Processed Chlorella vulgaris: Effects on Digestibility and Growth Performance in Nile Tilapia (Oreochromis niloticus)

Abstract

Microalgae are a promising feed ingredient in aquaculture due to their high nutrient content. This study evaluated the effects of different processing methods of Chlorella vulgaris on digestibility, retention, and growth performance in Nile tilapia (Oreochromis niloticus). A total of 270 mixed-sex tilapia (average weight: 2.8 ± 0.15 g) were randomly assigned and fed to one of three experimental diets—a basal diet, a diet containing freeze-dried Chlorella, or a diet containing spray-dried Chlorella—with three replicates each for 5 weeks. Results indicated that spray-dried Chlorella significantly enhanced protein and energy digestibility, nutrient retention, and growth performance compared to freeze-dried Chlorella. These findings underscore the critical role of processing methods in maximizing the nutritional potential of microalgae for aquaculture feeds. Further research is recommended to optimize processing techniques and inclusion levels for cost-effective and sustainable applications.

1. Introduction

The rapid expansion of global aquaculture has established it as the fastest-growing animal production sector, now surpassing capture fisheries in total output [1]. To sustain this growth and meet the rising demand for animal protein, the industry must identify and utilize high-quality feed ingredients. Microalgae have emerged as promising protein sources due to their remarkably high protein content, often exceeding 50% of dry weight, with well-balanced essential amino acid profiles that are comparable or even superior to conventional plant and animal proteins. Among the diverse microalgae taxa, Chlorella vulgaris is particularly noted for its nutritional profile—rich in protein (47.8 ± 0.05%), energy (1.42 MJ ± 0.04 kg⁻¹), lipids (13.3 ± 0.07%), polyunsaturated fatty acids, and bioactive pigments [2,3,4,5]. These attributes highlight Chlorella vulgaris’ potential for fish diets.

Chlorella vulgaris is well-suited for large-scale cultivation, and its inclusion in aquafeeds has been widely recognized [6,7]. Nevertheless, the commercial use of microalgae is constrained by technological and economic barriers, as well as variability in nutritional quality caused by differences in cultivation, harvesting, and downstream processing methods [8,9]. Freeze-drying, also known as lyophilization, is one of the most nutrient-preserving processing methods for feed ingredients. This process removes water at low temperatures under vacuum, minimizing the degradation of heat-sensitive nutrients [10]. However, because freeze-drying is a gentle drying process, it has a limited impact on cell wall rupture.

A critical challenge to the effective use of microalgae in aquafeeds is their rigid cell wall structure, which can hinder nutrient digestibility and bioavailability. For instance, the rigid cell walls of many microalgae species, including Chlorella vulgaris, restrict the access of digestive enzymes, thereby reducing the efficiency with which fish can utilize the nutrients contained within [11,12]. Recent studies have shown that processing methods such as spray-drying can significantly improve the digestibility and nutritional value of microalgal biomass by altering cell wall integrity [13].

Despite the recognized potential of microalgae, limited information is available on how different processing methods affect the digestibility and nutritional composition of Chlorella vulgaris in fish, particularly during the fingerling phase of economically important species like Nile tilapia (Oreochromis niloticus). Moreover, most studies to date have focused on the nutritional composition of microalgae or their use as feed additives, with fewer investigations addressing the interplay between processing methods, digestibility, and growth performance in fish.

This study aims to compare the digestibility of freeze-dried and spray-dried Chlorella vulgaris when fed to Nile tilapia (Oreochromis niloticus) and to evaluate its impact on growth performance. We hypothesized that the cell wall rupture caused by the spray-drying process would enhance digestibility, leading to improved growth performance.

2. Materials and Methods

2.1. Algae Processing and Feed Formulation

Two drying process methods were used to produce the tested Chlorella. The first method was freeze-drying, in which Chlorella was grown in a closed loop photobioreactor and then freeze-dried (FD Chlorella, 89% DM, 49% CP, and 20.8 MJ kg⁻¹). The second method was spray-drying followed by bead-milling (SD Chlorella, 93% DM, 56% CP, and 20.0 MJ kg⁻¹), in which the Chlorella was obtained from a local industry.

In total, three diets were formulated (

Table 1. Formulation (g kg⁻¹ feed) and proximate composition (g kg⁻¹ dry matter) of experimental diets.

	Basal	FD Chlorella	SD Chlorella
Ingredients
Soybean Expeller	425.0	290	290
Wheat Bran	253.5	170	170
Corn Gluten Meal	239.1	165	165
Soy Oil	40.8	40	40
Wheat	26.3	20	20
Freeze-dried Chlorella	0	300	0
Spray-dried Chlorella	0	0	300
Vitamin and Mineral Premix 1	10	10	10
Titanium Dioxide	5	5	5
Proximate composition
Dry matter	807.3	892.5	877.0
Crude protein	311.4	372.5	393.2
Ether extract	113.2	105.8	106.1
Ash	57.8	60.3	55.7
Gross energy (MJ kg−1)	18.6	18.4	18.6

¹ Vitamin and mineral premix nutritional composition: vitamin A (3a672a) 800,000 I.E.; vitamin D3 (3a671) 500,000 I.E.; Betaine (betaine anhydrate 3a920) 84,000 mg; Choline chloride (3a890) 80,000 mg; Vitamin Eall-rac-alpha-tocopheryl acetate (3a700) 40,000 mg; Vitamin C (L-ascorbic acid 3a300) 30,000 mg; Niacinamide (3a315) 30,000 mg; Calcium D-Pantothenate (3a841) 10,000 mg; Vitamin B2 (3a825) 4000 mg; Vitamin B6 (3a831) 3000 mg; Vitamin B1 (3a821) 3000 mg; Vitamin K3 (3a710) 2000 mg; Folic acid (3a316) 1,600,000 μg; Biotin (3a880) 160,000 μg; Vitamin B12 (3a) 10,000 μg; Iron (II) sulfate monohydrate (3b103) 30,000 mg; Zinc (zinc oxide 3b603) 12,000 mg; Manganese (manganese (II) oxide 3b502) 5000 mg; Iodine (calcium iodate, anhydrous 3b202) 800 mg; Copper (copper (II) sulfate, pentahydrate (3b405) 600 mg; Selenium (sodium selenite 3b801) 40 mg.

): a basal diet (80% DM, 31% CP, and 18.6 MJ kg⁻¹) and two test diets containing either freeze-dried or spray-dried Chlorella. The test diets were produced by replacing 30% of the basal diet with the respective Chlorella. All diets were supplemented with TiO₂ as an inert marker.

The diets were prepared using a hand pelleting machine with a 1 mm die. First, the ingredients (excluding Chlorella) were mixed in a stand mixer for 20 min at high rotational speed. Then, Chlorella was added and mixed for 5 min at low rotational speed. Water was added to facilitate mixing and binding of the ingredients. Finally, the pellets were dried in a forced-air oven at 60 °C for 3 h. All feeds were stored in a freezer at −18 °C until use.

2.2. Experimental Setup

The present trial was approved by the Austrian local authority (BAES-FMT-FV-2023-02). The fish were bred and housed at the facilities of Blue Planet Ecosystems, Vienna, Austria. In total, 270 mixed-sex tilapia (Oreochromis niloticus) with an average weight of 2.8 ± 0.15 g (mean ± standard deviation) were randomly distributed into nine identical glass tanks (30 fish per tank) with 72 L water capacity each. Water tanks had a flow-through system, each equipped with a sponge biofilter powered by an airlift, air stones for oxygenation, and a standpipe with an automated inflow system to regulate water filling.

Water quality parameters were measured three times a week and maintained within optimal ranges for tilapia: temperature (26–28 °C), pH (7.8–8.1), ammonium and nitrite (<0.1 mg/L), nitrate (<20 mg/L), and dissolved oxygen (>4 mg/L). A 12L:12D photoperiod was maintained throughout the trial.

2.3. Fecal Sampling and Retention Calculations

The bottom and glass walls of each tank, as well as the sponge biofilter, were cleaned daily before feeding. Uneaten pellets were removed after feeding to avoid mixture with feces. Feces were collected daily, 3 h after feeding, using a vacuum tube attached to an individual filter bag for each tank and stored at −18 °C until further analyses.

At the end of the experimental period, four fish per tank were sampled to calculate protein and energy retention using the following equations:

Retention = {[(Final Weight × Final Body Y) − (Initial Weight × Initial Body Y)]/Total Y Ingested} × 100

where Y refers to crude protein or energy.

2.4. Zootechnical Performance

Tilapia were fed twice daily (9:30 and 16:30) 1.5% of their body weight for 5 weeks. Tilapia were group-weighed every two weeks to adjust feeding and at the end of the trial to evaluate zootechnical performance. Uneaten pellets were collected from the tank, dried, and their weight used to adjust feed intake. Body weight gain (BWG), specific growth rate (SGR), feed intake (FI), feed conversion ratio (FCR), and survival rate were calculated using the following equations:

BWG (g) = (final weight − initial weight)/n° of fish

SGR (% day⁻¹) = ([ln (Wf) − ln (Wi)]/feeding days) × 100

where Wi and Wf refer to the initial and final weights, respectively

FI (g) = (total feed intake − uneaten feed)/n° of fish

FCR (g:g) = FI/BWG

Survival rate (%) = (final n° of fish/initial n° of fish) × 100

2.5. Chemical and Digestibility Analysis

Feed, faeces, and fish samples were analyzed in duplicate for the proximate composition of dry matter (DM), crude protein (CP), gross energy (GE), ether extract (EE), and ash using methods described by Naumann and Bassler (2012) [14]. For TiO₂ quantification, 0.5 g of dried samples were digested with 25 mL of H₂SO₄ (98%) and one Kjeldahl tablet for 60 min at 400 °C. After digestion, samples were left to crystallize overnight and then filtered into 100 mL PE bottles. A total of 5 mL of the filtrate was mixed with 1 mL of 1 M H₂SO₄ and 1 mL of H₂O₂. Samples were placed in disposable cuvettes and measured against distilled water on the spectrophotometer (wavelength of 405 nm). The concentration of TiO₂ was then calculated with the formula described below, in which y is the absorbance, factor 0.5994 represents the Ti concentration in TiO₂, and the volume of the test sample corresponds to the respective volume of the volumetric flask used after digestion [15]:

$$\mathrm{T}\mathrm{i}{\mathrm{O}}_2\hspace{0.33em}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\hspace{0.33em}(\mathrm{\%}\hspace{0.33em}\mathrm{o}\mathrm{f} \mathrm{D}\mathrm{M})={\displaystyle \frac{\left(\frac{\mathrm{y} - \mathrm{b}}{\mathrm{a}}\right)\times \mathrm{v}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{m}\mathrm{e}\hspace{0.33em}\mathrm{o}\mathrm{f} \mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\hspace{0.33em}(\mathrm{m}\mathrm{L})}{0.5994\hspace{0.33em}\times \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e} (\mathrm{g} \mathrm{D}\mathrm{M})\times 10000}}$$

Apparent total tract digestibility (ATTD) of nutrients was calculated according to methods established in the literature [16], after feed and faeces nutrient and marker analysis:

ATTD_{Nutrient,Diet} (%) = 100 × (1 − (Marker_Diet/Marker_Faeces) × (Nutrient concentration_Faeces/Nutrient concentration_Diet))

The formula for the calculation of ATTD of the test substance follows the described equation for fish in the literature [17]:

ATTD_{Test substance} = (ATTD_{Test diet} − ATTD_{Base diet}) × ((30 × Nutrient concentration_{Test diet} + 70 × Nutrient concentration_{Base diet})/30 × Nutrient concentration_{Test diet})

2.6. Statistical Analyses

The experiment followed a completely randomized design. Digestibility data were analyzed using R Studio (version 2022.12.0+353, R Development Core Team). Prior to group comparison, normality (Kolmogorov–Smirnov test) and homogeneity of variances (Levene’s test) were checked. For comparisons between the FD Chlorella and SD Chlorella groups, an independent samples t-test was used. Results are presented as means ± standard error of the mean (SEM), and differences were considered significant at p ≤ 0.05. The statistical model used was:

Y_ij = μ + T_i + ϵ_ij

where Y_ij is the dependent variable (e.g., nutrient digestibility, nutrient retention, or performance parameters) for the jth replicate in the ith treatment; μ represents the mean; T_i represents the effect of the ith treatment (FD or SD Chlorella); and ϵ_ij is the error.

3. Results

The proximate composition of Chlorella vulgaris, corrected for dry matter (DM) content, is presented in

Table 2. Proximate composition (g kg⁻¹ dry matter) of Chlorella vulgaris produced under different processing methods.

	FD Chlorella	SD Chlorella
Proximate composition
Dry matter	892.5	938.9
Crude protein	551.2	603.8
Ether extract	87.6	88.9
Ash	46.5	48.9
Gross energy (MJ kg−1)	2.08	2.00

. No significant differences (p > 0.05) were observed between freeze-dried (FD) and spray-dried (SD) Chlorella for DM, ether extract (EE), gross energy (GE), or ash content. However, FD Chlorella contained significantly less crude protein than SD Chlorella (551.2 vs. 603.8 g kg⁻¹ DM, respectively), representing a difference of 52.5 g kg⁻¹ DM or ~9%.

The apparent total tract digestibility (ATTD) of Chlorella produced using different processing methods is shown in

Table 3. Apparent total tract digestibility (ATTD, %) of Chlorella vulgaris produced under different processing methods.

	Dry Matter 1	Crude Protein 2	Energy 2
FD Chlorella	46.7 (b)	67.5	21.2 b
SD Chlorella	51.2 (a)	68.3	29.8 a
SEM	1.15	1.97	1.68
	p value
	0.06	>0.05	<0.05

^a–b Means within a column not sharing a common superscript differ regarding the effect of processing methods effect (p ≤ 0.05) according to an independent t-test. ^(a–b) Means within a column not sharing a common superscript tend differ regarding the effect of processing methods effect (p ≤ 0.1) according to an independent t-test. ¹ ATTD of dry matter of diet (%) = 100 × [1 − (dietary TiO₂/fecal TiO₂)]. ² ATTD of nutrient (%) = (ATTD test diet − ATTD basal diet) × ((30 × nutrient concentration test diet + 70 × nutrient concentration basal diet)/30 × nutrient concentration test diet). SEM Standard error of the mean.

. There was a tendency for higher DM digestibility in SD Chlorella compared to FD Chlorella (51.2% vs. 46.7%, p = 0.06). No significant difference was observed in protein digestibility between the two methods, with values of 68.3% for SD Chlorella and 67.5% for FD Chlorella (p > 0.05). Energy digestibility was significantly greater in SD Chlorella (29.8%) than in FD Chlorella (21.2%) (p < 0.05).

The protein and energy retention values are presented in

Table 4. Protein and energy retention (%) of Chlorella vulgaris produced under different processing methods.

	Crude Protein	Energy
FD Chlorella	73.8 b	50.3 b
SD Chlorella	91.3 a	67.9 a
SEM	8.74	8.79
	p value
	<0.05	<0.05

^a–b Means within a column not sharing a common superscript differ regarding the effect of processing methods effect (p ≤ 0.05) according to an independent t-test. SEM: Standard error of the mean.

. Protein retention was significantly affected by the processing method (p < 0.01). Fish fed SD Chlorella resulted in ~18% higher protein retention (91.63%) than FD Chlorella (73.81%). Similarly, energy retention also varied significantly between processing methods (p < 0.01). Energy retention was higher in fish fed SD Chlorella (67.90%) than in fish fed FD Chlorella (50.30%).

The zootechnical parameters are presented in

Table 5. Zootechnical performance of juvenile Nile Tilapia (Oreochromis niloticus) fed Chlorella vulgaris produced under different processing methods.

	BWG (g)	SGR (%)	FI (g)	FCR (g:g)	Survival Rate (%)
FD Chlorella	3.92 b	10.90 b	7.02	1.81 a	100
SD Chlorella	4.98 a	13.85 a	7.15	1.42 b	100
SEM	0.529	1.474	0.065	0.195	-
	p value
	<0.05	<0.05	>0.05	<0.05	>0.05

^a–b Means within a column not sharing a common superscript differ regarding the effect of processing methods effect (p ≤ 0.05) according to an independent t-test. BWG: body weight grain; SGR: specific growth rate; FI: feed intake; FCR: feed conversion ratio. SEM: Standard error of the mean.

. Fish fed the SD Chlorella presented significantly higher body weight gain (BWG; 4.98 g) and specific growth rate (SGR; 13.85%) compared to those fed FD Chlorella (BWG: 3.92 g; SGR: 10.90%) (p < 0.05). Similarly, the feed conversion ratio (FCR) was significantly better in fish fed SD Chlorella (1.42) in comparison to FD Chlorella (1.81) (p < 0.05). No statistical differences were observed in survival rate, as both groups achieved a 100% survival rate (p > 0.05).

4. Discussion

The present study demonstrates that the processing method of Chlorella vulgaris has a significant impact on its digestibility. This effect is primarily attributed to the robust structure of the microalgal cell wall, which consists of multiple polysaccharide layers—including cellulose, uronic acids, and chitin-like glucosamine polymers—that are highly resistant to digestive breakdown [18,19]. As reported by Weber et al. (2022) [19], Chlorella employs a three-layered wall structure that rapidly develops during growth, with an outermost layer conferring recalcitrance to both enzymatic hydrolysis and mechanical disruption. Because freeze-drying preserves cell integrity, it may further restrict enzyme access to encapsulated nutrients, explaining the limited digestibility observed in our study. Authors evaluated six processing methods (untreated, pasteurized, freeze-dried, frozen-thawed, commercially processed, and bead milled) for Nannochloropsis gaditana and found that processing methods that intensify the cell disruption consistently improved nutrient digestibility and, consequently, growth parameters in tilapia [20]. Similarly, studies evaluating physical processing techniques, such as spray-drying followed by bead milling, of Chlorella vulgaris reported enhanced digestibility, which was linked to cell wall disruption and increased substrate access for digestive enzymes [21,22].

Protein and energy ATTD in this study were notably lower (68.3–67.5% for protein and 21.2–29.8% for energy, for SD Chlorella and FD Chlorella, respectively) compared to values reported in the literature [20,23,24]. These discrepancies may arise from several factors: differences in processing intensity, life stage of the test fish (fingerlings vs. juveniles or adults), specific algal strain identification, and fecal collection interval. It is well-documented that digestive physiology—such as protease and carbohydrase activity, gut morphology, and nutrient absorption capacity—differs markedly with developmental stage, impacting feed utilization and measured digestibility. Additionally, different digestibility methodologies have been developed seeking more precise procedures for collecting the feces, such as dissection [25,26,27,28], stripping [25,26,28,29] suction [25,30] and an automatic faecal collection device [31] to overcome the leaching of nutrients [32]. In the current study, the tanks were cleaned before feeding, and feed residues were removed after feeding to avoid contaminating the fecal samples. Nevertheless, as fecal material stayed in contact with water for up to 3 h, the gap time between feeding and sampling, some nutrients might have leached, which could lead to an overestimation of ATTD [26,33,34]. Authors reported that leaching can result in over 10–20% loss of N from feces within 1–3 h post-defecation [26]. We opted to collect fecal samples using a vacuum tube due to infrastructure restrictions and fish size, which made handling for alternative sampling methods challenging.

The higher digestibility of SD Chlorella reflected in a better (p < 0.05) fish zootechnical performance. Tilapia fed SD Chlorella presented better BWG (4.98 g), and SGR (13.85%) compared to FD Chlorella (3.92 g and 10.90%, respectively). The higher digestibility could also be observed by the lack of significant difference for FI (p > 0.05), but with improved FCR for SD Chlorella compared to FD Chlorella (1.42 and 1.81, respectively). Protein and energy retention values were higher in SD Chlorella (91.6 and 67.9%, respectively) than in FD Chlorella (73.8 and 50.3%, respectively) as well. Similar results have been reported in multiple studies, where disruption of microalgal cell walls—through bead milling, enzymatic treatment, or other intensive processing—improved growth rates and FCR in tilapia and other fish species [13,20,35]. For instance, Teuling et al. (2019) [20] reported that Nile tilapia fed bead-milled Nannochloropsis gaditana had a 13% improvement in FCR compared to those fed untreated algae. These results support the hypothesis that microalgae cell wall disruption enhances nutrient utilization, supporting superior protein deposition and growth performance.

Although results regarding physical changes in microalgae structure caused by different processing methods can be transferred among different strains, their ATTD and subsequent performance impacts should be interpreted with caution. The proximal composition of microalgae can vary significantly, even within the same strain, depending on the cultivation medium to which it was subjected [36,37,38]. Furthermore, inherent characteristics of the microalgae cell wall, combined with altered composition due to growth phase and environmental stress, might result in misleading conclusions. An in vitro study evaluating 12 different microalgae strains (Arthrospira platensis F&M-C256, Klamath polvere, Nostoc sphaeroides F&M-C117, Chlorella sorokiniana F&M-M49, Chlorella sorokiniana IAM C-212, Chlorella vulgaris Allma, Tetraselmis suecica F&M-M33, Porphyridium purpureum F&M-M46, Phaeodactylum tricornutum F&M-M40, Tisochrysis lutea (T-ISO) F&M-M36, and Nannochloropsis oceanica F&M-M24) revealed that dry matter and protein digestibility varied up to 30% among algae strains [7]. These discrepancies reinforce the importance of reporting both strain and processing methods in digestibility studies.

While this study was limited to protein and energy digestibility analysis due to sample size, a more comprehensive understanding of nutrient release and utilization would require assessment of lipid digestibility. Cell wall disruption substantially increases the bioavailability of microalgal lipids, particularly those with high-value omega-3 profiles [21,22]. Beyond nutritional factors, cell wall disruption can impact the retention of heat-sensitive and functional compounds, including carotenoids, chlorophylls, and bioactive peptides, whose dietary efficacy relies on their liberation from the cell matrix. In this context, synergistic or sequential processing strategies may optimize digestibility and nutrient retention but must also be assessed for economic and energy tradeoffs. The industrial adoption of microalgae-based feeds requires cost-effective analyses to balance the energy input of physical cell disruption methods with the achieved improvement in nutrient bioavailability and growth performance to ensure economic feasibility at scale.

5. Conclusions

The results of this study confirm our original hypothesis that the cell wall rupture of Chlorella vulgaris caused by the spray-drying (SD Chlorella) process enhances digestibility, leading to improved growth performance in Nile tilapia (Oreochromis niloticus) compared to freeze-drying (FD Chlorella). SD Chlorella demonstrated an 8.6% higher energy digestibility than FD Chlorella, although no significant differences were observed in protein digestibility. Additionally, fish fed SD Chlorella exhibited significantly higher protein and energy retention values, which translated into superior zootechnical performance. These findings highlight the critical role of selecting appropriate processing methods, particularly during the early life stages of fish, to maximize the nutritional potential of microalgae in aquafeeds.