CO₂-Inorganic Carbon Auto-Buffering System for Efficient Ammonium Reclamation Coupled with Valuable Biomass Production in a Euryhaline Microalga Tetraselmis subcordiformis

Abstract

The performance of microalgae-based wastewater treatment processes for ammonium-N (NH₄⁺-N) removal depends on the maintenance of a favorable pH that is critical for minimizing nitrogen escape in the form of free ammonia (NH₃) and preventing high-NH₃ or extreme-pH stress. This study developed a CO₂-inorganic carbon (CO₂-IC) buffering system that automatically stabilized pH with the supply of a carbon source for efficient photosynthetic reclamation of NH₄⁺-N by a euryhaline microalga Tetraselmis subcordiformis. The soluble (NaHCO₃) and insoluble (CaCO₃ and MgCO₃) ICs were compared for this purpose. The pH was well controlled in the range of 6.5~8.5 in the CO₂-IC system, which was suitable for the photosynthetic growth of T. subcordiformis. The NH₄⁺-N (100 mg/L) was almost completely removed in three days, with the maximum removal rate of 60.13 mg N/L/day and minimal N escape of 19.65% obtained in the CO₂-NaHCO₃ system. The CO₂-IC system also restricted the release of extracellular organic matter by preventing stress conditions. The CO₂-NaHCO₃ system enabled the highest “normal” starch production suitable for fermentation, while the CO₂-CaCO₃/MgCO₃ system facilitated high-amylose starch accumulation that was conducive to producing bio-based materials and health-promoting ingredients. The proteins accumulated in T. subcordiformis were of good quality for animal feeds.

1. Introduction

With the acceleration of urbanization, the demand for metropolitan wastewater treatment is increasing [1]; at the same time, the reclamation of nutrients (mainly nitrogen, phosphorus, and carbon) from the wastewater is also crucial for the development of a sustainable life cycle following the circular economy principle [2,3]. Urban wastewater usually contains moderate amounts of ammonium-nitrogen (NH₄⁺-N, 27~100 mg/L) as the dominant nitrogen form [4,5,6,7], which has to be removed before discharging for the prevention of eutrophication to the water ecosystem. The current urban wastewater treatment techniques in the wastewater treatment plants (WWTPs) in China mainly include conventional activated sludge treatment, anaerobic-anoxic-oxic (A²/O), anaerobic-oxic (A/O), sequencing batch reactor (SBR), oxidation ditch, etc., which are efficient for COD removal yet have limited removal capacity for nitrogen and phosphorus [6]. In addition, the excess waste sludge discharge and substantial greenhouse gas (mainly CO₂ and N₂O) emission during the treatment remain a big challenge to meet sustainability standards [8].

Microalgae-based wastewater treatment processes have recently attracted increased attention because of their considerable benefits over traditional techniques, including highly efficient nutrient removal to a very low level with or without limited extra nutrient supplementation, reduction of greenhouse gas emission with the CO₂ fixation, and nutrient reclamation for generating value-added products [9]. As the main nitrogen source in the urban wastewater, NH₄⁺-N can be assimilated and converted to valuable proteins by microalgae, but the efficiency is highly dependent on the maintenance of a favorable environment for the microalgae [7]. The most critical challenge is the pH decrease during the NH₄⁺ assimilation owing to the excretion of H⁺, which causes the diminished photosynthesis and deteriorates the removal [10,11]. To stabilize the pH, organic buffering agents (e.g., Tricine, TAPS) or on-site pH monitoring/adjustment systems are usually applied, which can introduce extra CODs and increase operation facilities and costs [12,13,14]. The addition of alkaline inorganic bicarbonate (mainly NaHCO₃) is an alternative strategy to abate the acidification of the NH₄⁺ removal process by simultaneously providing a carbon source for microalgae to meet the proper C/N ratio in the wastewater treatment [12,15]. However, the utilization of bicarbonate by microalgae tends to release OH^- that exceeds the H⁺ excretion caused by the NH₄⁺ assimilation because of the inherent high C/N demand (100/14), leading to the alkalization of the water with pH > 9 [5]. A high pH, under which NH₄⁺ is prone to be converted to NH₃, is detrimental to the NH₄⁺ assimilation by microalgae because of the ammonia inhibition and probably the extreme pH itself [7]. In addition, NH₃ under a high pH is readily volatilized, resulting in a low bioconversion efficiency and potential air pollution [16]. CO₂ injection has been proposed to help control the pH variations and improve the treatment performance [5,6], but to what extent the application of CO₂ contributes to the reduction of NH₃ escape is largely unknown.

Aside from the soluble bicarbonate NaHCO₃, insoluble carbonates such as CaCO₃ have been shown to efficiently regulate pH for microalgae cultivated in NH₄⁺-rich wastewaters [17,18]. The insoluble nature of CaCO₃ restricted the unnecessary increase in pH [17]. However, since Ca²⁺ released into the water with the NH₄⁺ removal process can be inhibitory to microalgae [19], and the insoluble particles can shelter the microalgae from being exposed to light that is crucial for photosynthesis, the ultimate performance of the insoluble carbonate needs to be evaluated for comparison with traditional NaHCO₃.

This study aimed to establish a CO₂-inorganic carbon (CO₂-IC) buffering system to automatically stabilize the pH with the supply of a carbon source for efficient photosynthetic removal and reclamation of NH₄⁺-N by a euryhaline microalgal strain Tetraselmis subcordiformis. This strain is a euryhaline green microalga capable of acclimating to a wide salinity range (5.4~67.5 g/L NaCl) [20] and is assumed to be resistant to the fluctuations of salinity in urban wastewaters, especially those originated from coastal or island areas where seawater is used for toilet flushing or the intrusion of seawater into the wastewater often occurs [21]. The genus Tetraselmis has been reported to tolerate high NH₄⁺-N and have an excellent capability of apparent NH₄⁺-N removal from varied types of wastewater, including urban wastewater [4,13,22,23], but the processes were generally run without pH control (

Table 1. Photoautotrophic removal of NH₄⁺-N by microalgae with different inorganic carbon sources and CO₂ supplies in synthetic NH₄⁺-N-containing wastewater.

Algae Strain	Water Source	NH4+-N (mg/L)	Carbon Source	Air + CO2	NH4+-N Removal Efficiency (%) (Day)	Maximum NH4+-N Removal Capacity (mg N/L/Day)	NH3 Escaped (%)	Extra pH Control	Reference
Chlorella sp. L38	Freshwater	248	NH4HCO3	Air	80 (24)	8.67	NA a	No	[25]
		370	NH4HCO3	Air	55 (27)	10	19 b	Acid/base	[26]
		280	NH4HCO3	Air	45 (15)	8.31	21.43	Acid/base	[27]
		248	NH4HCO3	Air	44 (27)	12	24.2 b	Acid/base	[28]
Chlorella sp. L166	Freshwater	247	NH4HCO3	Air	87 (36)	13	76	No	[29]
		247	NH4HCO3	Air + 5%	52 (18)	10	42	No
		247	NH4HCO3	Air	84 (36)	9	73 b	No
Chlorella vulgaris	Freshwater	120	NH4HCO3	Air + 1%	100 (3)	53.4	NA	Acid/base	[30]
Chlorella strains	Freshwater	50	Na2CO3	Air	100 (4)	12.5	NA	No	[11]
Haematococcus pluvialis QLD	Freshwater	63	NaHCO3	Air + 1%	95.6 (5)	15.75	NA	No	[15]
Desmodesmus sp. F51	Freshwater	60.2	NaHCO3	Air + 2.5%	100 (1.1)	55.2	NA	No	[12]
		60.2	No	Air + 2.5%	100 (1.3)	50.9	NA	Acid/base
		60.2	NaHCO3	Air	25.25 (2)	20	NA	No
Botryococcus braunii	Freshwater	83.15	CaCO3	Air	68.55 (20)	2.85	NA	No	[17]
Arthrospira platensis	Semi-seawater	100	NaHCO3	Air	100 (2)	50	23~40	No	[31]
Tetraselmis chui	Seawater	13	NaHCO3	Air	73 (1)	9.5	NA	No	[32]
Nannochloropsis oculata	Seawater	13	NaHCO3	Air	32 (1)	4.2	NA	No	[32]
Tetraselmis sp.	Seawater	3.8	No	Air	100 (1)	3.8 c	NA	No	[33]
Tetraselmis subcordiformis	Seawater	100	NaHCO3	Air	99.6 (4)	49.27	48.60	No	This study
		100	MgCO3	Air	99.6 (4)	44.00	43.70	No
		100	CaCO3	Air	99.3 (4)	31.69	38.93	No
		100	NaHCO3	Air + 2%	99.5 (3)	60.13	19.65	No
		100	MgCO3	Air + 2%	99.5 (2)	54.38	20.33	No
		100	CaCO3	Air + 2%	99.8 (3)	45.95	26.86	No

Notes: ^a Not available. ^b Fed-batch mode for NH₄⁺-N supply. ^c Immobilized algae cells.

), or with a pH stat system or manual adjustment by acid/base, or buffered with organic agents (such as Tricine) [13,23,24]. The proportion of NH₄⁺-N that was incorporated into the microalgae biomass or escaped from the culture system, which was the essential index to assess the NH₄⁺-N bioconversion efficiency, remained undetermined in these processes. The present study compared different ICs, namely, soluble NaHCO₃ and insoluble CaCO₃ and MgCO₃, under both the IC and CO₂-IC systems in terms of the pH control, photosynthesis, cell growth, NH₄⁺-N removal and reclamation efficiency, and organic matter release to comprehensively illustrate the potential of T. subcordiformis used for wastewater treatment. In addition, the biomass production ability and the quality of the main components accumulated (starch and protein) were assessed within an algal biorefinery concept.

2. Materials and Methods

2.1. Algal Strain and Culture Conditions

Tetraselmis subcordiformis FACHB-1751, obtained from the Marine Bioengineering Group of the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, was previously cultured in artificial seawater (ASW, containing 27 g/L NaCl) [34] with the addition of 0.81 g L⁻¹ Tris and 0.33 mL L⁻¹ glacial acetic acid. Algae cells were collected in the late exponential phase and washed twice with nitrogen-free artificial seawater (ASW-N) to remove nitrate and organic carbon.

The washed cells were inoculated into a synthetic wastewater with a formula comprising the ASW-N medium containing 100 mg L⁻¹ of NH₄⁺-N provided as NH₄Cl, the concentration of which represented the common upper limit of the NH₄⁺-N in urban wastewaters [7]. The algal biomass concentration was adjusted to ensure an inoculation density of 0.9 g/L. In order to stabilize the pH in the process of NH₄⁺-N removal by the microalgae, 2% CO₂-rich air was injected into the culture at the rate of 0.4 vvm, with different inorganic carbons (NaHCO₃, MgCO₃, or CaCO₃) added with a carbon concentration of 12 mM (the minimal concentration of inorganic carbon salts required for maintaining favorable pH during the removal, data not shown), which constituted the auto-buffering CO₂-IC system. For reference, the 2% CO₂ was omitted, forming the IC system. As a negative control, both the 2% CO₂ and ICs were deprived from the cultures.

Microalgae cells were cultured in a cylindrical glass bubble photobioreactor (50 mm diameter, 400 mm height) with a working volume of 500 mL, as described by Yao et al. [35] at 25 ± 2 °C. A cold white fluorescent lamp was used to illuminate continuously from one side, providing an incident photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹.

2.2. pH, Growth Measurement and Biochemical Component Analysis

The pH was measured using a standard benchtop pH meter (ARK, pHS-4C+, Sichuan, China). MgCO₃ and CaCO₃ are insoluble particles in the culture, which interfered with the determination of biomass as cell dry weight. Therefore, the growth of microalgae was estimated by the increase in total main organic matter in the cultures (mg/L), calculated as the sum of volumetric concentrations of protein, carbohydrate, and lipid.

The total protein was extracted with 0.5 M NaOH at 80 °C for 10 min [36] and measured following a BCA method (BCA Protein Assay Kit, Beyotime, Nantong, China). The total carbohydrate content was determined with a sulfuric acid-anthrone method according to Yao et al. [37]. The total lipid content was analyzed with a sulpho-phospho-vanillin (SPV) colorimetric method as described in [38].

The starch in the microalgal biomass was extracted with 45% perchloric acid and stained with I₂-KI solution (1:2, v/v) at 25 °C for 15 min followed by a spectrophotometry analysis under 618 and 550 nm according to the previous study [39], which allowed the simultaneous determination of amylose (Am) and amylopectin (Ap) concentrations [40]. The total starch was estimated as the sum of amylose and amylopectin.

2.3. Photosynthetic Performance Analysis

The photosynthetic performance of the microalgae was measured as the maximum quantum yield of photosystem II with a chlorophyll fluorometer Os30p+ (Opti-sciences, Hudson, NH, USA) after dark adaption for 10 min [34]. The parameter expressed as F_v/F_m was calculated as described by Strasserf and Srivastava [41]:

F_v/F_m = (F_m − F₀)/F_m

(1)

where F_v represents the variation of chlorophyll fluorescence between maximal fluorescence (F_m) induced by saturating pulse and initial fluorescence (F₀).

2.4. Ammonium-Nitrogen (NH₄⁺-N) Analysis

The concentration of NH₄⁺-N in the culture system was determined by indophenol blue colorimetry [42] after proper dilution. The removal rate of NH₄⁺-N (R_N, mg N/L/day) was calculated as follows:

R_N = (N₀ − N_t)/t

(2)

where N₀ and N_t are the concentrations of NH₄⁺-N at culture times 0 and t, respectively.

2.5. Nitrogen Distribution and Total Organic Carbon (TOC) Analysis

In order to evaluate the proportion of N assimilated into the biomass (Biomass-N, %) or escaped from the culture system (Escaped-N, %), the total nitrogen (TN) in the water phase and the nitrogen element in the biomass phase were measured with a TN analyzer (TOC-L CPH/CPN, Shimadzu, Tokyo, Japan) and an elemental analyzer (Elemental Vario EL Cube, Hanau, Germany), respectively.

The Biomass-N (%) and Escaped-N (%) were estimated using the following equations:

Biomass-N (%) = TN_bimass(t)/TN₍₀₎ × 100

(3)

Escaped-N (%) = (TN₍₀₎ − TN_water(t) − TN_bimass(t))/TN₍₀₎ × 100

(4)

TN₍₀₎ = TN_bimass(0) + TN_water(0)

(5)

TN_bimass = ω_N × C_biomass

(6)

where TN_{bimass (t)/(0)} (mg/L) is the total N element in the biomass at time t/0 (day), TN_{water (t)/(0)} (mg/L) is the total N concentration in the water at time t/0 (day), TN₍₀₎ represents the initial total N (mg/L) in the culture system, ω_N (w/w) is the N content in biomass, and C_biomass (mg/L) is the apparent biomass concentration determined gravimetrically according to the previous study [39].

The total organic carbon (TOC) in the water was determined with a TOC analyzer (TOC-L CPH/CPN, Shimadzu, Japan). The carbohydrate concentration in the water was measured with the phenol–sulfuric acid method [43], and protein concentration was assayed following a BCA method (BCA Protein Assay Kit, Beyotime, China).

2.6. Amino Acid Analysis

The freeze-dried microalgae biomass was used for amino acid analysis according to the method described previously [44]. The relative proportion of each amino acid (AA) was expressed as g AA per 100 g of total AA. The essential amino acid index (EAAI) was calculated according to the following equation [45]:

$$EAAI=\sqrt[n]{\frac{a{a}_1\times a{a}_2\times \dots \times a{a}_n}{A{A}_1\times A{A}_2\times \dots \times A{A}_n}}$$

(7)

where aa_n represents the percentage of one kind of essential amino acid in the total essential amino acids in the sample; AA_n represents the percentage of one kind of essential amino acid in the total essential amino acids in reference samples (Penaeus monodon juvenile and ideal protein for dairy cow, respectively).

2.7. Statistical Analysis

All experiments were performed in duplicate, and SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. Two group comparisons were performed using a two-tailed distribution Student’s paired t test. Values of p < 0.05 were defined as statistically significant.

3. Results and Discussion

3.1. pH Variation, Photosynthetic Performance, and Cell Growth

Photosynthesis is indispensable for photoautotrophic removal of NH₄⁺-N in microalgae, which is affected by carbon availability and pH conditions surrounding the algal cells [11]. In order to verify whether the ICs could provide sufficient carbon and enable a suitable pH to sustain photosynthesis for algal cell growth, 12 mM of NaHCO₃, MgCO₃, or CaCO₃ were applied to the culture system with synthetic 100 mg/L NH₄⁺-N; simultaneously, the culture with no IC supply was set as a negative control. As shown in Figure 1a, the pH in the control group without the IC supply dropped sharply from 6.56 to 3.56 within three days, which was typically observed in the microalgae cultivation using ammonium as the sole nitrogen source owing to the release of H⁺ by algal cells after the assimilation of NH₄⁺-N [12]. In contrast, the supply of ICs led to a considerable increase in pH up to 9.0~9.6 in the first two days, which was indicative of the assimilation of soluble bicarbonate by the microalgae that generated hydroxyl ions in the medium. The increase in pH was previously reported in the freshwater microalgae Desmodesmus sp. F51 and Haematococcus pluvialis QLD adding NaHCO₃ as the inorganic carbon source for NH₄⁺-N utilization [12,15]. Here, it was similar in the marine microalga Tetraselmis subcordiformis. Because of the high C/N ratio in microalgae (CO_0.48H_1.83N_0.11P_0.01) [46], the bicarbonate consumption rate could be much higher than the NH₄⁺-N; therefore, the alkalization from the former exceeded the acidification in the latter process, causing an elevated pH. For the insoluble IC (MgCO₃ or CaCO₃) groups, the increased pH also suggested the partial solubilization of carbonate into the medium in the form of soluble bicarbonate as a carbon source for Tetraselmis subcordiformis. Similarly, the leach of Ca²⁺ into the medium was recorded in the Botryococcus braunii culture with CaCO₃ addition for the photoautotrophic NH₄⁺-N removal [17]. The release of H⁺ during the NH₄⁺-N assimilation could be the trigger for this solubilization. Accordingly, the photosynthetic performance revealed by the maximum quantum yield of photosystem II (F_v/F_m) decreased sharply (from 0.73 to 0.29) within four days in the control group without IC supply, while the groups with IC addition maintained above 0.6 (Figure 1c), which indicated the alleviation of the low-pH stress caused by the H⁺ release from the NH₄⁺-N assimilation. Consequently, the biomass accumulation as assessed by the sum of the main organic matter (proteins, carbohydrates, and lipids) also showed significant improvement in the IC groups compared with the control group, with the former reaching 1.0~1.2 g/L against the latter being only 0.30 g/L (Figure 1e). These results suggested that the bicarbonate and insoluble carbonate could be used as a pH regulator as well as a carbon source for NH₄⁺-N removal and biomass production.

It should be noted that the pH in the groups with IC addition fluctuated during the cultivation, and a high pH of up to 9.5 was reached, which could be unfavorable for the photosynthetic growth and NH₄⁺-N removal in Tetraselmis subcordiformis because the suitable pH for this alga is 6.5~8.5 [24,47], and the free ammonia (NH₃) generated under such a high pH could be toxic as well [16]. In fact, the F_v/F_m exhibited a temporary decline in the first two days when the pH increased to above 9 in the IC groups (Figure 1a,c), demonstrating a high-pH or ammonia stress present therein. To avoid the instability of the pH as well as the stress caused by the high pH, air enriched with 2% CO₂ was supplied to the IC groups (CO₂-ICs). As shown in Figure 1b, the pH levels in all the cultures with CO₂ and IC addition were maintained in the range of 6.5~8.5 throughout the cultivation, which matched the suitable pH range for T. subcordiformis. Correspondingly, the F_v/F_m also stayed at a relatively high level without significant decrease in the first four days (Figure 1d), and the biomass accumulation was enhanced to 1.7~2.1 g/L, which was 73~81% higher than the cultures without CO₂ addition (ICs, Figure 1e). These results demonstrated that the acidic CO₂ could form a buffering system with the alkaline bicarbonate/carbonate that was able to stabilize the pH and facilitate biomass accumulation in T. subcordiformis when NH₄⁺-N was used as the nitrogen source. It was reported that the addition of 1–2.5% CO₂ in the bicarbonate-NH₄⁺-N system was beneficial for the pH stabilization and biomass production in freshwater microalgae Desmodesmus sp. F51 and Haematococcus pluvialis QLD [12,15], which coincided with the case of T. subcordiformis herein.

Different ICs enabled diverse chemical environments, which in turn influenced the biomass accumulation and NH₄⁺-N removal. As shown in Figure 1e,f, the biomass accumulation in the cultures with MgCO₃ addition was generally inferior to the NaHCO₃ counterpart, regardless of the application of CO₂ buffering. Specifically, when no CO₂ was applied for buffering, the culture with MgCO₃ addition resulted in an overall higher pH (8.5~9.6) than NaHCO₃ and CaCO₃ (7.6~9.3, Figure 1a), and a lower F_v/F_m was obtained during the first three days (Figure 1c), indicating that the algal cells were exposed to severer high-pH or ammonia stress in the MgCO₃ culture, which finally led to a 25% decrease in biomass accumulation (Figure 1e) compared with the NaHCO₃ and CaCO₃ counterparts. Tetraselmis was reported to have a significantly higher growth rate under a pH of 7.5 rather than 8.5 [48], which was consistent with the results here. This indicated that the pH of the culture should be considered when choosing ICs as the carbon source for the photosynthetic removal of NH₄⁺-N. The addition of CaCO₃ did not affect the final biomass accumulation relative to the NaHCO₃ culture, but it caused an attenuation of biomass accumulation in the early phase (0–4 days) of the cultivation (Figure 1e,f). Considering that the pH levels of these two cultures were almost identical during this period, the decreased biomass production could not be ascribed to the pH difference but might be due to the inhibitory effect of Ca²⁺ released from the CaCO₃. In fact, the Ca²⁺ concentration increased from the initial level of 528 mg/L to 800 mg/L in the CaCO₃ culture when buffering with CO₂ (Figure S1), which can exert stress on T. subcordiformis. A high Ca²⁺ concentration in the medium can reduce the growth and photosynthesis in microalgae [19,49]. The lower F_v/F_m in the CaCO₃ culture was also observed compared to NaHCO₃ (Figure 1c,d), which supported this hypothesis. In addition, the insoluble nature of CaCO₃ could have partially impeded the light penetration in the culture, leading to relatively lower light exposure to the algae cells, compared with the NaHCO₃ counterpart, and consequently reduced biomass production. In all, the CO₂-ICs buffering system was efficient for T. subcordiformis to stabilize the pH and maintain photosynthesis along with the carbon supply for biomass accumulation in the synthetic NH₄⁺-containing wastewater, with the soluble NaHCO₃ performing the best.

3.2. NH₄⁺-N Removal and N Distribution

3.2.1. NH₄⁺-N Removal

To verify the NH₄⁺-N removal efficiency in the IC and CO₂-IC systems, the NH₄⁺-N concentration in the medium was tracked during the cultivation. As shown in Figure 2a, the addition of ICs significantly improved the NH₄⁺-N removal efficiency compared to the control group without IC addition, with more than 99% of NH₄⁺-N being removed within four days in the former as against only 38% achieved in the latter. The maximum NH₄⁺-N removal rates of 49.27, 44.00, and 31.69 mg N/L/day were obtained on the first day in the cultures with NaHCO₃, MgCO₃, or CaCO₃ addition, respectively, which was 1.7~3.3-fold higher than that in the control culture (Figure 2c). The supply of CO₂ to the IC cultures further accelerated the NH₄⁺-N removal, with very trace NH₄⁺-N detected in the culture medium on Day 3 (Figure 2b). The maximum NH₄⁺-N removal rate in the CO₂-IC cultures also showed 22~45% improvement relative to the IC counterparts (Figure 2d). The enhanced NH₄⁺-N removal efficiency in the IC system compared with the control and the further improvement in the CO₂-IC system were in accordance with the higher photosynthetic performance and biomass accumulation therein (as discussed in Section 3.1), demonstrating the effectiveness of pH control and carbon supply for the photosynthetic NH₄⁺-N removal by T. subcordiformis with these strategies. However, the NH₄⁺-N removal efficiency did not seem exactly the same when different ICs were applied. The addition of CaCO₃ led to a slower NH₄⁺-N removal (Figure 2a,b) and reduced the NH₄⁺-N removal rate (p < 0.05, Figure 2c,d) in both the IC and CO₂-IC systems compared with the NaHCO₃ counterparts. The maximum NH₄⁺-N removal rates in the CaCO₃ culture reached 31.69 mg and 45.95 mg N/L/day in the IC and CO₂-IC systems, respectively, which accounted for a 24–36% reduction relative to the cultures with NaHCO₃ addition (Figure 2c,d). The diminished NH₄⁺-N removal in the CaCO₃ culture was in accordance with the reduced photosynthesis and biomass accumulation (Figure 1c–f), which could be ascribed to the inhibitory effects of the high Ca²⁺ load as well as limited light penetration resulting from the insoluble nature therein, as discussed in Section 3.1. The highest maximum NH₄⁺-N removal rate reached 60.13 mg N/L/day in the CO₂-NaHCO₃ culture. In addition, the phosphorus (P) concentration was reduced from the initial level of 14.3 to less than 0.1 mg/L within one day in all the cultures (Figure S3), which perfectly met the P discharge standard (Grade I–A) for urban wastewater treatment in China [50], demonstrating the excellent ability of Tetraselmis subcordiformis for simultaneous NH₄⁺-N and P removal.

3.2.2. N Distribution

The removed NH₄⁺-N from the medium could have either been assimilated by the microalgae or stripped out of the culture system as NH₃ [26,27]. In order to gain further insight into the efficiency of NH₄⁺-N reclamation by the microalgal cultivation systems, the N balance was analyzed to explore the distribution. As shown in Figure 2e,f, the percentage of total N in the biomass generally increased during the first four (ICs) or three (CO₂-ICs) days when NH₄⁺-N was almost completely removed from the medium, yet with the maximum proportion of 55~58% achieved in the former and 73~81% reached in the latter. It suggested that although NH₄⁺-N was removed from the medium, only a part of it could be stored in T. subcordiformis, and the application of CO₂ significantly improved the bio-reclamation of NH₄⁺-N. Accordingly, N balance analysis showed that 39~50% of the initial N escaped from the culture system when only ICs were applied, whereas the N escape level was reduced to 20~27% when CO₂-IC systems were used (Figure 2g,h). It demonstrated that the supply of CO₂ in the IC systems could partially avoid the stripping of N from the culture and improve the NH₄⁺-N assimilation by the microalgae, which could be attributed to the pH reduction because of the acidic CO₂ supply and the enhanced photosynthetic activity that enabled a more efficient bio-assimilation of NH₄⁺-N. A higher pH was reported to result in more NH₃ escape in the cultivation of microalgae Chlorella sp. L38 [26] and Arthrospira platensis [31]. The pH of the IC systems reached 9~9.5 during the first two days (Figure 1a), under which the NH₄⁺ was very prone to be converted to ammonia (NH₃) in view of the dissociation constant (pK_a) of 9.25 for the reaction NH₄⁺↔NH₃ + H⁺, and it was easily stripped out of the culture system with the aeration [16,31]. Instead, in the CO₂-IC systems, the pH was kept at 6.5~8.5 throughout the cultivation, under which NH₄⁺ was dominant and hence greatly reduced the probability of the stripping. In fact, the estimation of free ammonia (NH₃) relative to the total ammonia (NH₃ and NH₄⁺) in the medium according to the formula of NH₃ (%) = 100/(1 + [H⁺]/K_a), where K_a is the dissociation constant of ammonia (4.36 × 10⁻¹⁰ at 25 °C in seawater) [13,51], showed that free ammonia accounted for maximally 42~65% in the ICs as against less than 8% in the CO₂-ICs (Figure S2a). Therefore, the CO₂-IC system allowed much less N escape than the IC systems. Meanwhile, the NH₃ generated under such a high pH in the ICs could be toxic to T. subcordiformis, which would diminish the photosynthesis and adversely affect the N bio-assimilation ability [16]. The estimated free ammonia (NH₃-N, mg/L) calculated as [NH₄⁺]/(1 + [H⁺]/K_a) reached as high as 18~23 mg/L in the ICs, which exceeded the reported maximum EC₅₀ (1258 μM, 17.6 mg/L in Scenedesmus obliquus) of NH₃ inhibition in microalgae [16], whereas it was less than 0.5 mg/L in the CO₂-ICs except for the initial day of 0.7~7.1 mg/L (Figure S2b). Higher photosynthetic activity was also recorded in the CO₂-ICs compared with ICs, as discussed in Section 3.1, which could enable a higher N bioconversion efficiency and less N escape. For the control group without IC addition, the percentage of total N in the biomass also showed a significant increase, and the maximum proportion reached 63% on Day 4, which was even slightly higher than that of the IC groups yet inferior to the CO₂-IC groups (Figure 2e,f). The N escape was less than 5% before four days because of the acidic environment (Figure 2g), which suggested that the removed NH₄⁺-N was predominantly assimilated by the microalgae. However, because of the low-pH stress, as discussed in Section 3.1, the overall NH₄⁺-N removal was impeded (Figure 2a). Collectively, the CO₂-IC auto-buffering system exhibited not only faster and complete NH₄⁺-N removal but also enabled the better ability of N bio-reclamation in T. subcordiformis.

It should be noted that different ICs caused different NH₄⁺-N bioconversion efficiency and N escape rates. Under the IC system on Day 4 when NH₄⁺-N was completely removed, MgCO₃ or CaCO₃ addition led to 23% higher N stored in the biomass than that of NaHCO₃ (58% vs. 47%), which was mirrored by the 22% decrease in N escape (39% vs. 50%) therein (p < 0.05, Figure 2e,g). The decreased N escape in the MgCO₃ or CaCO₃ culture could not be ascribed to the pH effect as discussed above. For instance, the MgCO₃ culture held the highest pH and free ammonia (Figure 1a and Figure S2b) but had a reduced N escape rate relative to the NaHCO₃ culture (Figure 2g). Instead, the higher N escape in the NaHCO₃ culture under ICs might be attributed to the N release from the biomass rather than the direct stripping of the NH₃ from the medium. It can be seen in Figure 2e that the percentage of N stored in the biomass decreased from Day 2 (55%) to Day 4 (47%) in the NaHCO₃ culture, suggesting a N release from the biomass, which was not observed in the MgCO₃ or CaCO₃ culture. Considering that ICs caused a high-pH environment and alkaline stress (Figure 1a,c), it can be speculated that metabolic changes including some deamination reactions (e.g., purine deamination [52]) were induced in the culture with NaHCO₃ addition that generated extra NH₃, which was further stripped out. Nevertheless, the reason for the mitigation of N escape in the MgCO₃ or CaCO₃ culture remained unclear. In contrast to the case under the IC system, the addition of NaHCO₃ under the CO₂-IC system led to the lowest N escape (19.7%) with the highest N stored in the biomass (77.2%) (Figure 2h). Comparatively, the addition of CaCO₃ herein caused an enhancement of N escape (26.9%) and reduction of N storage in the biomass (69.8%), which could be ascribed to the lower photosynthetic activity originated from the relatively less light exposure caused by the insoluble nature and the high-Ca²⁺ stress, as discussed in the previous sections.

The above results highlighted the CO₂-IC buffering system as an efficient strategy for the bio-reclamation of NH₄⁺-N by Tetraselmis subcordiformis. The NH₄⁺-N was completely removed from the medium within three days, with a maximum NH₄⁺-N removal capacity of 46~60 mg N/L/day, which was among the top performances compared with other microalgae under similar conditions reported in the literature (Table 1); meanwhile, the limited N escape rate (19.7~26.9%) in this auto-buffering system was also comparable to those obtained in Chlorella where an acid/base pH control system was applied [26,27,28] (Table 1). Among the three CO₂-ICs tested, the CO₂-NaHCO₃ culture system was particularly outstanding in the aspect of NH₄⁺-N removal, N bioconversion, and biomass production, which demonstrated the feasibility of eliminating pH real-time monitoring without affecting operation efficiency and could reduce instrument investment and operating cost. In addition, according to the emission standard for ammonia in China (GB 14554-93), the emission rate (G, kg/h) should not exceed 4.9 kg/h for each plant. Accordingly, in the context of influent NH₄⁺-N (N_in) of 100 mg/L with the minimum N escape of 19.65% obtained in this study, the maximum wastewater treatment capacity (Q, m³/day) has to be less than 5985 m³/day, as calculated with the formula Q < G × 1000 × 24/(N_in × 19.65%). This treatment scale can be classified as “large scale” [53], although the capacity is relatively small in China yet still acceptable [50]. Considering that the N_in in the urban wastewater from most of the cities in China was below 50 mg/L [50], the treatment capacity could be doubled. These analyses further signified the importance of the reduction of NH₃ release by using the established CO₂-IC systems during the wastewater treatment process to enable higher treatment capacity.

3.3. Extracellular Organic Matter Release during NH₄⁺-N Removal

The photoautotrophic cultivation of microalgae is always accompanied by extracellular organic matter (EOM) release, especially when algal cells are exposed to stressful conditions such as low light intensity, low nutrient availability, and extreme temperatures or pH [30,54,55,56]. The release of EOM is generally unfavorable to the wastewater treatment process since it increases the COD in water. Therefore, the variation of the total organic carbon (TOC) in the medium was investigated under IC or CO₂-IC systems. As shown in Figure 3a, the TOC in the medium increased throughout the cultivation in all the cultures, with the control group reaching the highest TOC release of 117.6 mg/L on Day 8. The addition of ICs remarkably reduced the TOC release, with the ΔTOC range at 67~77 mg/L (Figure 3a). Comparatively, the supply of CO₂ dramatically suppressed the release of TOC, with less than 20 mg/L of ΔTOC obtained in all the cultures (Figure 3b). Considering that stress is usually an important trigger for the EOM release [56], it could be inferred that the high ΔTOC in the control and IC cultures was ascribed to the extreme (low in the former and high in the latter) pH. It was reported that the dissolved organic carbon (DOC) was significantly augmented when the pH increased from 7.5 to 8.5 during the NH₄⁺-N removal by Chlorella vulgaris, and it was deemed to be raised by the free ammonia stress under the high-pH environment [30], a situation that could be applied in the present study when ICs led to a high pH and increased free ammonia concentration, as discussed in the previous section. The extremely high ΔTOC in the control group could be attributed to the low-pH stress, as was also recorded in the microalga Scenedesmus sp. LX1 exposed to a low pH of 5 [54]. It is worth noting (Figure 3b) that the CO₂-NaHCO₃ culture exhibited the lowest ΔTOC (1.3 mg/L), followed by CO₂-MgCO₃ (8.5 mg/L) and CO₂-CaCO₃ (15.3 mg/L). In view of the higher stress that the CO₂-CaCO₃ culture was subjected to compared with the CO₂-NaHCO₃ culture (discussed in Section 3.1), it was reasonable to have this higher ΔTOC. A high Ca²⁺ load was reported to improve extracellular secretions in the the NH₄⁺-N removal system by microalgae [19], which coincided with the present study.

In general, carbohydrate and protein are regarded as the main components in EOM [57]. Therefore, these two components were determined in IC or CO₂-IC systems. It was clear that the carbohydrate accounted for the dominant proportion of the EOM as against protein in all the cultures (Figure 3c,d), and the carbohydrate concentration in the IC cultures was 2.4~6.4 times higher than those in the CO₂-ICs cultures, which was in agreement with the TOC concentration (Figure 3). Interestingly, for the CO₂-IC system, while the ΔTOC was the highest in the CO₂-CaCO₃ culture, the carbohydrate concentration was the lowest therein (p < 0.05, Figure 3d), indicating that some other kinds of EOM, e.g., organic acids (humic acid, fulvic acid, glycolic acid, etc.), hormonal substances, or pigments [55,57], were present in this culture. Collectively, these results further highlighted the advantage of the CO₂-IC buffering system, especially the CO₂-NaHCO₃ culture, for enabling a favorable pH environment to avoid EOM generation, which benefited the maintenance of water quality during the NH₄⁺-N removal process. In particular, the ΔTOC of 1.3 mg/L in the CO₂-NaHCO₃ culture could be considered as a very low or even the lowest level, compared with other microalgae cultivation results reported previously [57].

3.4. Biomass Component Production

Microalgae assimilate NH₄⁺-N and convert it primarily to protein; at the same time, carbohydrates and lipids are the major products of the carbon fixation during the algal cell growth [58]. The accumulation of the biomass components can be varied depending on the environment that the microalgae are exposed to, especially when stress factors are present [59]. Tetraselmis subcordiformis mainly accumulated protein, starch, and lipid as the nitrogen and carbon reserves, which can be used for biorefinery and value-added products [44]. Therefore, the accumulations of these three components were assessed during the NH₄⁺-N removal process under IC or CO₂-IC systems.

As shown in Figure 4a, the protein accumulated mainly during the first four (ICs) or three (CO₂-ICs) days when NH₄⁺-N was assimilated (Figure 2e,f), demonstrating the conversion of the NH₄⁺-N into protein by the microalgae. The supply of CO₂ improved the protein production because of the favorable pH environment therein, with the maximum net increase in protein concentration reaching around 0.62 g/L in all the CO₂-IC cultures on Day 6, which represented 44% of the enhancement relative to the IC counterparts (Figure 4a). The protein accumulation generally showed no difference between the three ICs (NaHCO₃, MgCO₃, or CaCO₃) used under both IC and CO₂-IC systems, although a lag phase was observed in the MgCO₃ culture under the IC system (Figure 4a), probably because of the high pH and free ammonia inhibition in the first two days that suppressed the photosynthetic activity required for protein synthesis (Figure 1a,c and Figure S2b). Lower protein production was observed in Chlorella sp. L38 when the pH increased from 7 or 8 to 9 during the NH₄⁺-N removal, which was consistent with the present study [26].

The starch accumulation generally occurred on Day 4 and Day 2, respectively, in the ICs and CO₂-ICs systems (Figure 4b), which correlated to the exhaustion of NH₄⁺-N in the medium (Figure 2a,b), indicating a nitrogen limitation-triggered starch synthesis, as had been widely recognized in microalgae, including Tetraselmis subcordiformis [39,60]. Similar to the protein accumulation, the starch production was markedly improved in the CO₂-IC cultures compared to the IC cultures. However, different ICs led to enormous variations in terms of starch accumulation, with NaHCO₃ exhibiting the most prominent ability to induce starch synthesis in Tetraselmis subcordiformis, followed by CaCO₃ and MgCO₃; the maximum net increase in starch reached 1.3 g/L in the CO₂-NaHCO₃ culture on Day 6, which was 32% (p < 0.05) and 1.72-fold higher (p < 0.05) than that obtained in the CO₂-CaCO₃ and CO₂-MgCO₃ cultures, respectively (Figure 4b). The reduced starch accumulation in the CO₂-CaCO₃ and CO₂-MgCO₃ cultures could be attributed to the limited light exposure originated from the insoluble CaCO₃ and MgCO₃ particles. Light intensity is an important factor affecting starch production in microalgae; higher light exposure usually results in enhanced starch accumulation, especially under nutrient deprivation conditions [34,60,61]. In fact, the slower decline of F_v/F_m (Figure 1d), a sensitive indicator of photoinhibition, after the nitrogen deprivation from Day 4 in the CaCO₃ and MgCO₃ cultures relative to the NaHCO₃ culture implied the reduced light intensity per cell therein.

The lipid production, on the whole, mirrored the profile of protein, in which the accumulation started with the NH₄⁺-N assimilation and lasted until NH₄⁺-N was completely removed (Day 4), and there was no significant difference between the ICs applied in both cultivation systems (Figure 4c). It indicated that the accumulated lipid were largely polar lipids such as phospholipids and glycolipids, which were responsible for photosynthetic cell growth under nitrogen-replete conditions [62]. The maximum net increase in lipids reached 0.25~0.28 g/L in the CO₂-IC cultures and 0.13~0.16 g/L in the IC cultures, which showed a relatively minor contribution compared with the starch and protein accumulation (Figure 4). In all, Tetraselmis subcordiformis was able to assimilate NH₄⁺-N and store the N as protein, while starch and lipid could be simultaneously produced under both IC and CO₂-IC systems, with CO₂-NaHCO₃ culture showing the best overall biomass component production capacity, which manifested the superiority of this strategy for NH₄⁺-N removal and reclamation from wastewater.

3.5. Biomass Quality Evaluation

The main products obtained in the NH₄⁺-N removal process by Tetraselmis subcordiformis were starch and protein (Figure 4), as discussed above. Since the amylose/amylopectin ratio (Am/Ap) in starch and the amino acid profile of the protein are crucial indexes for the biomass quality of the microalgae that could determine the downstream applications, these two items were further evaluated in detail.

3.5.1. Starch Composition

In general, from the structural perspective, two types of starch, namely, amylose (Am), which is linear polymerized glucose linked by α-1,4 glycosidic bonds, and amylopectin (Ap), which has additional branching at the α-1,6 positions, can be synthesized by microalgae, including Tetraselmis subcordiformis [44,47,63]. As shown in

Table 2. Amylose (Am) and amylopectin (Ap) proportion in the starch of Tetraselmis subcordiformis under IC (NaHCO₃, MgCO₃ or CaCO₃, Day 8) or CO₂-IC (NaHCO₃, MgCO₃ or CaCO₃, Day 6) conditions for the NH₄⁺-N removal. The asterisk (*) indicates a significant difference (p < 0.05) compared with the corresponding NaHCO₃ culture.

Culture System	Bicarbonate/Carbonate Supply	Am (% of Total Starch)	Ap (% of Total Starch)	Am/Ap
IC	NaHCO3	39.46 ± 0.01	60.54 ± 0.01	0.65 ± 0.03
	MgCO3	41.76 ± 0.01	58.24 ± 0.01	0.72 ± 0.03
	CaCO3	37.10 ± 0.02	62.90 ± 0.02	0.59 ± 0.05
CO2-IC	NaHCO3	38.90 ± 0.01	61.10 ± 0.01	0.64 ± 0.02
	MgCO3	59.57 ± 0.00 *	40.43 ± 0.00 *	1.47 ± 0.00 *
	CaCO3	50.54 ± 0.00 *	49.46 ± 0.00 *	1.02 ± 0.01 *

, under the IC culture system, Ap constituted most of the starch stored in the algae, with a proportion of around 60% in all the IC conditions; Am/Ap ranged from 0.6 to 0.7, which was consistent with the starch obtained by adding the same dosage of NaHCO₃ under N starvation for four days (0.62) as reported previously in this microalga [47]. A similar starch composition was obtained under the CO₂-NaHCO₃ condition with a Am/Ap reaching 0.64 (Table 2), which could be classified as the “normal” starch that is prevalent in plants and algae [64,65]. Starch with a higher Ap proportion is considered to facilitate enzymatic hydrolysis for glucose release and hence improves the fermentation efficiency [66]. In view of the highest starch production under the CO₂-NaHCO₃ condition in this study, it was reasonable to apply this strategy for the NH₄⁺-N removal coupled with starch production that could be used as the feedstock for fermentation to manufacture biofuels (such as bioethanol) and bio-based chemicals [67]. Interestingly, the addition of MgCO₃ or CaCO₃ under the CO₂ supply significantly enhanced the relative Am proportion in starch (p < 0.05), with Am accounting for 50~60% and Am/Ap of 1~1.5 obtained (Table 2). Tracking the Am and Ap accumulation during the cultivation revealed that the increased Am/Ap could be largely ascribed to the stronger inhibition of Ap production relative to the Am (Figure 5b,d). For instance, in the CO₂-CaCO₃ culture, the Am production reached a similar level (0.48~0.49 g/L) as in the CO₂-NaHCO₃ culture (Figure 5b), but the Ap accumulation was remarkably reduced (0.47 for CO₂-CaCO₃ vs. 0.78 for CO₂-NaHCO₃, p < 0.05, Figure 5d), which accounted for the decrease in total starch accumulation (Figure 4b). In fact, the increase in Am/Ap in the CO₂-MgCO₃, CO₂-CaCO₃, and MgCO₃ cultures seemed to be correlated with the decrease in total starch accumulation (Table 2 and Figure 4b), suggesting that the low-light-caused reduction of starch accumulation (as discussed in Section 3.4) was manifested more in the Ap rather than the Am synthesis. Low light could have caused a reduced activity of the starch-branching enzyme responsible for the Ap synthesis [68], leading to the increased Am/Ap ratio. The starch obtained under the CO₂-MgCO₃ and CO₂-CaCO₃ conditions herein, with more than 50% of Am contained in the total starch (Am/Ap > 1), could be classified as high-amylose starch (HAS), which would be of high value and applied in bio-based material production (such as films, coatings, textiles, paper, medical devices, and biodegradable flexible packaging) because of the excellent material properties [64,69]; meanwhile, it could serve as the resistant starch supplemented in the feeds for promoting health in aquaculture [70]. The above application of starch produced in Tetraselmis subcordiformis during the NH₄⁺-N removal could improve the economic benefits for the wastewater treatment.

3.5.2. Amino Acid Profile

Table 3. Amino acid (AA) profiles (g AA/100 g total AA) in Tetraselmis subcordiformis under IC (NaHCO₃, MgCO₃, or CaCO₃, Day 8) or CO₂-IC (NaHCO₃, MgCO₃, or CaCO₃, Day 6) conditions for the NH₄⁺-N removal and comparison with traditional animal feed proteins in terms of the essential amino acid index (EAAI).

Animo Acids	IC			CO2-IC			Soybean Meal c	Fish Meal c	Penaeus monodon Juvenile c	Ruminant Diets c	Ideal Protein for Dairy Cow c
	NaHCO3	MgCO3	CaCO3	NaHCO3	MgCO3	CaCO3
Essential AA (EAA)
Arginine	4.65 ± 0.21	4.35 ± 0.12	4.92 ± 0.65	4.99 ± 0.26	4.50 ± 0.05	4.94 ± 0.17	8.30	6.75	8.00	14.8
Histidine	1.69 ± 0.06	1.94 ± 0.03	1.75 ± 0.02	2.23 ± 0.19	1.88 ± 0.04	2.03 ± 0.15	3.04	2.54	2.48	4.8
Isoleucine	4.22 ± 0.30	4.32 ± 0.09	4.39 ± 0.17	4.34 ± 0.12	4.49 ± 0.24	4.30 ± 0.01	5.22	4.32	4.46	3.6	4.8
Leucine	9.12 ± 0.23	9.16 ± 0.08	9.13 ± 0.27	9.16 ± 0.05	9.40 ± 0.23	8.91 ± 0.33	8.30	8.06	7.66	6.0	8.6
Lysine	7.39 ± 0.13	7.15 ± 0.01	7.45 ± 0.06	8.54 ± 0.57	8.31 ± 0.35	8.56 ± 0.28	6.95	8.76	7.58	7.6	6.7
Methionine	1.05 ± 0.62	2.14 ± 0.01	1.19 ± 0.07	0.92 ± 0.14	0.43 ± 0.20	1.45 ± 0.25	1.97	3.23	2.85	1.3	2.0
Phenylalanine	5.94 ± 0.01	5.86 ± 0.05	5.72 ± 0.12	5.71 ± 0.08	6.10 ± 0.23	5.75 ± 0.17	4.88	4.21	4.20	2.9	4.4
Threonine	6.66 ± 0.04	6.78 ± 0.33	6.33 ± 0.06	6.14 ± 0.06	6.15 ± 0.29	6.08 ± 0.04	4.09	5.12	3.96	3.8	5.2
Tryptophan a	-	-	-	-	-	-
Valine	6.56 ± 0.18	6.67 ± 0.02	6.63 ± 0.04	6.25 ± 0.67	6.71 ± 0.12	6.30 ± 0.03	3.85	5.07	5.16	5.1	5.3
TOTAL EAA	47.27	48.39	47.51	48.05	48.54	48.69	46.60	48.06	46.34
Non-essential AA (NEAA)
Alanine	8.16 ± 0.03	7.89 ± 0.04	8.15 ± 0.08	7.31 ± 0.00	7.29 ± 0.45	7.75 ± 0.23	4.61	6.45	6.11
Aspartic acid b	11.40 ± 0.34	11.24 ± 0.56	11.17 ± 0.39	11.85 ± 0.47	11.09 ± 1.32	11.15 ± 0.26	11.91	10.63	10.20
Cysteine	0.60 ± 0.06	0.63 ± 0.16	0.91 ± 0.11	0.60 ± 0.10	0.91 ± 0.23	0.63 ± 0.10	2.20	1.08	1.03
Glutamic acid b	13.74 ± 0.45	12.92 ± 0.30	13.94 ± 0.09	13.52 ± 0.22	13.18 ± 1.27	13.00 ± 0.33	18.07	15.38	16.07
Glycine	5.48 ± 00.35	5.51 ± 0.03	5.33 ± 0.06	5.23 ± 0.20	6.07 ± 0.54	5.70 ± 0.10	3.99	5.68	8.12
Proline	4.96 ± 0.15	5.00 ± 0.03	4.98 ± 0.21	4.83 ± 0.08	4.97 ± 0.59	5.02 ± 0.32	4.37	4.21	4.02
Serine	5.38 ± 0.13	5.45 ± 0.33	5.13 ± 0.23	5.16 ± 0.24	5.04 ± 0.11	5.18 ± 0.07	5.04	5.10	4.16
Tyrosine	3.00 ± 0.14	2.97 ± 0.02	2.87 ± 0.14	3.21 ± 0.15	3.46 ± 0.35	3.25 ± 0.08	3.22	3.41	3.94
EAAI for prawn	0.90	0.97	0.92	0.91	0.83	0.94	0.98	1.01
EAAI for ruminant	1.01	1.09	1.03	1.02	0.93	1.06				0.97

Notes: ^a Tryptophan was destroyed by acid hydrolysis. ^b Asparagine and glutamine were hydrolyzed to aspartic acid and glutamic acid, respectively. ^c Data acquired from Xiang et al. [44].

lists the amino acid (AA) profile (g AA/100 g total AA) in T. subcordiformis under IC (Day 8) or CO₂-IC (Day 6) conditions for the NH₄⁺-N removal. Generally, the AA profiles showed little variation among all the culture conditions. The dominant AAs were registered as glutamic acid, aspartic acid, leucine, and alanine, which accounted for more than 8% of the total AAs; the main essential amino acids (EAAs) were leucine, lysine, threonine, and valine, with the proportion being more than 6% of the total AAs. The AA profile character in T. subcordiformis cultivated in ammonium herein was similar to that cultivated in nitrate as nitrogen source [44] and was also consistent with other Tetraselmis species cultivated in urban wastewater that contained both nitrate and ammonium [71]. The most evident difference in the AA profiles among different ICs was the methionine, which was markedly increased in the MgCO₃ culture under the IC system and CaCO₃ under the CO₂-IC system compared with the respective NaHCO₃ cultures (Table 3). Methionine in protein was considered as an endogenous antioxidant in cells [72]. The increase in methionine indicated the oxidative stress that the microalgae were exposed to, which was in agreement with the reduced F_v/F_m in the MgCO₃ culture subjected to high-pH/ammonia inhibition and in the CO₂-CaCO₃ culture with high-Ca²⁺ stress, as discussed in the previous sections.

As for the nutritional concerns, compared to soybean meal, a widely used feed for animals and aquaculture [73], T. subcordiformis produced a higher proportion of EAAs (more than 47%) in both the IC and CO₂-IC systems with the removal of NH₄⁺-N (Table 3). In particular, the CO₂-IC system resulted in overall higher EAAs than the IC system (except for the MgCO₃ culture where methionine contributed to the atypically high EAAs), which could be caused by the enhancement of the histidine and lysine (Table 3), exemplifying the advantage of the CO₂-IC system for providing better AA nutrition for animals. Calculation of the essential amino acid index (EAAI) for prawn showed that the proteins synthesized in T. subcordiformis under the IC and CO₂-IC systems had EAAI scores higher than 0.90 (except for the CO₂-MgCO₃ culture), which could be classified as “good-quality” protein according to Peñaflorida [74]; the high proportion of valine (6.3~6.7) in T. subcordiformis replenished the insufficiency of this AA in soybean meal (actual/ideal = 3.85/5.16) and fish meal (actual/ideal = 5.07/5.16) for Penaeus monodon juvenile, demonstrating the good potential to be applied in aquaculture. Notably, the EAAI scores for the ruminant in this study were higher than 1.00 (except for the CO₂-MgCO₃ culture), which could be categorized as “high-quality” protein [45] and were even superior to that of ruminant diets (0.97). The shortage of phenylalanine (actual/ideal = 2.9/4.4), leucine (actual/ideal = 6.0/8.6), and threonine (actual/ideal = 3.8/5.2) in the ruminant diets could be completely compensated for by the proteins of T. subcordiformis (5.7~6.1 for phenylalanine, 8.9~9.4 for leucine, and 6.1~6.8 for threonine) with the removal of NH₄⁺-N under all the culture conditions herein, which manifested the great potential for livestock graziery. Collectively, together with the consideration of protein production ability (Figure 4a), the T. subcordiformis biomasses obtained under CO₂-NaHCO₃ and CO₂-CaCO₃ conditions were preferable for providing excellent alternative protein sources in livestock graziery and aquaculture, which could realize the bio-reclamation and valorization of waste NH₄⁺-N.

3.6. Future Works

The present study demonstrated a high NH₄⁺-N removal rate along with low N escape and high-quality biomass production in T. subcordiformis cultivated in synthetic wastewater under the CO₂-IC system. In real urban wastewater, although normally low contents of toxicant were present [6], the performance of the algae might be challenged because of the complexity of the composition of the wastewater, e.g., the variations of N/P ratio that could influence the photosynthetic growth and nutrient removal as well as the biomass component produced [6,9]. In addition, the implications of the microorganisms contained in the real wastewater might also be considered, although they could be both positive and negative [9]. Further investigation is needed to evaluate the feasibility of the established microalgal culture system herein for real urban wastewater treatment.

It should be noted that the present study performed the NH₄⁺-N removal under batch mode. For the best performance under the CO₂-NaHCO₃ culture system, the removal constant (k_N) was calculated to be 1.76 day⁻¹ when fitted with the first-order removal kinetics model (Table S1, [75]). It could be extrapolated to a continuous NH₄⁺-N removal process where a constant flow of wastewater is treated under a steady state. In this operation mode, the hydraulic retention time (HRT) could be predicted according the formula of (N_i − N_e)/HRT = k_N × N_e [76], where N_i and N_e are the influent and effluent NH₄⁺-N concentrations, respectively. For the N_i of 100 mg/L used herein and the N_e of 5 mg/L that meets the minimum discharge standard (Grade I–A) for urban wastewater treatment in China [50], the HRT has to be set at no less than 10.8 day. Further study needs to be conducted to dissect the effect of HRT on the NH₄⁺-N removal and biomass accumulation under the continuous treatment mode to make the process more practical. Moreover, for practical operations, flue gas could be used for the CO₂ supply with a circulation system to further improve the economic feasibility and sustainability of the treatment process.

4. Conclusions

In the present study, the continuous sparging of air containing 2% CO₂ along with inorganic bicarbonate/carbonate addition was used to achieve pH stabilization and maximize algae growth and NH₄⁺-N removal by Tetraselmis subcordiformis. The application of this culture system also significantly reduced the NH₃ escape into the air and restricted the extracellular organic matter release into the medium by the microalgae, which minimized the secondary pollution during the NH₄⁺-N removal process. Specifically, the addition of 12 mM NaHCO₃ with 2% CO₂-containing air sparging was demonstrated to exhibit the best performance, in which more than 98% of NH₄⁺-N was removed within two days and the maximum NH₄⁺-N removal capacity of 60.13 mg N/L/day achieved was the highest level compared with other microalgae under similar conditions reported hitherto. The NH₃ escape rate of 19.7% in this auto-buffering system was also comparable to that obtained in the acid/base-based pH monitoring algal culture system, which can comparatively reduce the instrument investment and operating cost; the total organic carbon release of 1.3 mg/L was also the lowest compared with other microalgae cultures reported previously. The starch accumulated in T. subcordiformis with the NH₄⁺-N removal was suitable for fermentation or bio-based material production and use as a health-promoting ingredient for aquaculture, and the proteins in the biomass were of good quality for animal feeds, both of which would valorize the NH₄⁺-N-containing wastewater treatment. Further study is needed to confirm the performance of the established CO₂-inorganic carbonate system for real urban wastewater treatment under both batch and continuous operation modes in terms of NH₄⁺-N removal and algal biomass production to make the process practical.