Biomass Production of Chlorella vulgaris var. vulgaris TISTR 8261 During Cultivation in Modified Food Industry Wastewater

Abstract

Industrial wastewater can serve as a low-cost nutritional source for sustainable microalgal biomass production. This study investigated the biomass of Chlorella vulgaris var. vulgaris TISTR 8261 grown in untreated wastewater collected from four food industry factories in Phra Nakhon Sri Ayutthaya Province, Thailand. Among them, wastewater from a processed food production plant (PFPP) supported the highest algal growth. Supplementation with 17.4 mM sodium acetate significantly improved algal biomass yield. Further optimization with 3.7 mM NH₄Cl, 1.0 mM KH₂PO₄, 0.2 mM MgSO₄, and a moderate concentration of trace minerals enhanced the specific growth rate and chlorophyll concentration. Scaled-up cultivation in 3.5 L culture bottles in optimized PFPP yielded a maximum biomass yield of 8.436 ± 0.378 g L⁻¹, comparable to 6.498 ± 0.436 g L⁻¹ in standard TAP medium. Biomass composition analysis after 15 days of cultivation revealed 42.70 ± 1.40% protein, 17.10 ± 1.60% carbohydrate, and 1.90 ± 0.10% lipid on a dry weight basis. These findings demonstrate that optimized PFPP wastewater can effectively support high-density cultivation of C. vulgaris var. vulgaris TISTR 8261, yielding nutritionally rich biomass, and offering a cost-effective and environmentally sustainable strategy for industrial-scale microalgal production.

1. Introduction

The food-processing industry is one of the major sources of high-strength wastewater due to its use of raw materials rich in organic matter and complex manufacturing processes. Food industry wastewater is typically characterized by high levels of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) [1], and it also contains significant amounts of macronutrients such as nitrogen, phosphorus, and sulfur, originating from biological materials including animal products, vegetables, and fruits. If not properly treated, this wastewater can lead to serious environmental issues, such as water pollution and eutrophication [2]. Nevertheless, such wastewater also represents a valuable nutrient source for microalgal cultivation, supporting circular economic strategies and sustainable development by enabling simultaneous biomass production and wastewater remediation [3,4].

Microalgae are photosynthetic microorganisms capable of absorbing high amounts of nutrients, with tolerance to environmental fluctuations. Microalgae can grow under mixotrophic or heterotrophic conditions by utilizing organic carbon compounds present in wastewater [5,6,7]. Many microalgal strains have been reported to grow efficiently in various types of wastewater and are widely applied in integrated systems for biomass production and wastewater treatment. These include Chlamydomonas sp. [8,9], Chlorella vulgaris [10,11], Desmodesmus sp. [12,13], Scenedesmus sp. [14,15], and Tetradesmus sp. [16]. Among these, Chlorella vulgaris is recognized as one of the most promising species due to its rapid growth, resilience to diverse environments, and high-value biochemical composition. Several studies have report that biomass composition typically contains proteins in the range of ~19–55% DW [17,18,19], lipids ~5–58% DW [20,21], carbohydrates ~10–50% DW [22,23], and pigments (chlorophylls and carotenoids) around ~1–2% DW [24,25]. These components have applications in food, feed, nutraceuticals, and biofuels [26].

In laboratory-scale cultivation, Chlorella vulgaris is often grown in nutrient-rich synthetic media such as CHU-10 [27], Bold’s Basal [28], Blue Green-11 [29], and Tris-Acetate-Phosphate (TAP) medium [30], which contains expensive macronutrient, micronutrient, and mineral sources. It is thus economically unfeasible for industrial-scale application. As a result, there is growing interest in cultivating C. vulgaris using alternative low-cost nutrient sources such as municipal, agricultural, and food-processing wastewater [31]. Studies have shown that C. vulgaris can reduce nutrient concentrations in wastewater while producing valuable biomass. For instance, C. vulgaris var. vulgaris TISTR 8261 cultivated in frozen food industrial treated wastewater supplemented with 17.4 mM sodium acetate yielded a biomass concentration of 18.80 g L⁻¹ [6]. Similarly, C. vulgaris UTX-265 cultivated in brewery wastewater with 10 g L⁻¹ glucose reached 3.20 g L⁻¹ biomass [32], while C. vulgaris cultivated in a 75% dilution of dairy industry wastewater yielded 2.43 g L⁻¹ [33]. Thailand has considerable potential for large-scale open-pond cultivation of microalgae due to its favorable climate, including year-round sunlight and suitable temperatures, as well as the abundance of food industry wastewater. These conditions could significantly reduce cultivation costs. However, knowledge gaps remain regarding the optimal nutrient composition of wastewater for supporting microalgal growth. Food-processing wastewater in Thailand often presents imbalanced nutrient profiles, with either deficiencies or excesses in key elements such as carbon, nitrogen, phosphorus, sulfur, and trace minerals, which can limit algal growth and photosynthetic efficiency [34]. Organic carbon scarcity, in particular, poses a major constraint, requiring the supplementation of external carbon sources like sodium acetate to boost biomass accumulation [35,36]. Additionally, trace elements such as iron, manganese, zinc, and copper—though required in minute quantities—play essential roles in enzymatic functions, pigment synthesis, and electron transport. Their deficiency or imbalance may adversely affect algal metabolism [37]. Recent studies have demonstrated that supplementation with trace minerals can restore physiological function, increase chlorophyll content, and enhance growth under nutrient-limited conditions [38,39].

Therefore, this study aims to investigate the feasibility of using food-processing wastewater as a nutrient source for the cultivation of Chlorella vulgaris var. vulgaris TISTR 8261. The focus is on optimizing carbon source concentrations in combination with nitrogen, phosphorus, sulfur, and trace mineral supplementation to improve algal growth and biomass production. The most favorable cultivation conditions will be further evaluated in 3.5 L culture bottles. The findings are expected to contribute to the development of a cost-effective and sustainable microalgae cultivation strategy utilizing available food industrial wastewater in Thailand.

2. Materials and Methods

2.1. Collection of Wastewater Samples

Wastewater samples were collected from four distinct food manufacturing facilities located in Phra Nakhon Sri Ayutthaya Province, Thailand. These included a beverage production plant (BPP), a dairy product production plant (DPPP), a frozen pizza dough production plant (FPDPP), and a processed food production plant (PFPP). The BPP facility produces beverages such as green tea and fruit juices. The DPPP facility manufactures a variety of dairy products including sweetened condensed milk, UHT milk, pasteurized milk, soy milk, and sterilized milk. The FPDPP facility produces frozen pizza dough, frozen pizzas, snacks, and fried chicken. The PFPP facility produces a variety of ready-to-eat meals, frozen foods, and chilled foods, including products such as steamed chicken breast, fried chicken, sausages, and other processed meat items. Wastewater was collected from the initial treatment stream of each facility during February 2024 and transported to the laboratory. To minimize variability in experimental conditions and ensure consistent comparison across treatments, a single sampling period was chosen.

2.2. Analysis of Wastewater

Immediately after collection, pH and dissolved oxygen (DO) levels were measured on-site using a portable multi-parameter meter (HQ40D, Hach, Loveland, CO, USA). Wastewater samples were filtered through Whatman No. 3 filter paper (90 mm diameter; Cytiva, Marlborough, MA, USA) to remove suspended solids before analysis. The wastewater samples were analyzed in their raw state without autoclaving to represent the original characteristics. Biochemical oxygen demand (BOD) was determined using a BOD analyzer (OxiDirect, Lovibond, Dortmund, Germany), while chemical oxygen demand (COD) and total nitrogen (TN) were analyzed following the standard methods of American Public Health Association (APHA) 5220 C and 4500-N C, respectively [40]. Total Kjeldahl nitrogen (TKN), representing organic nitrogen and ammonia nitrogen, was determined according to APHA method 4500-Norg B and C [40]. Total carbon (TC), total inorganic carbon (TIC), and total organic carbon (TOC) were measured using a TOC analyzer (TOC-VCSH, Shimadzu, Kyoto, Japan). Total phosphorus (TP), total sulfur (TS), and concentrations of metal ions (Ca²⁺, Mg²⁺, K⁺, Na⁺, Mn²⁺, Cu²⁺, Zn²⁺ and total dissolved Fe (TDFe)) were quantified via inductively coupled plasma–optical emission spectrometry (ICP-OES) (Optima 7300 DV, PerkinElmer, Waltham, MA, USA). Concentrations of nitrate (NO₃⁻), nitrite (NO₂⁻), phosphate (PO₄³⁻), and sulfate (SO₄²⁻) were determined using ion chromatography (Dionex ICS-5000+ DP, Thermo Fisher Scientific, Waltham, MA, USA). Prior to cultivation, wastewater samples were adjusted to pH 7.2 using NaOH or HCl and sterilized by autoclaving at 121 °C for 15 min to ensure no microorganism contamination. It is noted that for industrial applications, alternative sterilization methods such as UV treatment, membrane filtration, or lower-temperature heat treatment could be applied to reduce energy costs.

2.3. Green Algal Strain and Cultivation

The unicellular green alga Chlorella vulgaris var. vulgaris TISTR 8261 was obtained from the Thailand Institute of Scientific and Technological Research (TISTR), Pathum Thani Province, Thailand. To prepare for a starter culture, C. vulgaris var. vulgaris TISTR 8261 was cultivated in 250 mL Erlenmeyer flasks containing 100 mL of sterilized Tris-Acetate-Phosphate (TAP) medium [41]. Cultures were incubated at 25 ± 2 °C under continuous shaking at 120 rpm, illuminated with white fluorescent light at an intensity of 30 µmol photon m⁻² s⁻¹ for 36 h. Cells were harvested by centrifugation at 7000× g, 20 °C for 10 min, washed twice with sterile phosphate buffer, and resuspended in wastewater. The initial cell concentration was adjusted to an optical density at 750 nm (OD₇₅₀) of approximately 0.1, which corresponds to 7.5 × 10⁴ cells mL⁻¹. Cell culture was shaken under the previously described conditions for 14 days.

For laboratory-scale cultivation, C. vulgaris var. vulgaris TISTR 8261 was inoculated into transparent 3.5 L glass culture bottles at a working volume of 3000 mL, with an initial OD₇₅₀ of ~0.1. The cell cultures were cultivated at 25 ± 2 °C under continuous magnetic stirring to provide aeration and facilitate gas exchange, with white fluorescent light at an intensity of 100 μmol photons m⁻² s⁻¹ for 15 days. The pH of the cultures was measured every 2 days using a pH meter (Starter 2100, Ohaus, Parsippany, NJ, USA).

2.4. Growth, Total Cell Concentration and Dry Weight Determination

A total of 1 mL was sampled from 100 mL of the cell culture. The growth of C. vulgaris var. vulgaris TISTR 8261 was monitored from an algal sample by measuring the OD₇₅₀ using a UV–visible spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). Total cell concentration was assessed using a hemocytometer (BOECO, Hamburg, Germany) under a light microscope (CX23, Olympus, Tokyo, Japan), with three independent counts per sample [30]. To determine the dry weight of algal biomass, 10 mL of culture was filtered through a pre-weighed GF/C glass microfiber filter (47 mm diameter; Whatman, Maidstone, UK). The filter was washed twice with distilled water, dried at 85 °C in a hot-air oven for 16 h, and then transferred to a desiccator for 1 h prior to final weighing [6].

The specific growth rate of C. vulgaris var. vulgaris TISTR 8261 was calculated using the formula described by Sekine et al. [42] using Equation (1).

Specific growth rate (day⁻¹) = (ln (x₂/x₁))/t₂ − t₁

(1)

where x₁ and x₂ are the OD₇₅₀ at time t₁ and t₂ (day), respectively.

The doubling time of C. vulgaris var. vulgaris TISTR 8261 was performed according Jafarpour et al. [43] using Equation (2).

Doubling time (day) = 0.6931/μ

(2)

where μ is the specific growth rate (day⁻¹).

Biomass productivity was calculated as described by Elshobary et al. [44] using Equation (3).

Biomass productivity (g L⁻¹ day⁻¹) = x₂ − x₁/t₂ − t₁

(3)

where x₁ and x₂ are the dry weights of algal biomass (g L⁻¹) at time t₁ and t₂ (day), respectively. Both specific growth rate and biomass productivity calculations were performed using data obtained during the exponential growth phase of C. vulgaris var. vulgaris TISTR 8261.

2.5. Total Chlorophyll Concentration Determination

Total chlorophyll concentration was determined by harvesting algal cells via centrifugation at 7000× g for 10 min at 20 °C. Chlorophyll was extracted from the cell pellet by incubating cells with 90% (v/v) methanol at 70 °C under dark conditions for 4 h. The absorbance of the extracts was measured at 665 nm and 650 nm using a UV–visible spectrophotometer. Total chlorophyll concentration was calculated using the formula described by Lee and Shen [45] using Equation (4).

Total chlorophyll (mg L⁻¹) = (4.0 × A₆₆₅) + (25.5 × A₆₅₀)

(4)

where A₆₆₅ is absorbance at 665 nm and A₆₅₀ is absorbance at 650 nm.

2.6. Growth Optimization

In normal TAP medium, sodium acetate at a concentration of 17.4 mM is used as the carbon source. TAP also contains macronutrient components such as ammonium chloride (NH₄Cl), potassium dihydrogen phosphate (KH₂PO₄), and magnesium sulfate (MgSO₄) at concentrations of 3.73 mM, 1.0 mM, and 0.2 mM, respectively. In addition, TAP requires a trace mineral solution comprising 18.0 μM FeSO₄.7H₂O, 76.5 μM ZnSO₄.7H₂O, 184.3 μM H₃BO₃, 25.8 μM MnCl₂.4H₂O, 0.65 μM CuSO₄.5H₂O, 3.0 μM Na₂MoO₄.2H₂O, and 0.65 μM CoCl₂.6H₂O [41]. To optimize algal growth, C. vulgaris var. vulgaris TISTR 8261 was cultivated in wastewater supplemented with various sodium acetate concentrations ranging from 0 to 1.74, 3.48, 8.70, 17.40, and 34.80 mM. Algal growth was determined under the previously described conditions. Subsequently, C. vulgaris var. vulgaris TISTR 8261 was cultivated in wastewater containing sodium acetate with suitable concentration and supplemented with macronutrients including 3.73 mM NH₄Cl, 1.0 mM KH₂PO₄, and 0.2 mM MgSO₄. These concentrations were chosen to match those in the standard TAP medium. Additionally, the trace mineral solution containing essential elements was added at varying concentrations of 0.1×, 0.2×, 0.5×, and 1.0×, relative to the TAP formulation. The initial cell concentration was adjusted to an OD₇₅₀ of 0.1. The culture was transferred to a 250 mL Erlenmeyer flask and incubated under the previously described cultivation conditions. Algal growth was monitored daily for 5 days by measuring the optical density at 750 nm. Growth parameters, including chlorophyll concentration, specific growth rate, and doubling time, were also evaluated.

2.7. Biomass Characteristics of C. vulgaris var. vulgaris TISTR 8261

Cells of C. vulgaris var. vulgaris TISTR 8261 cultivated in 3.5 L culture bottles for 15 days were harvested by centrifugation at 7000× g at 4 °C for 10 min. The harvested cells were then dried in a hot-air oven at 85 °C for 16 h and subsequently transferred to a desiccator for 1 h prior to final weighing. A 10 g sample of the dried biomass was submitted for nutritional analysis at the Public–Private Collaborative Research Center (PPCRC), School of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Thailand. The proximate composition—including moisture, crude protein, crude fat, crude fiber, ash, carbohydrate, calcium, and phosphorus contents—was analyzed using the standard methods described by the Association of Official Analytical Chemists (2005) [46].

2.8. Statistical Analysis

All experimental results were expressed as mean values with their corresponding standard deviations (SD) calculated from three independent replicates. Error bars in graphs represent the standard deviation. Prior to performing one-way analysis of variance (ANOVA), data were tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene’s test, with p > 0.05 indicating that assumptions were satisfied. Statistical comparisons among different treatments were then performed using ANOVA, followed by Duncan’s multiple range test to identify significant differences. A significance level of p < 0.05 was applied to determine statistical relevance. Data processing and analysis were carried out using IBM SPSS Statistics software, version 24.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Chemical Characteristics of Wastewater from Food Industry Factories

The chemical characteristics of wastewater collected from Phra Nakhon Sri Ayutthaya Province, Thailand, are summarized in

Table 1. Chemical characteristics of wastewater collected from food industry factories in Phra Nakhon Sri Ayutthaya Province, Thailand, compared with TAP medium. All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates (except for TAP medium). Statistically significant differences among samples within the same row are denoted by distinct superscript letters (a–d) at a confidence level of 95%.

Parameters	Wastewater Sources				TAP
	Frozen Pizza Dough Production Plant (FPDPP)	Dairy Product Production Plant (DPPP)	Processed Food Production Plant (PFPP)	Beverage Production Plant (BPP)
pH	6.87 ± 0.03 b	7.38 ± 0.02 a	7.32 ± 0.01 a	4.81 ± 0.01 c	7.20
DO (mgO2 L−1)	0.080 ± 0.01 b	0.07 ± 0.01 b	0.07 ± 0.01 b	5.88 ± 0.11 a	N/A **
BOD (mgO2 L−1)	693.00 ± 4.90 b	575.00 ± 3.50 c	880.00 ± 1.10 a	605.00 ± 4.70 bc	N/A **
COD (mgO2 L−1)	1536.00 ± 60.30 c	1868.80 ± 11.10 b	2227.20 ± 168.10 ab	2739.20 ± 314.60 a	N/A **
TC (mg L−1)	148.30 ± 5.82 c	132.80 ± 4.12 c	176.90 ± 1.23 b	751.34 ± 6.78 a	417.50
TIC (mg L−1)	1.55 ± 0.52 b	1.62 ± 0.21 b	3.67 ± 0.75 ab	7.14 ± 0.59 a	N/A **
TOC (mg L−1)	146.80 ± 3.67 c	131.20 ± 4.34 c	173.20 ± 7.25 b	744.20 ± 9.87 a	417.50
TN (mg L−1)	17.45 ± 0.24 b	9.44 ± 0.12 c	27.88 ± 0.02 a	4.86 ± 0.02 d	104.80
TKN (mg L−1)	15.53 ± 0.11 b	8.49 ± 0.21 c	25.01 ± 0.15 a	3.06 ± 0.07 d	N/A **
TP (mg L−1)	4.45 ± 0.01 b	4.81 ± 0.01 a	4.99 ± 0.01 a	4.86 ± 0.01 a	15.47
TS (mg L−1)	3.58 ± 0.01 b	2.77 ± 0.01 c	4.17 ± 0.01 a	1.11 ± 0.01 d	16.06
NO3− (mg L−1)	1.66 ± 0.37 b	0.59 ± 0.29 c	2.72 ± 0.25 a	1.62 ± 0.37 b	N/A **
NO2− (mg L−1)	0.26 ± 0.02 a	0.36 ± 0.10 a	0.15 ± 0.07 b	ND *	N/A **
PO43− (mg L−1)	4.16 ± 0.25 a	4.02 ± 0.38 a	4.29 ± 0.10 a	3.92 ± 0.05 a	47.47
SO42− (mg L−1)	2.96 ± 0.07 b	1.97 ± 0.29 bc	3.34 ± 0.04 a	0.63 ± 0.07 d	48.18
Ca2+ (mg L−1)	38.78 ± 0.13 a	17.43 ± 0.11 c	28.62 ± 0.22 b	12.06 ± 0.05 d	16.64
Cu2+ (mg L−1)	0.013 ± 0.01 ab	0.02 ± 0.01 a	0.01 ± 0.00 b	0.01 ± 0.00 b	0.04
TDFe (mg L−1)	0.18 ± 0.01 d	0.42 ± 0.01 a	0.36 ± 0.01 b	0.24 ± 0.01 c	1.00
K+ (mg L−1)	15.61 ± 0.19 d	17.62 ± 0.23 b	16.80 ± 0.01 c	22.06 ± 0.12 a	31.25
Mg2+ (mg L−1)	8.31 ± 0.06 a	4.44 ± 0.02 c	8.60 ± 0.03 a	5.13 ± 0.02 b	9.87
Mn2+ (mg L−1)	0.04 ± 0.01 b	0.02 ± 0.01 c	0.04 ± 0.01 b	0.13 ± 0.01 a	1.41
Na+ (mg L−1)	250.20 ± 4.31 b	159.37 ± 0.03 d	197.07 ± 1.90 c	319.50 ± 2.82 a	0.08
Zn2+ (mg L−1)	0.14 ± 0.07 a	0.11 ± 0.01 ab	0.09 ± 0.01 b	0.09 ± 0.01 b	5.02

* ND: Not detected. ** N/A: Not available.

. The pH values of the wastewater ranged from slightly acidic in BPP to nearly neutral in FPDPP, DPPP, and PFPP. Dissolved oxygen (DO) concentrations in FPDPP, DPPP, and PFPP were markedly low (<0.1 mg O₂ L⁻¹), whereas the DO concentration in BPP was substantially higher (5.88 ± 0.11 mg O₂ L⁻¹). The BOD values were highest in PFPP (880.00 ± 1.10 mg O₂ L⁻¹). Although BPP exhibited the highest COD level (2739.20 ± 314.60 mg O₂ L⁻¹), its BOD was relatively moderate. In terms of carbon content, BPP wastewater showed the highest total carbon (TC), total inorganic carbon (TIC), and total organic carbon (TOC) values, with TOC reaching 744.20 ± 9.87 mg L⁻¹. Conversely, TC, TIC, and TOC levels were relatively similar in DPPP, FPDPP, and PFPP. In the case of macronutrients, total nitrogen (TN) concentrations ranged from 4.86 ± 0.02 mg L⁻¹ in BPP to 27.88 ± 0.02 mg L⁻¹ in PFPP. Total Kjeldahl nitrogen (TKN), representing the sum of organic nitrogen and ammonia nitrogen, followed a similar pattern, with the highest level observed in PFPP (25.01 ± 0.15 mg L⁻¹), followed by FPDPP (15.53 ± 0.11 mg L⁻¹), DPPP (8.49 ± 0.21 mg L⁻¹), and the lowest in BPP (3.06 ± 0.07 mg L⁻¹). Total phosphorus (TP) concentrations were relatively stable across all samples, ranging from 4.45 ± 0.01 mg L⁻¹ in FPDPP to 4.99 ± 0.01 mg L⁻¹ in PFPP. Total sulfur (TS) concentrations also varied among the samples. The highest TS level was observed in PFPP, followed by FPDPP, DPPP, and the lowest in BPP. Regarding major anions, the highest sulfate (SO₄²⁻) concentration was found in PFPP, while the lowest was observed in BPP. Nitrate (NO₃⁻) concentrations were highest in PFPP (2.72 ± 0.25 mg L⁻¹), whereas nitrite (NO₂⁻) was undetectable in BPP. Sodium (Na⁺) levels were highest in BPP and lowest in DPPP. Other key elements such as calcium (Ca²⁺), magnesium (Mg²⁺), potassium (K⁺), and total dissolved iron (TDFe) were present in all wastewater samples with varying concentrations. The highest Ca²⁺ concentration was recorded in FPDPP, while the highest Fe concentration was found in DPPP. Trace levels of manganese (Mn²⁺), copper (Cu²⁺), and zinc (Zn²⁺) were also detected across all samples. Importantly, it can be observed that wastewater from each factory exhibits distinct chemical compositions, with various concentrations of carbon, nitrogen, and other essential nutrients. The result indicates that this wastewater could be used as a culture medium for C. vulgaris var. vulgaris TISTR 8261 cultivation.

3.2. Growth of C. vulgaris var. vulgaris TISTR 8261 in Wastewater

Based on OD₇₅₀ measurements, the green alga C. vulgaris var. vulgaris TISTR 8261 exhibited robust growth in TAP medium starting from the second day of cultivation, with continuous growth until reaching the stationary phase at day 14. In contrast, when cultivated in wastewater from different sources, C. vulgaris var. vulgaris TISTR 8261 showed reduced growth performance compared to TAP medium (Figure 1A). Among the wastewater types, the highest growth was observed in PFPP wastewater. Chlorophyll concentration analysis further supported these findings. C. vulgaris var. vulgaris TISTR 8261 cultured in all four wastewater types showed significantly lower chlorophyll concentration compared to TAP medium (Figure 1B), with reductions ranging from 2.8- to 8.9-fold. Quantitative growth kinetics analysis confirmed these observations. Cultivation in TAP medium resulted in a specific growth rate of 0.549 ± 0.012 day⁻¹ and a doubling time of 1.262 ± 0.012 days. PFPP wastewater supported the second-highest specific growth rate at 0.329 ± 0.022 day⁻¹, with a doubling time of 2.104 ± 0.130 days. In contrast, growth in DPPP and BPP wastewater was slower, and the lowest growth rate was observed in FPDPP wastewater, which exhibited the longest doubling time (

Table 2. The specific growth rate and doubling time in C. vulgaris var. vulgaris TISTR 8261 grown in different media and wastewater. All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates. Statistically significant differences among samples within the same column are denoted by distinct superscript letters (a–e) at a confidence level of 95%.

Culture Media	Specific Growth Rate (Day−1)	Doubling Time (Day)
TAP	0.549 ± 0.012 a	1.262 ± 0.012 a
FPDPP	0.109 ± 0.013 e	6.387 ± 0.114 d
DPPP	0.196 ± 0.021 c	3.530 ± 0.023 c
PFPP	0.329 ± 0.022 b	2.104 ± 0.130 b
BPP	0.171 ± 0.010 d	4.047 ± 0.029 cd

). In addition, nutrient removal efficiencies were evaluated after 14 days of cultivation in PFPP wastewater. The removal rates of nitrate, nitrite, phosphate, and sulfate were 97.42%, 96.73%, 96.96%, and 91.31%, respectively. Based on these results, PFPP wastewater was selected for subsequent experiments to optimize biomass production of C. vulgaris var. vulgaris TISTR 8261. In this study, bacterial contamination was assessed in all cultures after cultivation using microscopic observation with the Gram staining method to confirm that no contamination occurred.

3.3. Growth of C. vulgaris var. vulgaris TISTR 8261 Cultivated in PFPP Wastewater Supplemented with Different Sodium Acetate Concentrations

To enhance the growth and chlorophyll accumulation of C. vulgaris var. vulgaris TISTR 8261 using PFPP wastewater as a culture medium, various concentrations of sodium acetate were added. Based on OD₇₅₀ measurements, the highest growth was observed when PFPP wastewater was supplemented with 17.4 mM, achieving a growth level comparable to that in TAP medium (Figure 2A). Lower concentrations of sodium acetate (1.74 and 8.70 mM) also improved growth, albeit to a lesser extent, while the highest concentration (34.80 mM) resulted in a slight inhibition of algal growth (Figure 2A). Chlorophyll concentration analysis showed that C. vulgaris var. vulgaris TISTR 8261 cultivated in PFPP wastewater with sodium acetate supplementation exhibited lower chlorophyll levels compared to TAP medium (Figure 2B). The maximum chlorophyll concentration was recorded in cultures grown in PFPP wastewater supplemented with 17.4 mM sodium acetate, though still approximately 1.8-fold lower than that observed in TAP medium. These results were consistent with the growth kinetics parameters presented in

Table 3. The specific growth rate, doubling time in C. vulgaris var. vulgaris TISTR 8261 grown under different sodium acetate concentrations in wastewater from a processed food production plant (PFPP). All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates. Statistically significant differences among samples within the same column are denoted by distinct superscript letters (a–d) at a confidence level of 95%.

Culture Media	Sodium Acetate Concentrations (mM)	Specific Growth Rate (Day−1)	Doubling Time (Day)
TAP	17.40	0.548 ± 0.011 a	1.265 ± 0.012 a
PFPP	0.00	0.330 ± 0.014 d	2.098 ± 0.051 d
PFPP	1.74	0.398 ± 0.022 c	1.743 ± 0.019 c
PFPP	8.70	0.439 ± 0.011 b	1.578 ± 0.013 b
PFPP	17.40	0.544 ± 0.021 a	1.274 ± 0.017 a
PFPP	34.80	0.392 ± 0.031 c	1.770 ± 0.013 c

. The specific growth rate of C. vulgaris var. vulgaris TISTR 8261 was highest in TAP medium (0.548 ± 0.011 day⁻¹), followed closely by PFPP wastewater with 17.4 mM sodium acetate (0.544 ± 0.021 day⁻¹). The corresponding doubling times were 1.265 ± 0.012 and 1.274 ± 0.017 days, respectively. The lowest growth rate occurred in unmodified PFPP wastewater (0.330 ± 0.014 day⁻¹), with a doubling time of 2.098 ± 0.051 days. Supplementation with 8.70 mM sodium acetate improved the specific growth rate to 0.439 ± 0.011 day⁻¹, whereas 34.80 mM sodium acetate did not further enhance growth, resulting in a decreased growth rate (0.392 ± 0.031 day⁻¹). Overall, sodium acetate particularly at 17.40 mM can substantially enhance algal growth in PFPP wastewater. However, the consistently low chlorophyll concentration suggests that additional nutrient supplementation is necessary. Future optimization should focus on enriching PFPP wastewater with key macronutrients such as nitrogen, phosphorus, and sulfur, along with trace elements contained in TAP medium.

3.4. Growth of C. vulgaris var. vulgaris TISTR 8261 Cultivated in PFPP Wastewater Supplemented with Macronutrients

To enhance chlorophyll concentration in C. vulgaris var. vulgaris TISTR 8261, macronutrients were supplemented into PFPP wastewater. The OD₇₅₀ measurements showed that the algal culture exhibited higher biomass accumulation when cultivated in PFPP wastewater supplemented with 17.4 mM sodium acetate, 3.73 mM ammonium chloride (NH₄Cl), 1.0 mM potassium dihydrogen phosphate (KH₂PO₄), and 0.2 mM magnesium sulfate (MgSO₄) (matching the concentrations in TAP medium), compared to cultivation in TAP medium and PFPP wastewater supplemented with sodium acetate alone (Figure 3A). Chlorophyll concentration analysis revealed that although the addition of macronutrients to PFPP wastewater increased algal growth, the chlorophyll concentration of C. vulgaris var. vulgaris TISTR 8261 remained lower than that observed in TAP medium (Figure 3B). However, the addition of macronutrients significantly improved the specific growth rate, yielding the highest rate of 0.570 ± 0.025 day⁻¹ and the shortest doubling time of 1.215 ± 0.015 days (

Table 4. The specific growth rate and doubling time in C. vulgaris var. vulgaris TISTR 8261 grown under different trace mineral concentrations in wastewater from a processed food production plant (PFPP). All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates. Statistically significant differences among samples within the same column are denoted by distinct superscript letters (a–d) at a confidence level of 95%.

Culture Media	Trace Mineral Concentration (×)	Specific Growth Rate (Day−1)	Doubling Time (Day)
TAP	1.0	0.546 ± 0.021 c	1.269 ± 0.011 c
PFPP	0.0	0.338 ± 0.024 d	2.050 ± 0.043 d
PFPP + Sodium acetate	0.0	0.550 ± 0.022 bc	1.261 ± 0.021 c
PFPP + Sodium acetate + NH4Cl + KH2PO4 + MgSO4	0.0	0.570 ± 0.025 bc	1.215 ± 0.015 bc
PFPP + Sodium acetate + NH4Cl + KH2PO4 + MgSO4	0.1	0.738 ± 0.021 ab	0.939 ± 0.012 ab
PFPP + Sodium acetate + NH4Cl + KH2PO4 + MgSO4	0.2	0.778 ± 0.035 a	0.891 ± 0.021 a
PFPP + Sodium acetate + NH4Cl + KH2PO4 + MgSO4	0.5	0.732 ± 0.019 ab	0.947 ± 0.017 ab
PFPP + Sodium acetate + NH4Cl + KH2PO4 + MgSO4	1.0	0.611 ± 0.010 bc	1.134 ± 0.019 b

). In contrast, PFPP wastewater without supplementation resulted in the lowest specific growth rate (0.338 ± 0.024 day⁻¹) and the longest doubling time (2.050 ± 0.043 days) (Table 4). To further increase chlorophyll accumulation, trace mineral supplementation was planned for subsequent experiments using PFPP wastewater as the culture medium.

3.5. Growth of C. vulgaris var. vulgaris TISTR 8261 Cultivated in PFPP Wastewater Supplemented with Different Trace Mineral Concentrations

To overcome the limitation in chlorophyll concentration observed in C. vulgaris var. vulgaris TISTR 8261, various concentrations of trace minerals were supplemented into PFPP wastewater along with macronutrients (17.4 mM sodium acetate, 3.73 mM NH₄Cl, 1.0 mM KH₂PO₄, and 0.2 mM MgSO₄). The results indicated a significant increase in growth, as measured by OD₇₅₀, across all tested trace mineral concentrations (Figure 3A), with the most pronounced effect observed at the 0.2× concentration. OD₇₅₀ values were consistent with chlorophyll concentration data. Cultures supplemented with 0.2× trace minerals exhibited the highest chlorophyll accumulation, reaching a level approximately 1.2-fold higher than that in TAP medium on day 5 of cultivation (Figure 3B). In contrast, cultures supplemented with 1.0× trace minerals showed a chlorophyll concentration slightly lower than that observed in the TAP control (Figure 3B). The highest specific growth rate was recorded in cultures with 0.2× trace minerals, reaching 0.778 ± 0.035 day⁻¹, with a corresponding doubling time of 0.891 ± 0.021 days (Table 4). These values were significantly higher than those observed in non-supplemented conditions and exceeded those obtained in the TAP control. Supplementation with 0.1× and 0.5× trace minerals also resulted in enhanced growth, with specific growth rates of 0.738 ± 0.021 and 0.732 ± 0.019 day⁻¹ and doubling times of 0.939 ± 0.012 and 0.947 ± 0.017 days, respectively (Table 4). Although supplementation with 1.0× trace minerals still promoted growth (specific growth rate = 0.611 ± 0.010 day⁻¹), the effect was slightly lower compared to the intermediate concentrations.

3.6. Scaled-Up Cultivation of C. vulgaris var. vulgaris TISTR 8261 in 3.5 L Culture Bottles

Following the optimization of nutrient conditions in 250 mL flask experiments, C. vulgaris var. vulgaris TISTR 8261 was cultivated on a larger scale using 3.5 L culture bottles to assess its growth performance and biomass production. The cultivation was conducted in three different media: (1) standard TAP medium (TAP), (2) PFPP wastewater (unmodified PFPP), (3) PFPP wastewater supplemented with 17.4 mM sodium acetate, 3.73 mM NH₄Cl, 1.0 mM KH₂PO₄, 0.2 mM MgSO₄, and 0.2× trace mineral solution (modified PFPP). The pH of the cultures was monitored and remained relatively stable (~7.0–7.5) throughout the cultivation period. The results found that visual observation revealed rapid algal proliferation in modified PFPP, with noticeable green coloration developing within the first 24 h and intensifying over time (Figure 4). In contrast, limited growth and pale color were observed in unmodified PFPP. By day 7, cultures grown in modified PFPP medium reached a deep green hue, indicating robust biomass accumulation (Figure 4). Growth kinetics monitored over 15 days showed that cells cultivated in modified PFPP entered the exponential phase on day 1 and maintained rapid growth until day 6, followed by a transition to the stationary phase on day 7 (Figure 5A). This pattern contrasted with slower and less pronounced growth in TAP and unmodified PFPP. Further analyses confirmed that modified PFPP supported significantly higher cell density (Figure 5B), chlorophyll concentration (Figure 5C), and dry biomass (Figure 5D) compared to other media. The maximum biomass yield achieved in modified PFPP medium was 8.436 ± 0.378 g L⁻¹ on day 6, which was 1.3-times and 6.7-times higher than in TAP and unmodified PFPP, respectively (Figure 5D). The growth parameters summarized in

Table 5. The specific growth rate, doubling time, maximum biomass yield, and biomass productivity in C. vulgaris var. vulgaris TISTR 8261 grown in different media in 3.5 L culture bottles. All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates. Statistically significant differences among samples within the same column are denoted by distinct superscript letters (a–c) at a confidence level of 95%.

Culture Media	Specific Growth Rate (Day−1)	Doubling Time (Day)	Maximum Biomass Yield (g L−1)	Biomass Productivity (g L−1 Day−1)
TAP	0.540 ± 0.028 b	1.281 ± 0.018 b	6.498 ± 0.436 b	0.985 ± 0.032 b
Unmodified PFPP	0.316 ± 0.015 c	2.189 ± 0.093 c	1.240 ± 0.245 c	0.107 ± 0.021 c
Modified PFPP	0.746 ± 0.070 a	0.928 ± 0.014 a	8.436 ± 0.378 a	1.154 ± 0.043 a

revealed that cultures in modified PFPP exhibited the highest specific growth rate (0.746 ± 0.070 day⁻¹), the shortest doubling time (0.928 ± 0.014 days), the greatest maximum biomass yield (8.436 ± 0.378 g L⁻¹), and the highest biomass productivity (1.154 ± 0.043 g L⁻¹ day⁻¹). These values were significantly higher (p < 0.05) than those obtained from TAP and unmodified PFPP (Table 5).

3.7. Nutritional Composition of C. vulgaris var. vulgaris TISTR 8261 Cultivated in Different Media

After 15 days of cultivation in 3.5 L culture bottles, the biochemical composition of C. vulgaris var. vulgaris TISTR 8261 was analyzed using the proximate analysis method to determine major nutritional components, including moisture, crude protein, crude fat, crude fiber, ash, carbohydrate, calcium, and phosphorus. The comparison was made between cells cultivated in standard TAP medium and optimized PFPP wastewater (modified PFPP). The results indicate that there were no significant differences in the moisture content, crude protein, crude fat, and crude fiber of cells cultivated between the two media (

Table 6. Nutritional compositions of C. vulgaris var. vulgaris TISTR 8261 cultivated in TAP medium and modified PFPP in 3.5 L culture bottles. All values are presented as the mean ± standard deviation (SD) from three separate experimental replicates.

Cell Content (% (w/w))	Culture Media
	TAP	Modified PFPP
Moisture	10.60 ± 1.20	14.40 ± 0.11
Crude protein	46.70 ± 1.30	42.70 ± 1.40
Crude fat	2.20 ± 0.10	1.90 ± 0.10
Crude fiber	15.70 ± 1.40	11.90 ± 1.30
Ash	3.50 ± 0.40	11.70 ± 1.60
Carbohydrate	20.90 ± 1.60	17.10 ± 1.20
Calcium	1.00 ± 0.10	2.70 ± 0.20
Phosphorus	0.80 ± 0.10	2.40 ± 0.30

). The protein content remained relatively high in both conditions, with 46.70 ± 1.30% in TAP and 42.70 ± 1.40% in modified PFPP. However, cells grown in modified PFPP showed a significantly higher ash content (11.70 ± 1.60%) compared to TAP (3.50 ± 0.40%), as well as elevated levels of calcium (2.70 ± 0.20%) and phosphorus (2.40 ± 0.30%), which were 2.7-fold and 3-fold higher than those found in the TAP-grown biomass, respectively.

4. Discussion

4.1. Wastewater Characteristics and Implications for Algal Cultivation

The wastewater collected from four food industry plants in Phra Nakhon Sri Ayutthaya exhibited distinct physicochemical profiles (Table 1), which are influenced by variations in industrial processes, raw material inputs, and temporal factors. In this study, wastewater was collected during a single sampling period to ensure consistency and minimize variability across all experiments. The pH values ranged from slightly acidic in BPP to nearly neutral in DPPP, underscoring the influence of raw materials and specific industrial processes on effluent chemistry. Moreover, the low dissolved oxygen concentrations (<0.1 mg O₂ L⁻¹) in FPDPP, DPPP, and PFPP indicate a high organic or microbial load, a typical feature of concentrated food industry effluents. In contrast, the elevated DO in BPP suggests a lower biological oxygen demand. High BOD and COD levels in PFPP and BPP reflect the presence of both biodegradable substrates and more recalcitrant organic compounds. This aligns with previous findings that industrial wastewater used in algal cultivation often contain mixed organic fractions, enabling both fast and sustained nutrient release [47]. Regarding carbon content, BPP wastewater exhibited the highest total carbon fractions, indicating a greater organic load than the other sources. Conversely, PFPP showed significantly elevated concentrations of total nitrogen (TN), total Kjeldahl nitrogen (TKN), and nitrate (NO₃⁻), reaching 27.88 mg L⁻¹, 25.01 mg L⁻¹, and 2.72 mg L⁻¹, respectively, higher than levels reported in many agro-industrial effluents [1]. This suggests that PFPP effluent is rich in both organic and inorganic nitrogen, offering a potentially valuable nutrient source for algal growth. While sulfur and phosphorus levels were relatively consistent across samples, PFPP wastewater showed the highest total sulfur and sulfate concentrations. Elevated sulfur content may present both opportunities and challenges: while sulfate supports algal metabolism, excessive concentrations can inhibit growth unless properly balanced [4]. The variability in macronutrients and trace metals (e.g., Ca, Mg, K, Fe) across the wastewater sources implies that each may differently support microalgal physiology. Previous studies have demonstrated that such nutrient disparities significantly affect algal growth, nutrient uptake efficiency, and biomass composition [48].

4.2. Growth of C. vulgaris var. vulgaris TISTR 8261 in Different Wastewater Sources

The growth analysis of C. vulgaris var. vulgaris TISTR 8261 indicated that this strain can grow in various types of food industry wastewater, albeit at lower rates compared to the synthetic TAP medium, which is known to contain optimal concentrations of essential nutrients and trace elements required for algal metabolism and chlorophyll biosynthesis [41]. Among the four wastewater types tested, the effluent from PFPP showed the highest growth potential, with a significantly greater OD₇₅₀, specific growth rate (μ), and doubling time (Td) than other wastewater treatments (Figure 1 and Table 2). This enhanced growth performance in PFPP wastewater can be attributed to its relatively higher concentrations of total nitrogen (27.88 mg L⁻¹), total phosphorus (4.99 mg L⁻¹), and sulfate (3.34 mg L⁻¹), all of which are critical macronutrients for cell growth and division [34]. In addition, COD analysis revealed higher organic and inorganic loads in BPP (2739.20 ± 314.60 mg O₂ L⁻¹) compared to PFPP (2227.20 ± 168.10 mg O₂ L⁻¹) (Table 1). Nevertheless, algal growth in BPP was markedly lower, highlighting that nutrient availability, particularly total nitrogen and phosphorus, was more critical than COD in supporting C. vulgaris var. vulgaris TISTR 8261 growth. COD and BOD values were determined from wastewater, as these parameters serve as indicators of the organic load in raw effluents. Although autoclaving may slightly reduce COD and BOD values [49], the reduction is generally minor and unlikely to significantly affect algal growth in subsequent cultivation. However, the chlorophyll concentration in all wastewater treatments remained lower than in the TAP control, suggesting that micronutrient imbalances or organic inhibitors in the wastewater may have impaired pigment synthesis. A similar trend was observed by Taikhao and Phunpruch [6], who reported lower chlorophyll concentration and biomass in untreated wastewater compared to synthetic media, likely due to insufficient nutrient availability and high concentrations of soluble organic matter that inhibit algal metabolism. In line with these findings, FPDPP wastewater resulted in the poorest growth performance, with the lowest biomass and chlorophyll concentration observed. This may be due to the presence of inhibitory substances or severe nutrient limitations commonly associated with carbohydrate-rich dough-processing wastewater [50]. Moderate algal growth was observed in DPPP and BPP wastewater, which may reflect more balanced but suboptimal nutrient availability. The lower concentrations of nitrogen, phosphorus, and trace elements, combined with elevated salinity or variable carbon fractions, likely contributed to slower growth rates in these treatments. Such variation in nutrient profiles and salinity levels is known to affect Chlorella spp. physiology and biomass productivity [51]. In addition to growth, the bioremediation potential of C. vulgaris var. vulgaris TISTR 8261 was clearly demonstrated in PFPP wastewater, where nitrate, nitrite, phosphate, and sulfate removal efficiencies exceeded 90% after 14 days of cultivation. These results are comparable to those reported by Taikhao and Phunpruch [6], who found similarly high removal rates of nutrients in treated wastewater using the same algal strain. The ability of C. vulgaris var. vulgaris TISTR 8261 to efficiently assimilate nitrogenous, phosphorous, and sulfurous compounds highlights its dual utility in biomass production and wastewater treatment. These findings are further supported by broader research on microalgal bioremediation. For instance, Lopez Ponte et al. [52] reported high nitrate and phosphate removal efficiencies by green algae such as Chlorella sp. (95% NO₃⁻ and 69.3% PO₄³⁻) and Desmodesmus sp. (96.5% NO₃⁻ and 88.3% PO₄³⁻) in domestic wastewater. These microalgae exhibit rapid growth, high nutrient uptake, and strong resistance to environmental fluctuations and pollutant stress, making them ideal candidates for large-scale bioremediation. Moreover, the metabolic processes of microalgae naturally release oxygen, which supports aerobic bacteria that degrade organic matter in wastewater—enhancing overall treatment efficiency through synergistic interactions [53]. Nevertheless, a closer inspection of chemical composition between PFPP wastewater and TAP medium revealed a substantial difference in total carbon content (176.90 mg L⁻¹ vs. 417.50 mg L⁻¹). Carbon is a fundamental element required for photosynthesis and biomass synthesis; thus, its limited availability in PFPP effluent likely constrained chlorophyll production and maximum growth potential [54]. To overcome this limitation, supplementation with an external carbon source such as acetate may enhance biomass accumulation and pigment synthesis, particularly in carbon-limited or unbalanced wastewater.

4.3. Effect of Sodium Acetate Supplementation on the Growth of C. vulgaris var. vulgaris TISTR 8261 in PFPP Wastewater

This study demonstrates that supplementation with sodium acetate significantly enhances the growth performance of C. vulgaris var. vulgaris TISTR 8261 in PFPP wastewater, a nutrient-limited effluent from the processed food industry. The addition of sodium acetate at 17.40 mM resulted in a substantial increase in biomass accumulation and specific growth rate (μ = 0.544 ± 0.021 day⁻¹), nearly equivalent to those achieved in the synthetic TAP medium (μ = 0.548 ± 0.011 day⁻¹). This improvement confirms the role of acetate as a viable external carbon source for supporting algal metabolism under suboptimal growth conditions. These results are consistent with previous findings by Taikhao and Phunpruch [6], who reported that sodium acetate supplementation at 17.4 mM in treated wastewater successfully restored the growth and biomass production of C. vulgaris var. vulgaris TISTR 8261 to levels comparable to those observed in TAP medium. They attributed the improved performance to the provision of an accessible carbon source that compensated for the low total carbon content in the wastewater—reported as only 32.50 mg L⁻¹ in frozen food effluent. Likewise, Liu et al. [5] observed that pretreated municipal wastewater contained only 20.52 mg L⁻¹ of acetate, which was below the level required to support efficient algal growth. Our study supports this explanation, as the PFPP wastewater used here contained only 176.90 mg L⁻¹ of total carbon—substantially lower than the 417.50 mg L⁻¹ found in TAP—and sodium acetate supplementation provided a crucial carbon boost to sustain cell proliferation. Moreover, acetate metabolism in algal cells is well-established as a dual pathway process involving both the tricarboxylic acid (TCA) cycle and the glyoxylate cycle, enabling the production of ATP and reducing power (NAD(P)H), which are essential for biosynthesis and cell division [35]. The enhancement of biomass accumulation in our study—despite the nutrient-limited profile of PFPP wastewater—underscores acetate’s role as a versatile energy source under mixotrophic conditions. Similar positive effects of acetate supplementation on Chlorella species have been documented in various studies, including cultivation in optimized synthetic media [36] and soybean-processing wastewater [55]. Furthermore, while moderate acetate concentrations (1.74 and 8.70 mM) yielded progressive growth enhancements, the highest concentration (34.80 mM) caused a slight growth inhibition. This is likely attributable to metabolic stress or pH shifts caused by excess acetate, which can alter intracellular redox balance or suppress photosynthetic pathways, as reported in other mixotrophic cultivation systems [54]. Interestingly, while biomass accumulation was markedly improved with acetate supplementation, chlorophyll concentration remained consistently lower in all PFPP-based treatments relative to TAP (Figure 2B). This suggests that pigment biosynthesis is not solely dependent on carbon availability but also requires adequate levels of essential nutrients such as nitrogen, sulfur, and trace metals (e.g., Fe, Mg, Zn), which are deficient in PFPP wastewater (Table 1). As chlorophyll synthesis involves nitrogen assimilation and iron-dependent enzymatic steps [56], the low pigment content despite improved growth may reflect a partial decoupling of the photosynthetic machinery from cell division under unbalanced nutritional conditions. These findings underscore the importance of a dual supplementation strategy for optimal algal cultivation in industrial wastewater. While carbon limitation can be mitigated through organic carbon sources such as sodium acetate, micronutrient and macronutrient enrichment remains critical to support balanced physiological functioning and pigment production. Future studies should explore combined supplementation with nitrogen, phosphorus, sulfur, and key trace elements to promote both biomass productivity and photosynthetic efficiency.

4.4. Effect of Macronutrients and Trace Mineral Supplementation on Growth of C. vulgaris var. vulgaris TISTR 8261 in PFPP Wastewater

The cultivation of C. vulgaris var. vulgaris TISTR 8261 in wastewater derived from a PFPP under various nutrient supplementation regimes revealed critical insights into nutrient limitations and strategies for medium optimization. When cultivated in PFPP wastewater, C. vulgaris var. vulgaris TISTR 8261 exhibited minimal growth and pale pigmentation, indicating a pronounced deficiency in available carbon and essential macronutrients required for cellular metabolism and pigment synthesis. Supplementation with sodium acetate significantly improved algal growth, with biomass levels approaching those achieved in the standard TAP medium (Figure 3). This result aligns with previous findings highlighting the importance of organic carbon supplementation for mixotrophic growth, especially under nutrient-deficient conditions such as those found in industrial wastewater [5]. Further chemical analysis showed that PFPP wastewater contained considerably lower levels of nitrogen (27.88 mg L⁻¹ vs. 104.80 mg L⁻¹), phosphorus (4.99 mg L⁻¹ vs. 15.47 mg L⁻¹), and sulfur (4.17 mg L⁻¹ vs. 16.06 mg L⁻¹) compared to TAP medium. These macronutrients play crucial roles in protein synthesis, nucleic acid metabolism, and enzyme function [57]. Their deficiency has likely contributed to the poor growth observed. However, supplementation with 3.73 mM NH₄Cl, 1.0 mM KH₂PO₄, and 0.2 mM MgSO₄ restored these essential elements and resulted in a substantial improvement in algal growth (Figure 3), likely due to re-establishing a more favorable C/N/P/S stoichiometric balance that supports cell division and metabolic activity. In addition to macronutrient limitations, PFPP wastewater was also deficient in several critical trace minerals. Compared to TAP, PFPP contained substantially lower concentrations of iron (0.36 mg L⁻¹ vs. 1.00 mg L⁻¹), manganese (0.04 mg L⁻¹ vs. 1.41 mg L⁻¹), copper (0.009 mg L⁻¹ vs. 0.0407 mg L⁻¹), and zinc (0.09 mg L⁻¹ vs. 5.02 mg L⁻¹). These micronutrients, although required in trace amounts, are essential cofactors in a wide range of biochemical processes, particularly those associated with photosynthesis and antioxidant defense [58]. Iron (Fe) is indispensable for chlorophyll biosynthesis and electron transport, serving as a core component of cytochromes and ferredoxin [59]. Manganese (Mn) is a vital constituent of the oxygen-evolving complex (OEC) in photosystem II, which facilitates water splitting and oxygen production [60]. Zinc (Zn) functions as a cofactor for carbonic anhydrase and other enzymes involved in DNA replication and protein synthesis [37]. Copper (Cu) is essential for plastocyanin-mediated electron transport and maintaining cellular redox balance [61]. Deficiencies in these elements can severely compromise photosynthetic performance and metabolic efficiency. This was corroborated by the low chlorophyll concentration observed in cultures grown in unmodified PFPP wastewater (Figure 3B). However, the addition of a trace mineral solution significantly improved both growth rate and chlorophyll accumulation, particularly at the 0.2× concentration. At this level, cultures achieved the highest specific growth rate (0.778 ± 0.035 day⁻¹) and shortest doubling time (0.891 ± 0.021 days), along with peak chlorophyll concentration (Table 4, Figure 3B). These findings support the role of micronutrients as indispensable enzymatic cofactors involved in key pathways such as photosynthesis, respiration, and nutrient assimilation [62]. Interestingly, while the 0.2× trace mineral concentration proved optimal, higher supplementation (e.g., 1.0×) led to a slight decline in growth (0.611 ± 0.010 day⁻¹) (Table 4). This dose-dependent response can be explained by the fact that the baseline PFPP wastewater already provided sufficient levels of essential trace metals, so only minimal supplementation was required to support growth. In contrast, the 1.0× concentration likely resulted in an excess of certain metals, causing potential toxicity or metabolic burden, such as oxidative stress or inhibition of enzymatic functions in microalgae. These observations are consistent with earlier reports demonstrating inhibitory effects of excessive trace metals on microalgal growth [63]. Overall, these results demonstrate that although PFPP wastewater in its native form is suboptimal for supporting robust algal growth, it can be effectively transformed into a suitable cultivation medium through minimal supplementation. The optimized formulation—comprising 17.4 mM sodium acetate, 3.73 mM NH₄Cl, 1.0 mM KH₂PO₄, 0.2 mM MgSO₄, and 0.2× trace mineral solution—offers a cost-effective and scalable nutrient strategy. Its application in 3.5 L culture bottles systems holds promise for sustainable biomass production, nutrient recovery, and value-added wastewater utilization.

4.5. Scaled-Up Cultivation of C. vulgaris var. vulgaris TISTR 8261 Using Optimized PFPP Wastewater

The successful scaled-up cultivation of C. vulgaris var. vulgaris TISTR 8261 in 3.5 L culture bottles using optimized PFPP wastewater underscores the effectiveness of targeted nutrient supplementation in transforming a low-nutrient waste stream into a viable growth medium. The significantly enhanced growth performance—characterized by a maximum biomass yield of 8.436 ± 0.378 g L⁻¹ and productivity of 1.154 ± 0.043 g L⁻¹ day⁻¹—demonstrates not only the physiological responsiveness of C. vulgaris var. vulgaris TISTR 8261 to optimized C/N/P/S/trace mineral stoichiometry but also the scalability of the optimized formulation for larger cultivation volumes. Compared to the control groups (TAP and untreated PFPP), cultures grown in modified PFPP exhibited a substantially faster growth rate (µ = 0.746 ± 0.070 day⁻¹) and a shorter doubling time (0.928 ± 0.014 days) (Table 5), indicating an early onset of exponential growth and a more efficient utilization of nutrients under mixotrophic conditions. While the optimized nutrient formulation clearly enhanced biomass yield, it is important to note that at high cell densities, light penetration was limited due to self-shading. This indicates that photosynthesis alone could not account for the observed productivity. Instead, the presence of sodium acetate in the medium supported mixotrophic metabolism, which played a decisive role in sustaining growth under light-limited conditions. The success of the system therefore highlights the synergistic interplay between photosynthesis and acetate-driven mixotrophy in achieving robust biomass accumulation. The observed robust chlorophyll accumulation and dark green pigmentation further support enhanced photosynthetic capacity (Figure 4 and Figure 5), which may be attributed to the sufficient availability of micronutrients (e.g., Fe, Mn, Cu, Zn) critical for chloroplast development and electron transport mechanisms [37,59,61]. When benchmarked against other Chlorella strains cultivated in various wastewater substrates (

Table 7. Biomass yield and biomass productivity of C. vulgaris var. vulgaris TISTR 8261 compared with other green algal strains cultivated in wastewater under different conditions.

Species	Substrate	Biomass Yield (g L−1)	Biomass Productivity (g L−1 Day−1)	Reference
Chlorella vulgaris var. vulgaris TISTR 8261	Wastewater from a processed food production plant	8.436	1.154	This study
Chlorella thermophila	Simulated dairy wastewater	2.1	0.175	[65]
Chlorella sorokiniana	Sugarcane molasses, lab-scale	-	0.128	[66]
Chlorella protothecoides	Expired fruit juices, fed-batch bioreactor	27.0	2.94	[64]
Chlorella pyrenoidosa	Swine wastewater	0.83	-	[69]
Chlorella pyrenoidosa	Domestic wastewater	1.44	-	[69]
Chlorella sp.	Brewery wastewater digestate	1.36	-	[70]
Chlorella minutissima	Saline aquaculture wastewater	4.77	0.55	[68]
Chlorella vulgaris	Oil industry produced water	1.69	-	[26]
Chlorella vulgaris	Municipal wastewater (seasonal variation study)	-	0.0215–0.0281	[51]
Chlorella vulgaris	Meat-processing wastewater	0.675–1.538	-	[71]
Chlorella vulgaris	Brewery wastewater	-	0.47	[67]
Chlorella vulgaris	Sugar industry wastewater	0.35	-	[72]

), C. vulgaris var. vulgaris TISTR 8261 cultivated in modified PFPP demonstrated a notably high biomass yield. While some systems such as C. protothecoides in fed-batch reactors using expired fruit juices reached higher yields (27.0 g L⁻¹) [64], these typically involve high-value substrates, energy-intensive operation modes, or specialized cultivation systems. In contrast, the use of minimally supplemented industrial wastewater (PFPP) offers a cost-effective and environmentally sustainable platform with comparably strong performance. For instance, the productivity observed here (1.154 g L⁻¹ day⁻¹) was significantly higher than in other studies using dairy (0.175 g L⁻¹ day⁻¹) [65], molasses (0.128 g L⁻¹ day⁻¹) [66], or brewery effluents (0.47 g L⁻¹ day⁻¹) [67]. Even when compared with saline aquaculture wastewater (0.55 g L⁻¹ day⁻¹) [68], the productivity of C. vulgaris var. vulgaris TISTR 8261 in PFPP was more than double, emphasizing its suitability for high-output systems. Notably, the disparity between unmodified PFPP and modified PFPP highlights the critical role of balanced nutrient supplementation. The dramatic improvements upon the addition of organic carbon and essential macro- and micronutrients validate the findings from previous flask-scale experiments, confirming that the original limitations in PFPP (low N, P, S, and trace minerals) were the primary bottlenecks for growth. These enhancements point toward the potential of PFPP wastewater to serve not only as a nutrient carrier but also as part of a resource recovery strategy, aligning with circular bioeconomy principles. A methodological limitation of this study is that the wastewater medium was sterilized by autoclaving. This process is known to alter the chemical composition of complex media, for example by causing the precipitation of phosphate and iron salts or by degrading heat-sensitive organic compounds. Consequently, the nutrient profile available to C. vulgaris var. vulgaris TISTR 8261 during cultivation may not have been identical to that measured prior to sterilization, potentially affecting nutrient bioavailability and algal growth outcomes. Although the present study demonstrates the feasibility of using sterilized wastewater as a growth medium, future work should consider sterile filtration as an alternative sterilization approach to maintain the native chemical characteristics of wastewater while preventing microbial contamination.

To further evaluate the economic feasibility of using PFPP wastewater compared with synthetic medium, a cost analysis was performed. The preparation of TAP medium required several defined chemical salts, resulting in an estimated cost of approximately 2.45 USD L⁻¹. In contrast, cultivation in modified PFPP wastewater, which only required minimal nutrient supplementation, was estimated at 0.12 USD L⁻¹. This represents an approximately 20-fold reduction in cultivation cost. These findings confirm that PFPP wastewater not only provides sufficient nutrients for algal growth but also offers a highly cost-effective platform compared with conventional synthetic media, supporting its potential for industrial-scale applications.

4.6. Nutritional Quality of Biomass of C. vulgaris var. vulgaris TISTR 8261 Biomass Cultivated in Modified PFPP Wastewater

The biochemical analysis of C. vulgaris var. vulgaris TISTR 8261 after 15 days of cultivation in 3.5 L bottles revealed valuable insights into the nutritional profile of biomass grown in different media. No significant differences in moisture, crude protein, crude fat, or crude fiber content between cultures grown in standard TAP medium and those grown in modified PFPP wastewater were found. Crude protein content was slightly reduced in biomass from modified PFPP (42.70 ± 1.40%) compared to TAP (46.70 ± 1.30%), though both values remain high, underscoring the suitability of C. vulgaris var. vulgaris TISTR 8261 as a protein-rich feedstock. Previous studies suggest that a protein content above 40% is considered favorable for applications in aquaculture and livestock feed [73]. The minor reduction in protein under PFPP conditions may be attributed to variations in the nitrogen form and availability, even after supplementation, or to metabolic adjustments induced by complex wastewater matrices [74]. In contrast, significant increases in ash (11.70 ± 1.60% vs. 3.50 ± 0.40%), calcium (2.70 ± 0.20% vs. 1.00 ± 0.10%), and phosphorus (2.40 ± 0.30% vs. 0.80 ± 0.10%) were observed in PFPP-grown biomass compared to TAP. These increases suggest enhanced mineral uptake under PFPP conditions, possibly due to the higher availability of ions such as Ca²⁺ and PO₄³⁻, or due to improved solubility and bioavailability facilitated by wastewater characteristics like pH and organic matter complexation. The elevated ash content aligns with reported ranges in other mineral-rich microalgae, such as Chlorella sp. and Arthrospira sp., which can range from 5.50 to 27.30% [75]. While high ash content reflects beneficial mineral accumulation—useful in feed fortification with essential elements like calcium, phosphorus, iron, and magnesium—excessive ash can sometimes negatively impact digestibility if not appropriately balanced. From an application perspective, the high ash content carries both advantages and limitations. On one hand, mineral-rich biomass is valuable for animal feed fortification, providing essential elements such as calcium, phosphorus, iron, and magnesium, and can also serve as a biofertilizer, improving soil fertility and crop performance by contributing nutrients and bioactive compounds [76,77]. On the other hand, excessive ash reduces the suitability of the biomass for biofuel applications, as high mineral fractions lower lipid quality, complicate transesterification, and increase fouling during thermochemical conversion. Furthermore, an excessively high ash content may impair digestibility in feed if not appropriately balanced. These findings underscore the importance of aligning cultivation strategies with the intended end use of the biomass: wastewater-grown microalgae are particularly promising for nutrient recycling and agricultural applications, but may require further downstream processing or selective cultivation strategies if the target product is high-quality biofuel. The phosphorus content observed in PFPP-grown biomass (2.40%) falls within the typical range reported for Chlorella species (0.70–2.50%) [20] and is nutritionally significant, as phosphorus plays a central role in energy metabolism and nucleic acid synthesis in animals [78]. Likewise, calcium concentrations in Chlorella vulgaris (3.50–12.80 g kg⁻¹) have been linked to improved bone development and immune responses in swine diets when included at levels of 3–5% [78]. These findings suggest that elevated calcium not only promotes skeletal growth but also supports cellular signaling and overall feed value [79]. These results demonstrate that cultivation in optimized PFPP medium not only maintains high protein yields but also enhances the mineral richness of the biomass.

5. Conclusions

This study demonstrates the potential of wastewater from the processed food production plant (PFPP) as an effective and economical medium for cultivating C. vulgaris var. vulgaris TISTR 8261. The addition of sodium acetate and subsequent optimization with selected macro- and micronutrients significantly enhanced algal growth, biomass productivity, and pigment content. Cultivation in scaled-up 3.5 L culture bottles yielded a high biomass concentration of 8.436 ± 0.378 g L⁻¹, with the resulting biomass rich in protein (42.70%), carbohydrate (17.10%), calcium (2.70%), and phosphorus (2.40%) and relatively low in lipids, making it particularly suitable for applications in animal feed fortification and biofertilizer production rather than for biofuel or general bio-based industries. Importantly, the optimized PFPP cultures achieved high nutrient removal rates, with substantial reductions in nitrogen and phosphorus concentrations, directly demonstrating the system’s bioremediation potential. These findings highlight the dual benefit of nutrient recovery and value-added biomass generation, supporting the sustainable integration of microalgal biotechnology into wastewater treatment systems. For future applications, large-scale production would typically require cultivation in open raceway ponds or closed photobioreactors, where parameters such as light penetration, mixing, aeration, and contamination control play a crucial role. Moreover, the variability in wastewater composition, cost-effectiveness of nutrient supplementation, and the need for continuous monitoring systems should also be addressed to ensure stable and efficient algal growth at the industrial scale.