Optimization of Cultivation Condition of Newly Isolated Strain Chlorella Sorokiniana pa.91 for CO2 Bio-Fixation and Nutrients Removal From Real Municipal Wastewater: Impact of Temperature and Light Intensity

The cultivation conditions of a newly isolated strain Chlorella sorokiniana pa.91 were optimized for the first time by performing sixty batch cultivation experiments at various temperatures (20, 25, 30 and 35 °C) and light intensities (1000, 3000, 4000, 5000 and 7000 Lux) in three different culture mediums of BG-11, real settled municipal wastewater (RMWW) and synthetic wastewater (SWW). Additionally, to evaluate the capability of C. sorokiniana pa.91 in CO 2 bio-fixation and wastewater treatment, the microalgae was cultivated in a flat-plate photobioreactor (CO 2 = 16% and 0.6 vvm aeration) under the optimal condition. The optimization results suggested that at the culture conditions of 30 °C, 4000 Lux and RMWW (COD 211 mgL -1 ) microalgae had the best performance in growth and biomass productivity. Maximum biomass concentration and productivity of 3.21 gL -1 and 0.31 gL -1 d -1 were achieved, respectively, by cultivation of C. sorokiniana pa.91 in the photobioreactor under the optimized condition. Experimental results showed that C. sorokiniana pa.91 has a high capacity of CO 2 bio-fixation (0.59 mgL -1 d -1 ) and CO 2 removal rate (35.6 %). Moreover, using C. sorokiniana pa.91 could efficiently remove 74% of NH 3 , 93% of NO 3- , 83% of PO 4-3 and 76% of COD from real municipal wastewater after eight days of cultivation in the photobioreactor. The suggested that the new specie


Introduction 1
The increasing use of water in urban area due to increased urbanization, industrialization 2 and population growth has leaded to generate large amount of municipal wastewater. The 3 discharge of untreated wastewater into the ecosystem has caused several environmental 4 problems due to its high pollutants concentrations such as nitrate, phosphate and chemical atmosphere is another global crises leads to serious environmental problems (Yin et al., 2020). 16 The total emission of CO2 has increased from 98 to 395 PPMV (parts per million volume) since 17 1990 and expected to reach 500 PPMV by 2050 if no reaction is taken (Shahid et al., 2020). 18 Several attempts have been made to reduce CO2 concentration such as carbon capture, storage 19 and utilization technologies (Nocito and Dibenedetto, 2020). 20 Meanwhile, microalgae has received great attention due to their enormous multiple 21 applications for wastewater treatment, sustainable CO2 bio-fixation and producing high-22 valuable bio-produces (Anto et al., 2020; Pugazhendhi et al., 2020). Microalgae can reduce the 23 nutrients such as nitrogen and phosphorus from wastewater by consuming them that promote 24 microalgae biomass growth rate (Watsuntorn et al., 2019). In addition to the wastewater 25 treatment, microalgae have a great potential for carbon mitigation and CO2 bio-fixation due to 26 their higher photosynthetic efficiency (40-50% more than earthy plants) (Chang et al., 2016). 27 It has been reported previously that microalgae contributed to almost 40% of global CO2 28 sequestration. For example, 1.83 kg of CO2 can be utilized by approximately 1 kg of microalgae 29 (Ng et al., 2017). Scenedesmus sp. (Xu et al., 2015). According to Kumar  needs to be optimized to achieving maximum biomass concentration at reasonable cost (Devi 54 and Parthiban, 2020). The key factors that have significantly influenced on photosynthesis 55 intensity and biomass productivity includes temperature, the light intensity, carbon dioxide and 56 pH (Bazdar et al., 2018). Among them, temperature is an important factor that affects 57 morphology and physiology of microalgae cell through changes in its metabolic rate (Qu et al.,58 2019). Therefore, finding optimal temperature for each microalgae species is vital to have a 59 best performance of growth in culture medium (Brindhadevi et al., 2021;Nogueira et al., 2015).    Table 1.

Photobioreactor experiments for CO2 bio-fixation and nutrient removal 101
After optimizing the growth conditions of Chlorella sorokiniana pa.91, the microalgae was 102 cultivated in a pilot-scale flat-plate photobioreactor (FP-PBR) at the optimum condition to 103 investigate its capacity for CO2 bio-fixation and nutrient removal from real municipal 104 wastewater. The PBR used in this study was 8 L glass flat-plate photobioreactor (340 mm 105 length×100 mm width×400 mm height), equipped with a gas sparger at the bottom of the PBR. 106 On the top of the photobioreactor, three holes were placed to measure CO2 output. Liquid 107 samples were also collected from a sample point at the side of the photobioreactor daily to 108 determined temperature, biomass concentration, pH and nutrient concentrations. The light 109 intensity was provided by white fluorescent lamp that exposed to back side of photobioreactor 110 with cycle of Light/dark 12/12h. In addition, CO2 gas (16%) was aerated by the sparger at the 111 rate of 0.6 vvm into the photobioreactor to providing mixing.
Where, is biomass productivity and and 0 are the final and initial biomass 125 concentration at time and 0 (day), respectively. removal percentage and removal rates were calculated as Eqs (3) and (4): Where, 0 and are the initial and final nutrient concentrations at day 0 and , respectively.  Lux was investigated (Fig. 1). As shown in Fig. 1a    Lux the optimal condition were obtained at 25 °C instead of 30 °C (Fig. 3) (Fig 6). showed a similar trend with that observed for ammonia. However, the final removal rate of the 313 phosphate (83%) was higher than ammonia (73%) and less than nitrate (93%) which might be 314 attributed to the initial concentration of the nutrients in the culture medium. As showed in Fig.   315 6c, the PO4 -3 concentration decreased markedly from 6.1 to less than 1 mg L -1 within 8 days of

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However, it seems that the wastewater in this study has provided sufficient sCOD to the 328 microalgae cells for metabolism process. The results suggested that using C. sorokiniana pa.91 329 for municipal wastewater could reduce COD concentration below the standard for agriculture 330 reuse water (50 COD-mgL-1) (Shoushtarian and Negahban-Azar, 2020).

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For comparison purposes, Table 2   Biomass concentration and productivity of C. sorokiniana p.91 cultivated in at-plate photobioreactor using real municipal wastewater under optimal condition of 30 ˚C temperature and 4000 Lux light intensity Figure 5 Variation of (a) CO2 removal rate and dissolved oxygen concentration, (b) CO2 bio-xation and pH variation during cultivation C. sorokiniana p.91 in at-plate photobioreactor using real municipal wastewater under optimal condition of 30 ˚C temperature and 4000 Lux light intensity