ppm Mg 2+ . The results of this study concludes when the higher the concentration of Na-HCO3 in microalgae culture, the efficiency of CO2 fixation will decrease.

Correlation Between Variables and Dry Biomass of Haematococcus pluvialis
The results of dry biomass of Haematococcus pluvialis in this study are shown in the Figure 6. When we harvesting the microalgae, 0.05 grams of chitosan flocculant was added. Harvesting microalgae cells by flocculation is considered better than conventional methods such as centrifugation or filtration because it can produce better biomass in quantity (Shaikh et al., 2020). The addition of the macronutrient Mg 2+ is an absolute essential element that must be available even in small amounts (Ermis et al., 2020). Magnesium metal cation is the core of the chlorophyll molecule which is absolutely needed by microalgae to increase chlorophyll production, but can have a negative impact on microalgae growth in conditions of Mg 2+ deficiency or surplus media, so that the right concentration is needed (Liu et al., 2022). Based on the research results obtained, the optimum dry biomass production was produced by the H2 variable, namely 0.728 grams with treatment of 0 ppm NaHCO3, 60 ppm urea, and 50 ppm Mg 2+ . Optimum biomass productivity is also produced by the H2 variable. This is because the higher the biomass productivity, the higher the dry biomass produced .

Conclusion
The results of this study concludes that every increase in the absorbance value (OD) will be followed by the number of microalgae Haematococcus pluvialis. When the higher the concentration of NaHCO3 and flow rate of CO2 in microalgae culture, it will decrease the growth rate, biomass productivity, and also the efficiency of CO2 fixation. The higher @The Author(s). 2022. Published by CBIORE of biomass productivity will also produce the higher yield of dry biomass. Suggestions given for further research, the volume of microalgae should be maintained during cultivation, keeping the aerator from being too tight, double check the equipment periodically to ensure that the tool settings are in the same condition during cultivation, and control the conditions that need to be considered to produce optimum biomass.