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Article

Enhancing Biomass Production of Chlorella vulgaris in Anaerobically Digested Swine Wastewater Using Carbon Supplementation and Simultaneous Lipid Production

by
Chenkai Zhou
1,2,†,
Mingmin Yuan
1,2,†,
Cuifeng Huang
1,2,
Qiqi Chen
1,2,
Jiamin Wang
1,2,
Xinting Chen
1,2,
Hua Yang
1,2,
Jun Fang
1,2,* and
Bo Yang
1,2,*
1
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
2
Yuelushan Laboratory, Changsha 410128, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Appl. Sci. 2025, 15(9), 5103; https://doi.org/10.3390/app15095103
Submission received: 10 April 2025 / Revised: 29 April 2025 / Accepted: 2 May 2025 / Published: 4 May 2025
(This article belongs to the Special Issue Application of Biological Processes in Waste Treatment and Valorisation)
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Abstract

:
This study investigated anaerobically digested swine wastewater (ADSW) as a nutrient source for Chlorella vulgaris FACHB-8 cultivation under mixotrophic conditions with carbon supplementation. The microalgal strain was grown in ADSW supplemented with six carbon sources, followed by concentration optimization. Under optimized conditions (20 g/L glucose), FACHB-8 demonstrated a high biomass productivity (271.31 mg/L/day) and a specific growth rate of 0.42 per day. The system achieved an 88.70% total nitrogen removal and an 82.93% total phosphorus removal. The biomass contained 45.59% lipids, 29.72% proteins, and 13.05% carbohydrates, with fatty acid methyl esters showing balanced proportions of saturated (50.77%) and unsaturated fatty acids (49.23%). These findings highlight the potential of glucose-based mixotrophic cultivation for simultaneous wastewater treatment, renewable biomass production, and value-added lipid production. This work proposes a scalable swine wastewater treatment system that synergizes bioremediation and renewable energy production via carbon-enhanced microalgae cultivation, offering a dual-functional strategy for sustainable livestock wastewater reuse.

1. Introduction

The global energy crisis represents a critical challenge characterized by diminishing fossil fuel reserves, volatile energy markets, and environmental degradation associated with conventional energy utilization [1]. Global economic expansion and population growth have accelerated the depletion of finite fossil fuel reserves, including petroleum and natural gas [2]. Current consumption patterns threaten to exhaust these resources within foreseeable timelines, potentially triggering socioeconomic instability through energy supply shortages. Furthermore, conventional energy exploitation and utilization generate substantial pollutant emissions, particularly CO2 and sulfur compounds, exacerbating environmental damage and accelerating climate change [3]. These pressing issues necessitate an urgent development of sustainable energy alternatives.
Microalgae represent a diverse group of unicellular photosynthetic organisms inhabiting aquatic environments, comprising both prokaryotic (e.g., Cyanobacteria) and eukaryotic species (e.g., green algae and diatoms) [4]. Oleaginous microalgae strains exhibit exceptional lipid accumulation capacities [5], typically containing 20~50% of lipids by dry cell weight, with some species containing a lipid content of up to 75% [6]. Their rapid growth rates under optimal conditions make them promising candidates for biofuel production. Microalgae biodiesel, produced through the biochemical conversion of algal biomass [7], presents a renewable alternative to petroleum diesel with comparable combustion properties but a reduced carbon footprint [8]. However, the current production costs of microalgae biodiesel are still relatively high, primarily due to the higher cultivation expenses of algal cells [9,10]. Therefore, cost reduction strategies focusing on low-cost nutrient sources could enhance the economic viability [11]. Recently, the use of wastewater as a nutrient source for microalgae cultivation has been suggested as a viable approach to effectively lower the production costs [12]. The microalgae-based wastewater treatment process enables microalgae to utilize nitrogen and phosphorus nutrients from low-cost wastewater for cell growth and bioremediation, simultaneously accumulating high-value bioactive compounds such as lipids [12,13,14,15]. Recent advances highlight the potential of algae–bacteria symbiotic systems, where algal oxygen supports bacterial organic matter oxidation while bacterial respiration provides CO2 for photosynthesis, achieving a lower energy consumption than axenic algal processes [16]. Emerging low-cost strategies seek to enhance these bioremediation and bioproduction processes through nutrient modulation (e.g., nitrogen deprivation) and organic carbon supplementation, though wastewater-derived contaminants necessitate a strain-specific adaptability for effective cultivation [11].
Microalgae can be cultivated under photoautotrophic, heterotrophic, and mixotrophic conditions [17]. Photoautotrophic cultivation relies on light-driven photosynthesis for energy conversion, while heterotrophic cultivation utilizes organic carbon substrates (e.g., simple sugars) under dark conditions. Mixotrophic cultivation synergistically combines both photosynthetic and heterotrophic modes [18,19]. Although photoautotrophic cultivation remains the predominant commercial-scale method, due to its operational simplicity and cost-effectiveness, it presents critical limitations in wastewater treatment applications. The reduced light penetration caused by shadowing effects within the cultivation medium lead to a lower biomass productivity [20,21]. The application of photoautotrophic cultivation in the wastewater treatment process is less feasible due to the reduced light penetration caused by large wastewater volumes, particularly when handling dark-colored wastewater. Heterotrophic cultivation offers a cost-effective scalability in simple bioreactors with an enhanced productivity over photoautotrophic cultivation but faces certain drawbacks such as the species specificity, contamination risks, and increased substrate costs [22]. In contrast, mixotrophic cultivation can simultaneously assimilate organic carbon, nutrients, and CO2 through combined respiration and photosynthesis, addressing the limitations of single-mode systems. When applied to wastewater treatment, mixotrophic cultivation effectively enhances the biomass production with reduced production costs and provides additional advantages such as a lower light requirement and simultaneous production of photosynthetic biocompounds (e.g., phycocyanin and carotenoids), which is not attainable in the heterotrophic cultivation mode [17,23].
The performance of the microalgal wastewater treatment process depends critically on both the microalgae strain selection and wastewater characteristics. It is therefore crucial to optimize the treatment process to maximize the biomass productivity. Selecting an algal species with a rapid growth and high wastewater tolerance is critical for optimizing treatment efficiency [24]. Promising candidates like Chlorella and Scenedesmus (Chlorophyta) demonstrate such traits, which is exemplified by C. vulgaris achieving an 89.5% TN and 85.3% TP removal in undiluted anaerobic swine wastewater within open raceway systems [25]. Microalgae, as unicellular cell factories, only primarily utilize simple molecules like sugars, nitrogen compounds, and amino acids. Although most sources of wastewaters contain abundant dissolved organic matter suitable for bacterial assimilation, most remain inaccessible as carbon sources for microalgae-mediated nutrient removal [26,27]. Hence, supplementing with suitable carbon sources may be an essential strategy to enhance the efficiency of the wastewater treatment process [28]. The supplementation of both inorganic and organic carbon sources can promote microalgae growth, shorten the cultivation cycle, and enhance biomass accumulation [29]. In this regard, using carbon-supplemented mixotrophic cultivation in wastewater may effectively increase the microalgal biomass, thereby enhancing the nutrient removal efficiency and promoting the simultaneous production of high-value products such as lipids. Numerous studies have shown that glucose, a simple sugar, serves as the most effective carbon source for microalgae cultivation [17], demonstrating an 85% assimilation efficiency in synthesizing oligosaccharides and polysaccharides [29,30]. It provides a higher energy content (approximately 2.8 kJ mol−1) than other carbon sources like acetate (0.8 kJ mol−1). Also, glucose supplementation was found to enhance the C. vulgaris cell size and improve starch and lipid accumulation [29].
This study aims to explore the effect of carbon supplementation on the biomass production, nutrient removal, and potential lipid production of the oleaginous microalgal strain C. vulgaris FACHB-8 in anaerobically digested swine wastewater (ADSW). In our previous work, we have evaluated the excellent ability of this strain in ADSW treatment and potential lipid production [31]. Moreover, C. vulgaris has been demonstrated to be viable in integrated systems for various types of wastewater treatment alongside a simultaneous lipid production [11,32].Therefore, C. vulgaris FACHB-8 was selected and used in this study. To our knowledge, limited studies have investigated the effect of nutrient supplementation in the wastewater cultivation medium on the microalgal biomass production and lipid yield. Therefore, in our study, FACHB-8 was cultured in wastewater supplemented with various kinds of carbon sources to select the optimal carbon source. Afterwards, the suitable concentration of the selected carbon source was ascertained to enhance the biomass production and nutrient removal in wastewater. The nutrient removal efficiencies were studied in more detail under optimized wastewater conditions. Finally, the biochemical composition, including the carbohydrate and protein content, and lipid productivity as well as fatty acid profiles were examined. This study uniquely explores carbon supplementation strategies in C. vulgaris cultivation using real ADSW, targeting a simultaneous biomass enhancement and high lipid productivity under optimized mixotrophic conditions.

2. Materials and Methods

2.1. Wastewater Collection, Pretreatment, and Analysis

Samples of anaerobic digested swine wastewater (ADSW) were collected from Xin Wu Feng Co., Ltd. (Branch office: Yong’an Town, Changsha, Hunan Province, China). The raw ADSW sample was centrifuged at 8000× g for 10 min, followed by immediate filtration through 0.45 µm nylon membranes to remove insoluble solids. Sterilization was performed via autoclaving at 121 °C for 20 min. For physicochemical analysis, the pH value was measured using a glass electrode. Total nitrogen (TN) was quantified by ultraviolet spectrophotometry after oxidation with alkaline potassium persulfate. Total phosphorus (TP) was determined by ammonium molybdate spectrophotometry. Ammonium nitrogen (NH4+-N) was measured using sodium chloride reagent-based spectrophotometry. Chemical oxygen demand (COD) was quantified using the dichromate method. Total organic carbon (TOC) and inorganic carbon (IC) were analyzed according to the Chinese National Standards. The compositions of ADSW are summarized in Table 1.

2.2. Strain and Cultivation Conditions

The oleaginous microalgal strain Chlorella vulgaris FACHB-8 used in this study was obtained from the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB). This strain was cultivated and maintained in undiluted ADSW (after pretreatment). Prior to autoclaving, undiluted ADSW was pH-adjusted to 7.2. In all cases, all cultures were prepared in 250 mL Erlenmeyer flasks containing 120 mL of the liquid medium, inoculated at 10% (v/v). The algal cells were cultured at 25 °C, 150 r/min, under strictly controlled illumination of 5000 lux (measured at the culture medium surface center of all flasks) under a photoperiod of 12 h:12 h for 14 days.

2.3. Experimental Setup

The schematic of the experimental setup is shown in Figure 1. Six carbon sources (sodium carbonate, sodium bicarbonate, sodium acetate, glucose, sucrose, and glycerol) were supplemented to ADSW at 10 g/L (based on preliminary experimental results) to prepare test media. A control (CK) group with no carbon addition was included. After 14-day microalgae cultivation, biomass (dry cell weight), pigment concentration (chlorophyll a and carotenoids), and nutrient removal rates (TN and TP) were assessed to identify the optimal carbon source.
Subsequently, the optimal carbon source was added to ADSW at seven concentrations (0, 2, 5, 8, 10, 20, and 30 g/L). After 14 days of cultivation, biomass, pigment concentration, and nutrient removal of algal cells were also measured to determine an optimal supplementation concentration for subsequent experiments.
On this basis, the nutrient removal efficiencies were thus studied in more detail under such optimized wastewater conditions. The biochemical composition (lipid, carbohydrate, and protein content), lipid productivity, and fatty acid profiles were also analyzed.

2.4. Analytical Procedures

2.4.1. Measurement of Microalgal Growth

Microalgal growth was quantified gravimetrically through dry cell weight (g/L) measurements. Empty 2 mL centrifuge tubes were pre-dried in an 80 °C oven for more than 8 h, cooled in a desiccator for 30 min, and their tare weights recorded. A 2 mL aliquot of the algal suspension was transferred to the pre-weighed tubes, and centrifuged at 8000× g for 10 min. The pellet was washed twice with distilled water (2 mL per wash) using identical centrifugation parameters. Washed algal pellets were dried overnight at 80 °C, cooled in a desiccator for 30 min, and reweighed. Final biomass was calculated by subtracting the tare weight from the gross weight [33].
Algal biomass productivity (g/L/d) and specific growth rate (d−1) were calculated using the following Equations (1) and (2), respectively.
P = ( X 1 − X 0 ) / ( t 1 − t 0 )
μ = ( ln X 1 − ln X 0 ) / ( t 1 − t 0 )
where t1 and t0 (d) are the final and initial time points, respectively, and X1 and X0 (g/L) are the dry cell weight of algal cells at t1 and t0, respectively [33].

2.4.2. Nutrient Removal Analysis

Two milliliters of culture suspension were sampled from each flask for nutrient removal assessment. Following centrifugation at 9000× g for 10 min, supernatants were diluted to appropriate concentrations and analyzed for TN and TP using the methods described in Section 2.1.

2.4.3. Determination of Pigments

For pigment quantification, chlorophyll a and carotenoids were extracted from microalgal biomass using 1 mL methanol at 4 °C for 5 h, followed by centrifugation. The supernatant’s absorbance was measured at 750, 665, 652, and 480 nm using a UV-3600 Plus spectrophotometer (Shimadzu, Kyoto, Japan). The pigment concentrations were calculated using the following Equations (3) and (4), respectively [31].
Chlorophyll   a   ( mg / L ) = 16.5169   ×   A 665 − 8.0962   ×   A 652
Carotenoids mg / L = 4   ×   A 480  
Absorbance values at 652, 665, and 480 nm were corrected by subtracting the 750 nm absorbance.

2.4.4. Determination of Carbohydrate and Protein Content

To determine the carbohydrate content, freeze-dried algal biomass samples (10 mg) were triple-extracted with boiling distilled water (30 min per cycle). The combined extracts were filtered into a 25 mL volumetric flask and diluted to mark. Total carbohydrate content was analyzed via the phenol–sulfuric acid method [31].
To determine protein content, freeze-dried biomass (10 mg) was treated with 200 μL 1 M NaOH, hydrolyzed at 80 °C for 10 min, then diluted with 1.8 mL distilled water. After centrifugation (12,000× g, 30 min), supernatants from duplicate extractions were pooled and quantified using a Bradford protein assay kit (P0006, Beyotime, Shanghai, China) [31].

2.4.5. Lipid and Fatty Acid Methyl Ester (FAME) Analysis

Total lipids were extracted from freeze-dried biomass via methanol–chloroform solvent partitioning [34]. Briefly, 20 mg lyophilized powder was homogenized with 3 mL methanol–chloroform (2:1, v/v) in pre-weighed 10 mL tubes, vortexed for 2 min, and centrifuged (10,000× g, 5 min). The chloroform phase was collected after triplicate extractions, evaporated under nitrogen gas, and dried to constant weight at 80 °C for gravimetric quantification.
FAMEs were synthesized by suspending 20 mg biomass in 1 mL toluene, 2 mL 1% methanol sulfate, and 0.8 mL hexane containing 0.8 mg heptadecanoic acid (internal standard) at 50 °C overnight. Hexane-extracted FAMEs were analyzed via GC-MS (QP 2010 SE, Shimadzu, Japan) equipped with a Stabilwax-DA capillary column (30 m × 0.25 mm × 0.25 µm) (Shimadzu, Japan). The injector temperature was set at 250 °C, with the column initially held at 150 °C. The temperature was first ramped to 200 °C at 10 °C/min, then to 220 °C at 6 °C/min, and finally maintained at 220 °C for 10 min. FAMEs were identified by matching mass spectra with the NIST reference library.

2.4.6. Statistical Analysis

Experimental data were statistically analyzed using Excel 2016 (Microsoft Excel, Microsoft Corporation, Redmond, WA, USA) and SPSS 24.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation (SD). Group comparisons were performed using one-way ANOVA and Student’s t-test, with statistical significance defined as p < 0.05. Graphical representations were generated using GraphPad Prism 8.0.2.

3. Results and Discussion

3.1. Effect of Different Carbon Sources on Microalgal Growth, Pigments, and Nutrient Removal

As an essential nutrient, carbon sources significantly affect the growth rate, biomass accumulation, physiological metabolism, and target product synthesis in microalgae. Carbon sources serve as fundamental components for photosynthesis and the cellular metabolism, where appropriate carbon supplementation ensures a sufficient carbon availability to enhance the microalgal proliferation and biomass production [35]. Under mixotrophic cultivation, microalgae perform photosynthesis while simultaneously utilizing both CO2 and organic carbon sources for biomass synthesis. This cultivation mode enables microalgae to integrate phototrophic and heterotrophic metabolic pathways. In other words, they assimilate organic compounds alongside CO2 during heterotrophic phases, while respired CO2 is subsequently captured and reutilized through photosynthetic activity [36]. Mixotrophic cultivation faces two key limitations: extreme light levels (low/high) or an insufficient organic carbon supply, both of which restrict cell growth. Many algal species exhibit metabolic flexibility, utilizing both autotrophic and heterotrophic pathways for biomass synthesis. The capacity of mixotrophs to assimilate organic substrates demonstrates that cellular proliferation is not exclusively reliant on photosynthesis, thereby reducing the absolute dependence on light energy as a growth-limiting factor [37]. Representative species with mixotrophic capabilities include the cyanobacterium Limnospira platensis (formerly Spirulina platensis) and the green microalga Chlamydomonas reinhardtii. Notably, glucose supplementation during both light and dark phases directly affects cell growth [27]. Accordingly, mixotrophic cultivation in wastewater with carbon supplementation seems to be an effective way to boost the algal biomass, achieving dual benefits: an efficient nutrient removal and a simultaneous production of high-value products, such as lipids.
Studies have shown that the type and concentration of carbon sources can modulate cellular metabolic pathways, probably altering the energy allocation and carbon flux within microalgal cells [38]. The supplementation of either inorganic or organic carbon sources can improve the microalgae growth and shortens the cultivation time. Typically, microalgae require CO2 to maintain stable pH levels in their growth medium. Recent research has studied how CO2 impacts the algae biomass, lipid and hydrocarbon production, and fatty acid composition [39]. Microalgae can also use soluble carbonate (CO32−) for growth by either directly absorbing it or converting it to CO2 through the enzyme carbonic anhydrase [40].
Hence, to determine the optimal carbon source for biomass production, FACHB-8 was cultivated in ADSW supplemented with six different inorganic/organic carbon sources: sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), sodium acetate (CH3COONa), glucose, sucrose, and glycerol (10 g/L each). The biomass growth profiles of FACHB-8 under these carbon sources are shown in Figure 2. Table 2 summarizes the biomass production, biomass productivity, maximum biomass productivity, specific growth rate, and maximum specific growth rate of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Figure 2 illustrates that the algal cells in all six carbon-supplemented groups and the control group (CK) exhibited a lag phase during days 0~2. From days 2~6, cells cultured in glucose- or sodium acetate-supplemented ADSW rapidly transitioned to the logarithmic phase, whereas other groups remained in the lag phase (Figure 2). This indicates that the optimal carbon source supplementation shortens the lag phase, accelerates entry into the exponential growth phase, and enhances biomass accumulation. By day 8, growth rates in glucose and sodium acetate groups began to decline as cells entered the stationary phase. Meanwhile, other groups exhibited gradual increases in growth rate, yet their biomass production remained significantly lower than those of the glucose and sodium acetate groups. After 14 days of cultivation, the glucose group achieved the highest biomass (3.90 g/L), followed by the sodium acetate group (3.33 g/L), while the remaining four groups showed a relatively lower biomass production (Figure 2). As shown in Table 2, the glucose group also demonstrated a superior biomass productivity (258.10 mg/L/d) and maximum biomass productivity (830.00 mg/L/d). Previous studies on carbon-supplemented mixotrophic cultivation in microalgae report biomass productivities often ranging from 200 to 500 mg/L/d [41,42,43], within which our glucose-supplemented group (258.10 mg/L/d) falls. Additionally, the specific growth rates in the glucose (0.19/d) and sodium acetate (0.17/d) groups significantly exceeded those of other treatment groups (p < 0.05, Table 2). These results suggest that, aside from glucose and sodium acetate, the other tested carbon sources, especially inorganic carbon sources, exhibited limited efficacy in enhancing the microalgal biomass production in ADSW under mixotrophic cultivation. Among all tested carbon sources, glucose exhibited a superior efficiency in enhancing biomass production for FACHB-8, a finding consistent with Gupta et al.’s report of glucose as the optimal carbon source for enhancing Chlorella biomass in wastewater systems [18].
In previous studies, glucose was identified as the optimal carbon source for enhancing biomass production in microalgae [17,44]. The rapid biomass accumulation in glucose-supplemented cultures is attributed to its efficient assimilation by most microorganisms. It has been recognized that carbon source utilization (e.g., glucose, acetate) through heterotrophic or mixotrophic pathways varies across species and depends on the presence of specific transporters. Notably, glucose is usually preferred in Chlorella species due to dedicated glucose transporters that facilitate cellular uptake, followed by mitochondrial oxidative phosphorylation to produce ATP [26].
Photosynthetic pigment profiles correlate with microalgal physiology and are modulated by strain-specific responses to carbon sources. Therefore, assessing the effect of different carbon sources on pigment profiles is essential. Chlorophyll a, the dominant photosynthetic pigment in microalgae, is widely recognized as a reliable estimator for quantifying phytoplankton biomass. Generally speaking, higher chlorophyll a concentrations typically reflect a more active photosynthesis and increased uptake of nutrients, such as nitrate (NO3−), ammonium (NH4+), and phosphate (PO43−). Thus, chlorophyll a is often positively correlated with nutrient removal efficiency and biomass accumulation [45]. As shown in Figure 3a, the chlorophyll a concentrations increased moderately in all groups after 14 days of cultivation. However, the sodium carbonate and sodium bicarbonate groups exhibited a lower chlorophyll a content compared to the control (CK). Notably, these two groups showed no significant difference in the dry cell weight relative to CK (Figure 2), indicating their inability to enhance the chlorophyll a accumulation or biomass productivity in ADSW. In contrast, glycerol-supplemented cultures achieved higher chlorophyll a levels by day 14 despite slower initial accumulation rates (Figure 3a). The glucose and sodium acetate groups displayed the most rapid chlorophyll a synthesis between days 2 and 4, followed by stabilization and a secondary increase after days 12~14 (Figure 3a). This two-phase trend corresponds to their growth dynamics, as both groups initiated the exponential phase between days 2 and 4 and subsequently entered the stationary phase (Figure 2). The late-phase increase in chlorophyll a could be attributed to the algal adaptation to ADSW and the continuous biomass accumulation supported by the exogenous carbon supplementation. Carotenoids, which function as photoprotectants and antioxidants to mitigate oxidative stress [46], exhibited concentration trends similar to chlorophyll a across various groups (Figure 3b). The levels of carotenoids often increase under high light or environmental stress conditions, helping to maintain a stable metabolism and cell function [46]. As a result, carotenoids are related to the adaptability and long-term productivity of microalgae. Collectively, these results demonstrate that glucose and sodium acetate were the most effective carbon sources for enhancing FACHB-8 growth and pigment synthesis in ADSW.
Nitrogen is a key component of microalgal biomass (~10% of dry weight) [47]. Microalgae efficiently absorb nitrogen under varying substrate concentrations [48]. In terms of nutrient removal, the efficiency of FACHB-8 in ADSW supplemented with different carbon sources is summarized in Table 3. The control group (CK, wastewater only) showed a TN removal of 88.58% and a TP removal of 44.16%. However, the sodium acetate group only achieved a comparative TN removal of 87.65% and a TP removal of 48.32%. The highest TN removal of 90.62% and TP removal of 76.14% were observed in the glucose-supplemented group. As a readily assimilable carbon source, glucose supported an optimal algal growth in ADSW, thereby maximizing the nutrient removal efficiency.
Consistent with these findings, the glucose group also exhibited the highest dry cell weight, chlorophyll a, and carotenoid concentrations, demonstrating its superior capacity to enhance both biomass production and wastewater remediation. This trend aligns with prior studies [49]. Consequently, glucose was identified as the optimal supplemented carbon source and served as the foundation for subsequent investigations.
Microalgae biodiesel, a renewable alternative to petroleum diesel with a reduced carbon footprint [7,8], faces high production costs primarily due to the expensive cultivation process [9,10]. Utilizing wastewater as a nutrient source for microalgae offers dual benefits, cost-effective nutrient recycling and bioremediation [13], while accumulating lipids for biodiesel [13,14,18,19]. However, the limited pollutant removal efficiency hinders scalability. Mixotrophic cultivation with carbon supplementation in wastewater can enhance biomass productivity [31,33], as demonstrated through optimal carbon source screening in ADSW systems in our study.
In our carbon source screening experiments, six inorganic and organic carbon sources—sodium carbonate, sodium bicarbonate, sodium acetate, glucose, sucrose, and glycerol—were selected based on literature reports of mixotrophic microalgae cultivation. According to ICIS Chemicals [50], the market prices (USD/ton) for industrial-grade sodium bicarbonate, sodium carbonate, glucose, sodium acetate, glycerol, and sucrose are 120~150, 100~130, 300~380, 800~1000, 600~800, and 450~550, respectively. Our experimental results identified glucose as the optimal carbon source for ADSW supplementation. However, despite its moderate price ranking among the six candidates, glucose contributes approximately 80% of the total medium costs in mixotrophic systems [51,52], creating economic barriers for large-scale implementation. To address this, agricultural and food industry byproducts rich in glucose or fermentable sugars (e.g., cane molasses, sugarcane bagasse hydrolysates, and starch-processing wastewater) have emerged as viable low-cost alternatives [17,53]. For instance, in the Chinese market, cane molasses costs 130~160 USD/ton [54], significantly lower than industrial glucose (300~380 USD/ton). This means simply substituting glucose with such waste molasses would reduce cultivation costs by approximately 50%. This integrated approach enables a cost-effective swine wastewater remediation coupled with the production of value-added algal biomass, thereby enhancing the scalability of this technology through the utilization of locally available waste streams as carbon feedstocks [11].

3.2. Effect of Different Glucose Concentration on Microalgal Growth, Pigments, and Nutrient Removal

The carbon source concentration significantly impacts microalgal growth rates in mixotrophic cultivation. However, excessive concentrations may inhibit biomass accumulation and lead to substrate inefficiency [55]. Consequently, following the identification of glucose as the optimal carbon source, the effect of varying glucose concentrations (0, 2, 5, 8, 10, 20, 30 g/L) on the biomass growth of FACHB-8 was systematically assessed. ADSW without glucose supplementation (0 g/L) served as the control. As shown in Figure 4, the algal growth was severely inhibited in this control group, with all glucose-supplemented groups exhibiting a higher biomass accumulation than the control after 14 days. Also, enhancing the initial glucose concentration in the ADSW significantly enhanced the biomass growth. During days 0~4, all treatment groups (except the 2 g/L and control groups) showed comparable growth rates. A marked acceleration in biomass accumulation occurred in the 20 g/L group from days 4 to 6, reaching 3.56 g/L by day 6 (Figure 4). This group ultimately achieved the highest biomass production (3.81 g/L) after 14 days of cultivation (Table 4), as compared to 0.58 g/L in the control group. According to Table 4, the 20 g/L group also reached the highest biomass productivity (271.31 mg/L/day) and maximum biomass productivity (1350.00 mg/L/day). Kong et al. [41] reported that culturing C. vulgaris 31 in a modified Bristol medium supplemented with 10 g/L of glucose under mixotrophic cultivation achieved a biomass productivity of 0.226 g/L/day, which is similar to our results. Furthermore, this group showed the highest specific growth rate of 0.42 d−1 (Table 4). In contrast, as readily seen in Figure 4, the 30 g/L group displayed a suppressed growth (1.92 g/L). This suppression was likely caused by excessive glucose inhibiting cellular respiration, thereby impairing growth, a phenomenon aligning with previous studies [29,56]. As shown in Figure 5a, chlorophyll a concentrations in ADSW-supplemented cultures increased compared to the control. Chlorophyll a levels peaked on day 6 across all glucose treatments, which was followed by stabilization. The 2 g/L glucose group achieved the highest chlorophyll a accumulation (27.32 mg/L), while the 20 g/L group showed a slightly lower but comparable value (22.85 mg/L). Figure 5b reveals that carotenoid concentrations increased significantly by day 4 in all groups except the 2 g/L and control treatments, mirroring the chlorophyll a trend. Carotenoid accumulation began on day 4 and stabilized by day 6, indicating a robust algal growth during this period. The 20 g/L glucose group yielded the highest carotenoid concentration (13.57 mg/L) after 14 days of cultivation. These results aligned with the aforementioned growth data, demonstrating the superior growth performance of FACHB-8 in ADSW supplemented with 20 g/L of glucose.
As shown in Table 5, intriguingly, the 2 g/L glucose group achieved the highest TN removal rate (92.15%), while the 20 g/L glucose group showed the highest TP removal rate (82.93%). Numerous studies have reported that microalgae-mediated wastewater treatment often achieves removal efficiencies of 60~99% for TN and 50~95% for total phosphorus TP [14,18,42]. Notably, TN removal rates remained similar (~85%) across all glucose-supplemented groups except the control and the 2 g/L glucose group (Table 5). The 20 g/L glucose group exhibited a relatively higher TN removal rate (88.70%). TP removal rates generally increased with higher glucose concentrations. Nitrogen is critical for cell growth, as microalgae assimilate NH4+-N and NO3−-N to synthesize proteins and nucleic acids [13]. The limited improvement in the TN removal with the increasing glucose may result from an energy reallocation toward biomass accumulation rather than nitrogen assimilation. In contrast, the phosphorus (essential for ATP and nucleic acids) uptake improved significantly in glucose-supplemented groups, likely due to the enhanced biomass production (Table 5). The 20 g/L glucose group achieved the highest TP removal (82.93%). Given its balanced TN removal (88.70%) and optimal TP removal, the 20 g/L glucose group demonstrated a superior wastewater purification efficiency.
In summary, the 20 g/L glucose supplementation group demonstrated an optimal algal growth and superior nutrient removal efficiency. Therefore, 20 g/L was selected as the optimal glucose concentration for ADSW treatment and was applied in subsequent studies.

3.3. Biomass Growth and Nutrient Removal Capacity of FACHB-8 in ADSW with Optimized Glucose Supplementation

Based on the previous results, this study evaluated the growth of FACHB-8 in ADSW with an optimized glucose supplementation (20 g/L). Compared to the control (CK, no glucose), the optimized group exhibited a dramatic biomass accumulation on day 4, entering the exponential phase (Figure 6a). The specific growth rate under optimized conditions (0.42/d) exceeded that of the control (0.31/d). After 14 days of cultivation, the biomass productivity reached 271.31 mg/L/day in the optimized group—6.72 times higher than the control (40.36 mg/L/day). At the same time, a TN removal of 88.70% and a TP removal of 82.93% was observed (Figure 6b). These findings confirm the efficacy of glucose supplementation in enhancing the FACHB-8 growth and wastewater treatment efficiency, while enabling the co-production of high-value products like lipids.
To further evaluate the nutrient removal trends of FACHB-8 under an optimal glucose supplementation (20 g/L), the time course changes in TN and TP concentrations in ADSW were measured. As shown in Figure 7a, the TN was rapidly removed within the first 8 days and stabilized afterward. The TN assimilation by Chlorella cells primarily occurred through chemical synthesis and enzymatic reactions within cells. Ammonium (NH4+-N) and nitrate (NO3−-N) served as the main nitrogen sources. These were converted into ammonia and nitrate through ammonification and nitrite reduction, followed by the nitrification and enzymatic synthesis of amino acids [15,57]. Consequently, TN concentrations decreased steadily as microalgae grew. The TN removal reached 62.68% by day 6 and 88.70% at the end of cultivation, indicating a robust TN removal performance.
For the TP removal (Figure 7b), TP concentrations increased during days 0~2 but declined rapidly in the subsequent 12 days. Reports have shown that microalgae possess a unique phosphorus storage mechanism that enables them to absorb excess phosphorus beyond immediate growth requirements, storing it as phosphate within intracellular polymeric substances [58]. During the initial 0~2 days, low metabolic activity might trigger phosphate release from the intracellular region into the medium, probably explaining the observed TP concentration increase in ADSW (Figure 7b). After day 4, cells entered the exponential growth phase. The elevated phosphorus levels and restored photosynthetic efficiency might promote excessive phosphorus uptake [59], driving a sharp TP decline (about 17 mg/L reduction from days 4 to 8, Figure 7b). After day 10, TP concentrations stabilized at a low level of approximately 4 mg/L (Figure 7b). Most of the phosphorus was consumed (~80%). This suggests that phosphorus might be the limiting factor in this study. This finding is similar to the results observed in previous studies [31]. After 14 days of cultivation, the TP decreased to 3.32 mg/L, compliant with China’s livestock wastewater discharge standard (GB 18596-2001; ≤8 mg/L) [60]. These results confirm the strong TP removal capacity of FACHB-8 under an optimized glucose supplementation. In this study, the significant phosphorus removal might not only result from algal assimilation through metabolic pathways but also from external environmental conditions. Previous studies suggest that phosphorus may precipitate when the pH exceeds 8.0 and dissolved oxygen levels are high [14]. Under optimized conditions, microalgae with a robust growth were likely to absorb atmospheric CO2 during growth, increasing the culture medium’s pH [61,62]. This might lead to promoted phosphorus precipitation, which was thus removed during sampling and centrifugation, leading to a significant increase in the TP removal efficiency. In other words, the notable improvement in the TP removal is not entirely due to microalgal metabolic assimilation, a phenomenon also observed in related studies [31,57]. Taken together, these findings indicate that the glucose added to the wastewater was efficiently assimilated by C. vulgaris cells, promoting biomass accumulation and nutrient utilization.

3.4. Lipid Production, Biomass, and FAME Composition in ADSW with Optimized Glucose Supplementation

Microalgae can thrive in wastewater containing various pollutants and produce bioactive compounds, such as lipids, proteins, and carbohydrates. This approach is eco-friendly and cost-effective for wastewater treatment. This study evaluated the biomass composition and lipid productivity of the oleaginous strain FACHB-8 under an optimal carbon supplementation to assess its potential for a simultaneous lipid production. After 14 days of cultivation in ADSW, cellular components included 45.59% lipids, 29.72% proteins, and 13.05% carbohydrates (Figure 8). Lipids were the most abundant component, followed by proteins and then carbohydrates. The lipid production reached 1.73 g/L with a productivity of 123.86 mg/L/day, highlighting its good potential for lipid production. The lipid content obtained in our studies (45.59%) surpasses that reported in previous studies, in which a 19~27% lipid content was observed in Chlorella species using different carbon supplement strategies [18,41]. Our results position C. vulgaris FACHB-8 as a promising oleaginous microalgal feedstock for renewable biodiesel production. Furthermore, the biomass exhibited a protein content of 29.72%, suggesting its viability as a feedstock for animal feed production. In contrast, the biomass of the control group contained 33.86% lipids, 10.63% proteins, and 8.30% carbohydrates (Figure 8).
Lipid accumulation is influenced by multiple factors, including the growth conditions (autotrophic, heterotrophic, or mixotrophic cultivation), culture age, algal strain, light intensity, pH, and nutrient supply [63]. The carbon source in the culture medium contributes to 17~65% of the dried cell weight of microalgae [64]. Microalgae utilize glucose to generate ATP, with glucose metabolism relying on cellular energy sources, enzymes, metabolites, and flux requirements. Glucose is converted into pyruvate and Acetyl-CoA, which is transported to the plastid for triacylglycerol synthesis; the resulting triacylglycerol and fatty acids are then returned to the cytosol for lipid assembly. Therefore, carbon supplementation significantly enhances lipid accumulation, as shown in this study. Nitrogen accounts for 1~14% of the microalgae dried cell weight [64]. While nitrogen significantly enhances the biomass growth, it has a limited impact on lipid accumulation, and a high biomass productivity does not correlate with a high lipid content [65]. Lipid accumulation typically initiates after nutrient depletion for biomass growth. Strategies such as nitrogen starvation have been demonstrated to improve the lipid and carbohydrate production [66]. Notably, lipid accumulation may also be driven by excess carbon availability during nutrient depletion, rather than solely by nutrient starvation [29]. In this study, an enhanced lipid accumulation was observed under nutrient deficient conditions after day 8 (Figure 7), while the residual carbon in the medium might therefore be utilized for lipid synthesis rather than biomass growth. In addition, as aforementioned, although wastewater sources generally contain abundant dissolved organic matter, most of these compounds are not readily available as carbon sources for microalgae-driven nutrient removal. Previous studies indicate that ADSW contains significant amounts of refractory organic matter, which is challenging for microalgae to degrade and utilize [67]. Therefore, the presence of more carbon substances is desirable to improve both the biomass and lipid accumulation. In addition, the optimal performance of strain FACHB-8 was achieved with the 20 g/L glucose supplementation, resulting in a maximum biomass productivity of 271.31 mg/L/d, alongside a maximum lipid content (45.59%) and lipid productivity (123.86 mg/L/d). The improvement was notably significant when additional carbon sources (e.g., glucose) were supplemented.
The fatty acid composition determines the quality of biodiesel derived from microalgae. Lipids extracted from FACHB-8 were esterified and analyzed via gas chromatography (Table 6). C16~C18 fatty acids accounted for ~80% of the total fatty acids, similar to prior studies (80~90%) [68,69]. The primary fatty acids in FACHB-8 included palmitic acid (C16:0, 26.99%), linoleic acid (C18:2, 25.51%), oleic acid (C18:1, 15.97%), and arachidic acid (C20:0, 13.00%). Saturated (SFA, 50.77%) and unsaturated fatty acids (UFA, 49.23%) were nearly balanced. Tan et al. [70] also found that the FAME composition of C. pyrenoidosa cultured in ADSW supplemented with 0.8% glucose was 51.14% SFA and 48.86% UFA, which is consistent with this study. Microalgae with balanced SFA/UFA ratios are generally regarded as ideal for biodiesel due to their oxidative stability and cold flow properties [71]. Thus, FACHB-8 lipids are suitable for biodiesel production.

4. Conclusions

This work establishes a viable mixotrophic algal cultivation strategy using glucose-supplemented ADSW to synergistically enhance biomass production, nutrient removal, and lipid synthesis. The optimization with 20 g/L of glucose in ADSW maximized the biomass yield (271.31 mg/L/day), pigment accumulation, and nutrient removal efficiency, achieving an 88.70% TN and 82.93% TP removal. The process simultaneously generated high-value bioproducts: 45.59% lipids, 29.72% proteins, and 13.05% carbohydrates. Notably, the lipid productivity (123.86 mg/L/day) and balanced saturated (50.77%) and unsaturated (49.23%) FAME profiles validate the potential of C. vulgaris FACHB-8 for both wastewater treatment and biodiesel feedstock applications.
However, these findings are constrained by laboratory-scale conditions without the verification of industrial-scale robustness or interference from complex contaminants in raw wastewater. Future research should focus on scaling the proposed cultivation process in pilot systems to validate its technical and economic feasibility under real-world conditions. Additionally, evaluating the adaptability of this glucose-enhanced mixotrophic strategy across diverse wastewater streams (e.g., municipal and industrial) could broaden its applicability. Exploring algae–bacteria symbiotic consortia may further enhance the nutrient recovery efficiency and system stability by leveraging microbial cross-feeding mechanisms. Further investigation into the integration of carbon supplementation with other nutrient optimization strategies (e.g., nitrogen–phosphorus ratios) may unlock synergistic effects to maximize both the biomass productivity and pollutant removal efficiency.

Author Contributions

Conceptualization, B.Y. and J.F.; data curation, C.H., Q.C., J.W., X.C., and H.Y.; writing—original draft preparation, C.Z. and M.Y.; writing—review and editing, B.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Scientific Research Fund of Hunan Provincial Education Department (No. 22A0151), the National Natural Science Foundation of China (No. 31800076), the Scientific Research Project of Hunan Provincial Department of Science and Technology (No. 2023NK4146), and Natural Science Foundation of Hunan province, China (No. 2019JJ50245).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data of this study are available from the correspondence author upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The schematic of the experimental setup.
Figure 1. The schematic of the experimental setup.
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Figure 2. Biomass growth of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Figure 2. Biomass growth of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
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Figure 3. Time course profile of chlorophyll a concentration (a) and carotenoid concentration (b) for FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Figure 3. Time course profile of chlorophyll a concentration (a) and carotenoid concentration (b) for FACHB-8 cultivated in ADSW supplemented with different carbon sources.
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Figure 4. Biomass growth of FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
Figure 4. Biomass growth of FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
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Applsci 15 05103 g005
Figure 5. Time course profile of chlorophyll a concentration (a) and carotenoid concentration (b) for FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
Figure 5. Time course profile of chlorophyll a concentration (a) and carotenoid concentration (b) for FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
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Applsci 15 05103 g006
Figure 6. Biomass growth (a) and nutrient removal capacity (b) of FACHB-8 before and after carbon source optimization. Note: Asterisks show significant difference from the data before optimization (t test, * p < 0.05).
Figure 6. Biomass growth (a) and nutrient removal capacity (b) of FACHB-8 before and after carbon source optimization. Note: Asterisks show significant difference from the data before optimization (t test, * p < 0.05).
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Figure 7. Changes in removal of TN (a) and TP (b) by FACHB-8 cultivated in ADSW with optimized glucose supplementation within 14 days.
Figure 7. Changes in removal of TN (a) and TP (b) by FACHB-8 cultivated in ADSW with optimized glucose supplementation within 14 days.
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Applsci 15 05103 g008
Figure 8. Cellular components of FACHB-8 before and after carbon source optimization.
Figure 8. Cellular components of FACHB-8 before and after carbon source optimization.
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Table 1. Compositions of ADSW.
Table 1. Compositions of ADSW.
ParametersNon-Autoclaved ADSWAutoclaved ADSW
TN (mg/L)370.67 ± 26.42332.47 ± 41.37
TP (mg/L)16.68 ± 0.8112.400 ± 0.91
NH4+-N (mg/L)221.60 ± 3.60100.73 ± 12.59
COD (mg/L)1050.19 ± 18.83702.25 ± 12.18
TOC (mg/L)356.45 ± 10.72168.01 ± 5.74
IC (mg/L)301.32 ± 10.67157.98 ± 6.65
pH7.45 ± 0.628.47 ± 0.53
Table 2. Biomass growth profiles of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Table 2. Biomass growth profiles of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Carbon SourceBiomass Production (g/L)Biomass Productivity (mg/L/d)Maximum Biomass Productivity (mg/L/d)Specific Growth Rate (/d)Maximum Specific Growth Rate (/d)
CK0.92 ± 0.09 e43.81 ± 9.67 e166.67 ± 16.27 e0.08 ± 0.02 c0.31 ± 0.02 b
NaHCO30.99 ± 0.17 de50.95 ± 15.68 de261.25 ± 51.27 cd0.09 ± 0.02 c0.27 ± 0.11 b
Glucose3.90 ± 0.15 a258.10 ± 15.24 a830.00 ± 45.89 a0.19 ± 0.01 a0.86 ± 0.04 a
Na2CO30.88 ± 0.12 e42.26 ± 12.00 e185.00 ± 45.21 ed0.08 ± 0.02 c0.28 ± 0.08 b
CH3COONa3.33 ± 0.23 b216.31 ± 18.63 b542.50 ± 4.33 b0.17 ± 0.01 a0.70 ± 0.01 a
Glycerol1.62 ± 0.17 c94.52 ± 10.82 c301.25 ± 37.12 c0.12 ± 0.01 b0.29 ± 0.04 b
Sucrose1.31 ± 0.10 cd70.83 ± 3.93 cd235.83 ± 46.46 cde0.10 ± 0.01 bc0.40 ± 0.19 b
Note: Values with different letters represent significant difference (p < 0.05) between treatments.
Table 3. Nutrient removal of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Table 3. Nutrient removal of FACHB-8 cultivated in ADSW supplemented with different carbon sources.
Carbon SourceTNTP
Initial Concentration (mg/L)Final Concentration (mg/L)Removal Efficiency (%)Initial Concentration (mg/L)Final Concentration (mg/L)Removal Efficiency (%)
CK332.47 ± 41.3737.77 ± 5.8688.58 ± 2.26 b12.40 ± 0.916.93 ± 0.4144.16 ± 1.13 b
NaHCO3110.20 ± 6.0035.40 ± 2.6967.84 ± 3.00 c21.47 ± 1.606.23 ± 0.8943.76 ± 4.38 b
Glucose453.13 ± 11.3542.43 ± 3.8490.62 ± 1.19 a17.53 ± 0.345.78 ± 1.0076.14 ± 0.76 a
Na2CO3189.80 ± 18.3724.95 ± 3.6086.56 ± 3.89 ab20.33 ± 1.004.18 ± 0.1323.06 ± 6.78 c
CH3COONa189.20 ± 22.8423.48 ± 3.6987.65 ± 0.62 a24.53 ± 3.8712.44 ± 0.9048.32 ± 9.76 b
Glycerol233.87 ± 17.5045.37 ± 6.1580.32 ± 5.02 b12.60 ± 0.7112.03 ± 0.2450.63 ± 7.82 b
Sucrose281.00 ± 37.5431.367 ± 1.47985.25 ± 5.29 ab19.00 ± 1.8815.81 ± 0.7769.93 ± 3.78 a
Note: Values with different letters represent significant difference (p < 0.05) between treatments.
Table 4. Biomass growth profiles of FACHB-8 cultivated in ADSW supplemented with varying concentrations of glucose.
Table 4. Biomass growth profiles of FACHB-8 cultivated in ADSW supplemented with varying concentrations of glucose.
Glucose Concentration (g/L)Biomass Production (g/L)Biomass Productivity (mg/L/d)Maximum Biomass Productivity (mg/L/d)Specific Growth Rate (/d)Maximum Specific Growth Rate (/d)
00.58 ± 0.07 e40.36 ± 6.84 e75.00 ± 14.14 f0.31 ± 0.07 a0.88 ± 0.24 b
21.40 ± 0.06 d98.45 ± 4.17 d240.00 ± 13.23 e0.35 ± 0.09 a0.85 ± 0.01 b
51.70 ± 0.02 c119.17 ± 1.09 c724.17 ± 92.38 b0.30 ± 0.06 a1.24 ± 0.15 ab
81.84 ± 0.03 c129.29 ± 1.29 c383.75 ± 90.16 d0.33 ± 0.08 a1.62 ± 0.29 a
103.04 ± 0.12 b213.21 ± 13.27 b506.67 ± 62.77 cd0.31 ± 0.07 a0.99 ± 0.47 ab
203.81 ± 0.21 a271.31 ± 18.02 a1350.00 ± 91.92 a0.42 ± 0.05 a1.00 ± 0.17 ab
301.92 ± 0.10 c135.71 ± 8.92 c601.67 ± 25.66 bc0.37 ± 0.07 a1.45 ± 0.56 ab
Note: Values with different letters represent significant difference (p < 0.05) between treatments.
Table 5. Nutrient removal of FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
Table 5. Nutrient removal of FACHB-8 cultivated in ADSW supplemented with varying glucose concentrations.
Glucose Concentration (g/L)TNTP
Initial Concentration (mg/L)Final Concentration (mg/L)Removal Efficiency (%)Initial Concentration (mg/L)Final Concentration (mg/L)Removal Efficiency (%)
0329.50 ± 50.7727.03 ± 0.9691.60 ± 1.00 ab19.21 ± 0.8613.87 ± 3.3637.38 ± 9.56 e
2349.90 ± 13.7127.90 ± 2.4192.15 ± 0.63 a21.53 ± 1.9712.82 ± 1.9940.58 ± 5.73 de
5347.93 ± 14.0141.95 ± 4.8587.97 ± 0.93 c18.80 ± 1.004.13 ± 0.6078.03 ± 2.86 a
8312.33 ±12.9949.25 ± 2.1784.76 ± 0.62 d19.95 ± 0.939.32 ± 0.4953.21 ± 3.63 cd
10347.47 ± 29.2247.42 ± 3.8986.22 ± 2.34 cd22.18 ± 0.888.95 ± 1.0459.65 ± 4.52 bc
20329.80 ± 23.5537.23 ± 4.4488.70 ± 1.17 bc19.73 ± 2.543.32 ± 0.5682.93 ± 4.18 a
30297.40 ± 34.4746.33 ± 0.7484.27 ± 1.96 d22.25 ± 1.266.55 ± 1.2870.70 ± 4.23 ab
Note: Values with different letters represent significant difference (p < 0.05) between treatments.
Table 6. Fatty acid methyl ester (FAME) composition (% total fatty acids) of FACHB-8 after carbon source optimization.
Table 6. Fatty acid methyl ester (FAME) composition (% total fatty acids) of FACHB-8 after carbon source optimization.
Fatty AcidPercentage (%)
C14:04.51 ± 0.29
C14:12.96 ± 0.16
C16:026.99 ± 0.72
C16:14.16 ± 0.63
C18:06.26 ± 0.15
C18:1 n-915.97 ± 1.11
C18:2 n-625.51 ± 1.09
C20:013.00 ± 0.42
C20:5 n-30.64 ± 0.11
SFA a50.77 ± 0.97
UFA b49.23 ± 0.97
MUFA c23.09 ± 1.63
PUFA d26.14 ± 0.98
Note: a, saturated fatty acids; b, monounsaturated fatty acids; c, unsaturated fatty acids; and d, polyunsaturated fatty acids.
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Zhou, C.; Yuan, M.; Huang, C.; Chen, Q.; Wang, J.; Chen, X.; Yang, H.; Fang, J.; Yang, B. Enhancing Biomass Production of Chlorella vulgaris in Anaerobically Digested Swine Wastewater Using Carbon Supplementation and Simultaneous Lipid Production. Appl. Sci. 2025, 15, 5103. https://doi.org/10.3390/app15095103

AMA Style

Zhou C, Yuan M, Huang C, Chen Q, Wang J, Chen X, Yang H, Fang J, Yang B. Enhancing Biomass Production of Chlorella vulgaris in Anaerobically Digested Swine Wastewater Using Carbon Supplementation and Simultaneous Lipid Production. Applied Sciences. 2025; 15(9):5103. https://doi.org/10.3390/app15095103

Chicago/Turabian Style

Zhou, Chenkai, Mingmin Yuan, Cuifeng Huang, Qiqi Chen, Jiamin Wang, Xinting Chen, Hua Yang, Jun Fang, and Bo Yang. 2025. "Enhancing Biomass Production of Chlorella vulgaris in Anaerobically Digested Swine Wastewater Using Carbon Supplementation and Simultaneous Lipid Production" Applied Sciences 15, no. 9: 5103. https://doi.org/10.3390/app15095103

APA Style

Zhou, C., Yuan, M., Huang, C., Chen, Q., Wang, J., Chen, X., Yang, H., Fang, J., & Yang, B. (2025). Enhancing Biomass Production of Chlorella vulgaris in Anaerobically Digested Swine Wastewater Using Carbon Supplementation and Simultaneous Lipid Production. Applied Sciences, 15(9), 5103. https://doi.org/10.3390/app15095103

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