CO2 Uptake and Domestic Wastewater Treatment by Chlorella vulgaris

Various anthropogenic activities worsen climate conditions and cause water scarcity. As result, many studies have been conducted using microalgae to address these problems. Chlorella vulgaris is known to thrive in different type of environmental condition, making it beneficial for utilization. The objectives of this research were to combine the use of domestic wastewater (DWW) while at the same time inject CO2 in order to analyze C. vulgaris’ ability to perform CO2 uptake and wastewater treatment. Industrial CO2 gas cylinder was used to supply CO2 into the airlift photobioreactor (PBR) containing 40% and 60% DWW. C. vulgaris was inoculated into the biosystem and cultivated for 7 days. The results show that C. vulgaris can grow under high CO2 supply conditions and used DWW as a nutrient source. The efficiency of pollutant absorption, such as ammonia, phosphate, MBAS ranges from 82.28% to 98.91%. However, its productivity is still low, and the organic matter (BOD and COD) treatment in DWW is not yet optimal.


Introduction
The 26th Conference of the Parties (COP26), which took place in Glasgow, Scotland in 2021, marked a collective commitment by various countries to limit the global temperature increase to a maximum of 1.5 °C and achieve net zero emissions (NZE) by 2050 [1].According to Stated Policies Scenario (STEPS) projection, CO2 emissions from energy and industry sector will rise by 2 Gt in the coming decade, going from 34 Gt in 2020 to 36 Gt in 2030 which is expected to have a significant global impact, resulting in a temperature rise of 2.7 °C [2].Consequently, it will impact thermal comfort of urban areas in the future [3], disrupt agricultural ecosystems [4], lead to changes in the distribution, structure, and function of plants [5,6], a surge in infectious diseases [7], and have economic implications on Gross Domestic Product (GDP) growth [8] that can contribute to "eco-anxiety" about climate change [9].
Driven by various global economic sectors, including energy supply, industry, agriculture, forestry and land use change, transportation, and energy use in buildings, greenhouse gases (GHGs) have been continuously emitted (primarily CO2) and contributing to a global increase in temperatures [10][11][12].Therefore, it is essential to implement mitigation measures to reduce CO2 emissions [13].One effective strategy for carbon sequestration is the biological approach, by functioning microalgae as a biological 1313 (2024) 012007 IOP Publishing doi:10.1088/1755-1315/1313/1/012007 2 agent due to its high growth rate and its ability to efficiently fix carbon, surpassing plants by 10 to 50 times [14].
The cultivation of microalgae for carbon sequestration requires water as a cultivation medium.However, considering water scarcity issues, wastewater can be utilized as both a cultivation medium and wastewater treatment.Various pollutants, such as chemical oxygen demand (COD), nitrate, phosphate, sulfate, chromium, cadmium, copper, lead, and more, have been demonstrated to be effectively absorbed in wastewater sourced from diverse settings, including laboratories [15], textile [16], domestic waste [17], and industrial waste [18].
In this case, as the most studied eukaryotic algae, C. vulgaris exhibits the capability to fix CO2 while concurrently reducing pollutant concentrations in wastewater.This ability is attributed to its rapid growth, short reproduction time, and adaptability to diverse physical and chemical conditions [18].This study focuses on wastewater treatment and CO2 uptake by microalgae in domestic wastewater.The research objectives is to analyze the uptake of CO2 by C. vulgaris when cultivated using DWW sourced from the Sewon Wastewater Treatment Plant (WWTP) in Bantul.Additionally, the study aims to examine the variations in characteristics of the DWW, both before and after the cultivation period of C. vulgaris.

The cultivation of C. vulgaris
The research was conducted from February -April 2023.The DWW was obtained from a WWTP in Sewon Subdistrict, Special Region of Yogyakarta, Indonesia, while the microalgae originated from Balai Perikanan Budidaya Air Payau (BPBAP) in Situbondo, Indonesia.The cultivation process involved three variations (40%, 60%, and 100% DWW), which occupied 3 L of an airlift photobioreactor (PBR) for 7 days.Industrial CO2 gas was injected into the PBR at a rate of 0.2 L/min.The light intensity was maintained within the range of 2000 lux and the pH was kept between 7and 9 [15].

CO2 concentration measurement
To assess the efficiency of carbon uptake by the biosystem, the concentration of CO2 was measured at the gas outlet.These measurements were performed three times throughout the cultivation period, specifically on the 1st, 4th, and 7th days, using gas chromatography with a thermal conductivity detector (GC-TCD).

DWW characteristics
The characteristics of the raw DWW (100% DWW) were measured before its use.Subsequently, the characteristics of 40% and 60% DWW were also measured before and after the cultivation period.The parameters measured included total suspended solids (TSS), pH, ammonia, nitrate, phosphate, sulfate, Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), and Linear Alkylbenzene Sulfonates (LAS).All these parameters were determined at the Balai Besar Teknik Kesehatan Lingkungan dan Pengendalian Penyakit (BBTKLPP) Yogyakarta.Additionally, Water Pollution Index (WPI) analysis was conducted to assess the overall water quality, and statistical analysis using Wilcoxon test was performed to evaluate the significance of the observed changes.

CO2 concentration in the gas outlet
The concentration of CO2 in 100% DWW steadily increases, reaching 37% on the 7th day.The lower concentration of CO2 on the 1 st and 4 th days may be attributed to the activity of photosynthetic organisms in the PBR other than C. vulgaris (table 1).Ultimately, the CO2 concentration increases on the 7 th day due to the rising level of dissolved and toxic CO2, which inhibits the process of photosynthesis.The concentration of CO2 in 40% DWW initially decreases but experience a subsequent increases on the 7 th day.The initial decline in CO2 concentration in the effluent is likely a result of consumption by microalgae.However, as the density of microalgae decreases on the 7th day, the COs2 concentration in the effluent also increases.This indicates that C. vulgaris did not consume much CO2.Furthermore, the high concentration of total coliforms resulting from metabolic activities, is one of the factors contributing to the increased CO2 production within the PBR [19].Additionally, the temperature in this treatment varied from 31 to 39 °C.Elevated temperatures contribute to a reduction in CO2 solubility in water, leading to heightened concentrations in the outlet [20].In contrast, 60% DWW continued to decrease until it reaches 0% on the 7 th day.When compared to the two previous treatments, 60% DWW demonstrated the highest uptake efficiency.The low CO2 concentration in the effluent indicates that more CO2 is consumed by C. vulgaris in this treatment.
CO2 uptake by the microalgae can take place through both passive diffusion and active transport into the cell.Active transport facilitated by transporter proteins, including carbonic anhydrase.In this process, CO2 is converted into bicarbonate ions (HCO3-), facilitating the easier passage of these gases across the cytoplasmic membrane.Once inside the microalgae cell, HCO 3-is reconverted into CO2 for utilization in the photosynthesis process [21].

Changes in wastewater characteristics
Raw DWW were measured before being utilized as the growth media for C. vulgaris.The characteristics of the raw DWW can be seen in Table 2.All parameters have met the standard requirements of DWW (Minister of Environment and Forestry Regulation Num.68 of 2016).However, when compared to the water quality standards for Class 1 rivers as specified in Appendix VI of Government Regulation Num.22 of 2021 concerning Environmental Protection and Management, several parameters (BOD, COD, ammonia, and total coliform) exceed the standard.
The 100% DWW was then combined with C. vulgaris, resulting in two of variations (40% DWW and 60% DWW).Both variations exhibited changes DWW characteristics changes after 7 days of cultivation, as stated in Table 3.According to that table, TSS increased in both treatments.In field applications, Vovk et al. (2020) [22] used C. vulgaris in a WWTP system in Ukraine.The results showed TSS removal efficiencies of 93.57-96.84%at 14 and 30 days after microalgae inoculation.In this case, the TSS concentration on day 30 was lower compared to day 14.
Ammonia is readily taken up microalgae than nitrate.Similar results were shown in a study conducted by Lizzul et al. (2014) [23], which also observed a faster absorption of ammonia compared to nitrate.This is evident in the significant decrease in ammonia concentration compared to nitrogen.
The absorption efficiency of ammonia reached 91.88% (in 40% DWW) and 94.58% (in 60% DWW).Ammonia (NH4 + ) and nitrate (NO3) are inorganic compounds that serve sources of nitrogen for microalgae growth.In dissolved form in wate.These compound absorbed by microalgae for nitrogen assimilation to form amino acids, proteins, and other cellular components.Conversely, the nitrate concentration increased after the cultivation period.The rapid decrease in ammonia concentration is attributed to active transport mechanisms into the cells, where absorption and assimilation processes within microalgae are faster and require less energy [23,24].The results of this study showed that phosphate absorption efficiency in 60% DWW reached 82.28%, while it increased in the case of 40% DWW.Previous research has shown a reduction in phosphate concentration with absorption efficiencies reaching 100% after 32 days of microalgae cultivation.Li et al. (2011) [25] also reported an 89.1% decrease in phosphate concentration after 14 days of cultivation.Additionally, Mayhead et al. (2018) [26] discovered higher phosphate absorption efficiency when the wastewater was pre-filtered, reaching 97.69% after 12 days of cultivation.Hence, a longer cultivation period can enhance nutrient absorption efficiency, including inorganic phosphate, consequently leading to a further reduction in phosphate concentrations.Conversely, the increase in phosphate concentration is caused by the high cell death rate in DWW 40% at the end of the cultivation period, resulting in the conversion of organic phosphate components into inorganic phosphate.Microalgae then absorb this inorganic phosphate for the synthesis of phosphoproteins, nucleic acids, phospholipids, and ATP.
Microalgae can absorb phosphate beyond their immediate needs and store it within the cells in the form of polyphosphates [27].
In contrast to phosphate and ammonia, sulfate concentration increased drastically after the cultivation period, particularly in DWW 40%.Initially, the sulfate concentrations in DWW 40% and 60% were 16 mg/L and 24 mg/L, respectively.These values increased to 61 mg/L and 585 mg/L.A drastic increase in sulfate concentration was similarly observed in a study conducted by Ponnuswamy et al. (2013) [28].
The COD and BOD concentrations were higher in DWW 40% compared to DWW 60%.The COD concentration exceeded the BOD concentration, which align with theoretical expectations.Both values indicate that the level of organic pollution in the wastewater [29].The high COD and BOD concentrations are likely the result of microalgae decomposition.Microalgae possess the ability to transform inorganic compounds into organic matter [30][31][32].Whe microalgae cells die, they settle and decompose, contributing to these high organic concentrations.Additionally, the high organic content may be attributed to the exopolysaccharides synthesized by microalgae.The decomposition process can lead to the breakdown of complex organic compounds into inorganic forms [33,34].
In the study's design, which involves the use of DWW in the airlift PBR, medium circulation by an aerator prevents sedimentation.This allows for the detection of organic content from dead microalgae in the measurements and calculations of COD and BOD concentrations.A study by Bayu et al. (2020) [17] resulted in BOD and COD concentration reductions with efficiencies of 37.50% and 7.02%, respectively.The BOD treatment efficiency for tofu wastewater using C. vulgaris was found to be 51.4% [35], while COD can be processed at 80-90% by Chlorella sp.cultivated with wastewater [26].
C. vulgaris is capable to significantly reduce the concentration of LAS.Among all the parameters tested in this study, LAS concentration was the second highest before cultivation period.Nevertheless, these concentration can be reduced to less than 1 mg/L with treatment efficiencies of 97.99% (in 40% DWW) and 98.91% (in 60% DWW).Similar results were found in a study conducted by Serejo et al. (2020) [36], where absorption efficiencies reached 90-97%, much higher than those for nitrogen and phosphorus.Several other microalgae species, such as Scenedesmus sp., Chlamydomonas sp., Chlorococcum humicola, and Botryococcus braunii, have also showed performance above 90% in eliminating surfactants when applied to urban wastewater with a retention time of 10 days in a batch system hena [37].Detergents containing LAS.The sulfur present in LAS serves as a nutrient source for C. vulgaris growth [38].Therefore, the reduction in MBAS concentration can be attributed to detergent degradation and sulfur bioaccumulation within C. vulgaris cells.Table 4 depicts the WPI analyses, indicating a worsening in water quality post-treatment.The shift in water quality is evident, transitioning from mild pollution at 100% DWW to moderate pollution at 40% and 60% DWW.The results of the Wilcoxon test suggest no significant difference in wastewater concentrations before and after cultivating C. vulgaris (p < 0.005) for both 40% and 60% DWW.This implies that the overall research design has not achieved efficient wastewater treatment.

Conclusion
C. vulgaris can survive under conditions of high CO2 supply and can utilize DWW as its nutrient source.The pollutant absorption efficiency was high, ranging from 82.28-98.91%for several parameters, such as ammonia, phosphate, and LAS.Nevertheless, organic removal, such as BOD and COD along with some nutrients was no yet optimal.Thus, several processing stages are still necessary to meet the required quality standards in field applications.Nevertheless, in WWTP, microalgae C. vulgaris can be considered for used as pollutants remover.

Table 1 .
Concentration of CO2 inlet, outlet, and absorption efficiency

Table 2 .
Raw domestic wastewater characteristics

Table 3 .
Changes in water quality in DWW 40% and 60%

Table 4 .
Changes in water quality in 40 and 60% DWW