Simultaneous Biological Nutrient Removal from Municipal Wastewater and CO2-bio xation using Chlorella kessleri

Mohammed Omar Faruque Chemical Engineering Department, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia Kazeem Ayodeji Mohammed Chemical Engineering Department, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia Mohammad Mozahar Hossain Chemical Engineering Department, King Fahd University of Petroleum and Minerals, Dhahran, Saudi Arabia Shaikh Abdur Razzak (  srazzak@kfupm.edu.sa ) King Fahd University of Petroleum & Minerals https://orcid.org/0000-0003-4316-7882


Introduction
Rapid industrialization and urbanization are generating enormous quantities of wastewater that must be treated before safe disposal. The wastewater (WW) associated with over population and industrialization are the major wastes, which pose an immense challenge to ensuring environmental sustainability around the world (Eze et al. 2018). The presence of excessive nutrients, such as nitrogen and phosphorous in the wastewater, can lead to the eutrophication in receiving streams and disturb the stability of the ecosystem (Cai et al. 2013). Hence, the removal of nutrients such as nitrogen and phosphorous from tertiary wastewater is vital to reduce the oxygen requirement of the receiving streams, save aquatic life, and prevent eutrophication in lakes and streams to protect human health. At present, a broad range of methods, including ltration, membrane technology, precipitation, the advanced oxidation process (AOP), and biological nutrient removal (BNR) using activated sludge are available for the removal of nutrients from tertiary wastewater. However, these techniques generally entail higher energy and maintenance costs, hazards associated with the disposal of chemicals, and the production of a large volume of waste sludge (Rajasulochana and Preethy 2016). In addition, the inability to eliminate nitrogen (N) and phosphorus (P) from tertiary wastewater simultaneously during the treatment process is a major drawback of these processes (Arbib et al. 2014).
Several studies have con rmed that microalgae can remove nitrogen and phosphorous during the treatment of tertiary municipal wastewater, which have gained much interest in recent years (Xin et (Olguín 2003). As the absence of one nutrient inhibits the removal of the other and vice versa, the nutrients N and P must be simultaneously present in culture media for microalgae growth. The concentration of nitrogen and phosphorous in wastewater varies, with nitrogen concentration varying between 15 and 90 ppm and phosphorous concentration varying between 4 and 20 ppm in municipal wastewater (Beuckels et al. 2015). As the main route of removal of nutrients by microalgae is through their uptake during growth, the microalgal growth rate directly in uences the rate of removal of nutrients. Also, nitrogen and phosphorous can be concurrently consumed and removed effectively only if the nitrogen to phosphorous (NP) ratio of wastewater is in an appropriate range (Xin et al. 2010b).
Microalgae cultivation in wastewater through photosynthesis can overcome the aforesaid problems and offers many advantages, including the following: i) adds an economic value to tertiary wastewater e uents in terms of water and nutrient recovery (Arbib et al. 2014 However, to the best of our understandings, a detailed study of the removal of nutrients by the microalgae Chlorella kessleri sp. as a function of the nitrogen to phosphorous ratio and the bio-xation of CO 2 under photoautotrophic condition s has not been conducted.
Finding the most appropriate nutrient ratio for the growth of microalgae is vital for the effective coupling of advanced wastewater treatment with bio-xation of CO 2 . Hence, this study was conducted using a set of synthetic tertiary municipal wastewater samples based on the modi ed BBM with varying NP ratios. In this study, Chlorella kessleri sp. was cultivated in modi ed BBM to evaluate the growth, nutrient uptake, and CO 2 bio-xation at different nutrient ratios.

Microalgae strain
The experiments were conducted using the microalgae strain Chlorella Kessleri (UTEX 2229) found from the University of Texas, Austin, USA.

Culture medium
The modi ed BBM (MBBM) was utilized as the medium of microalgae growth in the experiments. A set of synthetic wastewater media was prepared with varying nitrogen to phosphorous (NP) ratios, with the purpose of analyzing the removal of nutrients by growing microalgae in them to apply the results for the treatment of municipal tertiary wastewater. Nitrate (NO 3 -N) and phosphate (PO 4 3− -P) were utilized as the nitrogen and phosphorus supplies, correspondingly. Besides, nitrogen and phosphorus, the cultivation medium consisted of the following: 25

Experimental procedure and design
Chlorella Kessleri was cultivated in batch photobioreactors (2 L Erlenmeyer asks) and 1500 mL of the working liquid medium was added to each photobioreactor. The pre-cultured microalgae sp. was used to avoid the lag phase affecting the results. The asks were capped with foam stoppers to protect the content from contamination. The photobioreactors were placed in a fume hood under continuous illumination with four perpendicularly oriented Grolux uorescence (Sylvania F18W/T8/GRO) tube lights to cover a maximum area of the photobioreactor surfaces. The light intensity recorded at the reactor surface was in the range of 62-74 µmol/m 2 /s, applying a Fisher Scienti c™ Traceable™ Dual-Display Light Meter. Figure 1 depicts a schematic diagram of the experimental set-up employed in this experiment.
The air-CO 2 mixture was delivered to the bottom side of the photobioreactor precisely with the help of an air-CO 2 mixer device FC-SH (Live Cell Instruments, South Korea). Gas bubbles rise through the liquid medium and leave at the top side of the reactor, ensuring a well-mixed culture medium, thereby preventing cell sedimentation, and providing inorganic carbon. During photosynthesis, algae utilize carbon dioxide as the carbon source for cell growth. Chlorella kessleri is a photosynthetic microorganism with the capacity to grow in aqueous suspensions consuming nutrients from wastewaters, while simultaneously combining water and CO 2 photosynthetically to produce value added biomass.
The mechanism of conversion of CO 2 to biomass has been well explained by Sorensen et al. During the experiments, the temperature of the media was monitored daily applying a temperature sensor (Fisher Scienti c™ Thermometers with a Stainless Steel Probe on Cable) in the Celcius scale and maintained at 25 ± 2 C. In addition, the pH of the culture media was observed daily utilizing a pH meter (Fisher Scienti c Accumet R Basic AB15 plus Meter) and recorded in the range of 6.7 to 7.9 in all experimental runs.

Effect of the nitrogen to phosphorus (NP) ratios
Chlorella kessleri was cultured in media prepared with varying NP ratios to examine how the concentration of nitrogen and phosphorus in uences the microalgae growth, the extent of nutrient The speci c growth rate, µ g , described as the growth of the dry biomass weight per day, was computed applying the subsequent equation (Das et al.2011;Abreu et al.2012 where, X 1 and X 2 is the dry biomass weight at time t 1 and t 2 of the exponential growth phase.

Biomass productivity
The biomass productivity (P b ), which is also described as the rate of biomass production, was computed where C carbon carbon content of the microalgae species Chlorella Kessleri, M CO2 is the molecular weight of CO 2 ,M c is the atomic weight of carbon, and P B is the productivity of biomass.

Biomass concentration and the speci c growth rate at different NP ratios
Healthy growth of microalgae determines the e ciency of the treatment process. Understanding and controlling the essential factors that directly impact microalgae growth is important to maintain a healthy growth of microalgae. Several environmental issues in uence the rate of nutrient consumption by various species of microalgae. Together with other environmental issues, nutrient concentration and the NP ratios have a direct impact on the removal of nutrients, ultimately in uencing the microalgal growth (Cai et al. 2013). Figure 2 depicts the time pro les of the biomass concentration and the speci c growth rate of Chlorella kessleri cultivated at various NP ratios at a temperature of 25 C and a CO 2 concentration of 2% in air supplied to the culture media. Different NP ratios have a signi cant impact on the microalgae growth, which is con rmed by the biomass concentration and the speci c growth rate depicted in Fig. 2. The biomass concentration increases at all investigated NP ratios of 2:1, 4:1, and 6:1 during the cultivation period of 13 days. However, the maximum biomass concentration of 606.79 mg/L was gained for the NP ratio of 2:1 at the end of the cultivation period. After reaching its highest value at each NP ratio in the rst few days, the speci c growth rate gradually decreases during the cultivation period. The speci c growth rate at the NP ratio of 2:1 is higher than other two NP ratios (4:1 and 6:1) over the entire cultivation period, with a maximum speci c growth rate of 1.74 d − 1 attained on day 1.

Kinetic model based on a single substrate factor for microalgae growth
At constant temperature, light intensity, and homogeneous mixing, the substrate concentration and nutrient accessibility normally determines the rate of biomass accumulation during microalgae growth. The Monod model, as expressed in Eq. (4), is usually applied to analysis the microalgae growth restricted by a single nutrient (in this study nitrogen). This model has been used to predict and control algae cultivations in wastewater media. Figure 3 (a, b, and c) shows the experimentally determined speci c growth rate and its Monod model predictions plotted as a function of the available nutrient (nitrogen) concentration in media for the cultivation of Chlorella kessleri at NP ratios of 2:1, 4:1, and 6:1 and a 2% concentration of CO 2 in the supplied air. The microalgae kinetic model was standardized utilizing experimental Chlorella kessleri sp. cultivation results to establish the maximum speci c growth rate (µ m ) and half saturation constant (k s ). The kinetic model was validated utilizing supplementary experimental data for the cultivation of Chlorella kessleri in wastewater media.

Effect of the NP ratio on the inorganic nutrient removal
Page 9/20 The nitrogen to phosphorous (NP) ratio has been found to in uence the removal of nutrients from wastewater. In this study, the synthetic wastewater media with different NP ratios were prepared using a constant total nitrogen (TN) concentration and varying the total phosphorous (TP) concentration. Figure 4a shows the time pro les of the total nitrogen (TN) concentration and the percent total nitrogen (TN) removal by Chlorella kessleri cultivated under different NP ratios at a temperature of 25 C and a concentration of 2% CO 2 in the supplied air. Corresponding time pro les of the total phosphorus (TP) concentration and the percent total phosphorus (TP) removal are depicted in Fig. 4b. In general, the concentration of total nitrogen and total phosphorous decreases steadily during the cultivation period (13 days) at all evaluated NP ratios due to the consumption of N and P nutrients needed for the microalgae growth. The corresponding removal e ciency of nitrogen and phosphorous at the end of the 13-day cultivation period is shown in Table 1. The removal e ciency of nitrogen is more than 95% for all NP ratios evaluated except 8:1, at which the e ciency is only 72.4%. The lower nitrogen removal e ciency at an NP ratio of 8:1 may be due to the unavailability of the required amount of phosphorous in the culture media (Xin et al. 2010b). On the other hand, the NP ratio has a large effect on the removal e ciency of phosphorous. The maximum phosphorous removal e ciency at the NP ratio of 6:1 is about 97%.
However, the maximum removal e ciency of phosphorous at the NP ratio of 2:1is about 20%, which is the lowest among the NP ratios evaluated. The signi cant variation of the removal e ciency of phosphorous at different NP ratios may be due the variation of the initial concentration of phosphorous and the limited availability of nitrogen in the culture media (Beuckels et al. 2015).

Carbon content and biomass concentration under different NP ratios
The time-course pro les of the carbon content and biomass concentration of Chlorella kessleri cultivated under different nutrient levels at a temperature of 25 C and a concentration of 2% of CO 2 in the supplied air are presented in Fig. 5. The carbon content of the microalgae increases in the rst ve days of the cultivation period and starts decreasing after reaching a maximum at all NP ratios. On the other hand, the biomass concentration steadily increases during the entire cultivation period at all NP ratios. The increased percentage of carbon content in microalgae during the early stages of the cultivation period may be due to the uptake of high C-rich metabolites during cultivation (Beuckels et al. 2015). The percent carbon content of microalgae reaches the highest value of 56% at the NP ratio of 2:1.

Effect of the NP ratio on the CO 2 bio-xation rate (R CO2 )
A lot of research and development studies is carried out around the world on a variety of methods to mitigate the effects of CO 2 on the environment. Among them, using microalgal photosynthesis for biological CO 2 xation has been found to be an e cient method (Shaikh A. Razzak  bio-xation rate when Chlorella kessleri is cultured in a modi ed Bristol medium. However, in this study, the bio-xation rate of CO 2 by Chlorella kessleri at different NP ratios was evaluated in synthetic wastewater media. Figure 6 shows the time-course pro les of carbon dioxide bio-xation by Chlorella kessleri cultivated under different nutrient levels at a temperature of 25 C and with 2% CO 2 in the supplied air. The rate of bio-xation of CO 2 increases in the rst 7 days of the cultivation period at all NP ratios, reaching its highest value before gradually decreasing during the remainder of the cultivation period. The maximum CO 2 bio-xation rate of 89.36 mg/L/d during the 7 days of cultivation period was obtained at an NP ratio of 6:1 and a concentration of 2% CO 2 in the air supplied to the culture media.

Comparison analysis
The bar chart in Fig. 7 depicts the overall productivity of the biomass (PB), rate of bio-xation of CO 2 (R CO2 ), and TN and TP removal by Chlorella kessleri under different NP ratios (2:1 to 6:1) in a cultivation period of 13 days at a temperature of 25 C and a concentration of 2% CO 2 in the supplied air. PB undergoes a minor variation when the NP ratio increased from 2:1 to 6:1, with the maximum PB observed at an NP ratio of 2:1. The rate of bio-xation of CO 2 (R CO2 ) also undergoes insigni cant changes when cultivated at NP ratios ranging from 2:1 to 6:1. The removal of TN is more than 95% at all NP ratios, whereas the removal of TP signi cantly increases when the NP ratio is varied from 2:1 through 4:1 to 6:1.

Conclusion
The growth of the microalgae Chlorella kessleri sp. was evaluated in synthetic wastewater under different photoautotrophic conditions for the simultaneous CO 2 bio-xation and the removal of nitrogen and phosphorous. The synthetic wastewater is considered as equivalent to municipal tertiary wastewater for cultivating the microalgae sp. under different NP ratios and a concentration of 2% CO 2 in the supplied air.
The maximum speci c growth rate (µ m ) of Chlorella kessleri sp. and the concentration of the nutrients are in accordance with the Monod model. In this study, the NP ratio was maintained in a suitable range (2:1 to 6:1) to increase the removal e ciency of nitrogen by Chlorella kessleri sp. However, the removal e ciency of phosphorous is signi cantly affected by the variation of the NP ratio. According to the results of this study, the following conclusions can be drawn: 1. Biomass concentration increased at all studied NP ratios and the maximum biomass concentration of 606.79 mg/L was found at an NP ratio of 2:1. 2. The NP ratio and initial nutrient concentration are substantial factors affecting the e ciency of the removal of nutrients in wastewater. More than 95% of nitrogen is removed for at all evaluated NP ratios except 8:1, at which the percent removal is only 72.4%. On the other hand, the maximum 97% phosphorous removal e ciency is observed at an NP ratio of 6:1. 3. For all NP ratios, the bio-xation rate of CO 2 gradually increases over the rst seven days of the cultivation period and the maximum bio-xation rate of CO 2 of 89.36 mgL − 1 d − 1 is observed at an NP ratio of 6:1.
In terms of CO 2 bio-xation rates as well as both TN and TP removal e ciency, Chlorella kessleri has the potential to be used for low-cost tertiary municipal wastewater treatment.

Declarations
Ethics approval and consent to participate   The time-course pro les of the biomass concentration ( lled markers) and speci c growth rate (empty markers) of Chlorella kessleri cultivated under different NP ratios at a temperature of 25 C and a CO2 concentration of 2% in the supplied air.  The time-course pro les of (a) total nitrogen (TN) concentration ( lled markers) and total nitrogen (TN) removal (empty markers), (b) total phosphorus (TP) concentration ( lled markers) and total phosphorus(TP) removal (empty markers) during cultivation of Chlorella kessleri under different NP ratios at a temperature of 25 C and a CO2 concentration of 2% in the supplied air. The time-course pro les of the capacity of bio-xation of carbon dioxide during the cultivation of Chlorella kessleri under different NP ratios at a temperature of 25 C and a CO2 concentration of 2% in the supplied air. Biomass productivity (PB), rate of CO2 bio-xation (RCO2), and total nitrogen (TN) and total phosphorus (TP) removal using Chlorella kessleri cultivated under different NP ratios at a temperature of 25 C and a CO2 concentration of 2% in the supplied air.

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