Introduction

Microalgae have received increasing attention because of their ability to fix CO₂, remove nutrients from wastewater and generate biomass of industrial value [1]. They are a heterogeneous group of photosynthetic microorganisms capable of growing in marine, fresh, brackish, residual and edaphic environments under a wide range of temperatures, pH values and nutrient availability [2].

The chemical composition of microalgae is characterized by the presence of lipids (7-23%), carbohydrates (5-23%) and proteins (6-52%) [3], in addition to mineral salts, vitamins and amino acids. These biomolecules make microalgae a versatile resource with applications in food, bioenergy and high value-added compounds [4]. Biotechnological advances have promoted its use in the cosmetic, pharmaceutical, agricultural, and aquaculture industries and in bioremediation processes [5]. Moreover, algal biomass has historically been used as a fertilizer and as food for both humans and animals [6].

In the context of the environment, vinasse is one of the most important types of liquid waste produced in the sugar and biofuel industry. It is estimated that for each liter of ethanol produced, between 10 and 15 liters of vinasse are generated. Vinasse is characterized by its high organic load, expressed as chemical oxygen demand (COD > 40 000 mg/L), and a high mineral content [7, 8]. Its untreated discharge results in serious impacts on water bodies, such as decreased dissolved oxygen and eutrophication, and on aquatic biodiversity. In this context, the recovery of vinasse by growing microalgae is proposed as an alternative to reduce environmental impacts and simultaneously generate biomass with biotechnological potential.

Among microalgae, Chlorella vulgaris is notable for its rapid growth and high efficiency in the removal of nutrients, which makes it a model organism for integrating purification processes and the production of bioactive compounds [9]. Recent studies have reported that the modulation of parameters such as pH and mineral availability can optimize both the accumulation of biomass and the removal efficiency of pollutants in agro-industrial effluents [10, 11]. This allows the mitigation of pollution and movement toward value chains of the bioeconomy by transforming waste into resources for integrated biorefineries.

In this context, the objective of this study was to evaluate the growth of the microalga Chlorella vulgaris in vinasse from ethanol and digestate production and its removal efficiency of organic and mineral contaminants from these byproducts.

Materials and Methods

Materials

Two liquid substrates were used. The primary vinasse was obtained during ethanol distillation, and digestate was generated during the production of biogas from the same stillage (Figure 1). Both byproducts were selected because of their high organic load and mineral content, characteristics that make them suitable media for the growth of Chlorella vulgaris. In addition, aeration, spectrophotometry, cell counting, and the determination of minerals by atomic absorption spectrophotometry were performed in the laboratory, which allowed the characterization of the behavior of the culture and evaluation of the level of purification achieved.

Preparation of the Substrate Media

Media preparation involved the filtration of the vinasse and the digestate through a 100 μm mesh to remove suspended solids that could interfere with algal growth. The initial pH was adjusted to 6.5 ± 0.2 using diluted solutions of NaOH to maintain conditions close to neutrality [12]. In certain treatments, sterilization was performed in an autoclave at 121 °C for 20 minutes, whereas in others, nonsterile substrates were used, with the purpose of simulating scenarios of the direct application of these byproducts. Finally, both the vinasse and the digestate were diluted with distilled water to concentrations of 50% and 100% [13], in accordance with the experimental design.

Preparation and Inoculation of Chlorella vulgaris

The inoculum used in the tests was provided by the Center for Fisheries Research, La Habana. It was previously maintained in BG-11 medium under conditions of 25 ± 2 °C, with a photoperiod of 12:12 hours light:dark and continuous aeration of 0.45 L/min [14]. For the experiments, a culture in the exponential growth phase was selected, which made it possible to obtain an active inoculum with high adaptation capacity. The initial density used was close to 1×10⁶ cells/mL, and the inoculation volume represented 10% of the total volume of each bioreactor. The cells were cultured in 1000 mL Erlenmeyer flasks, which were used as photobioreactors [15]. The transfer was carried out under aseptic conditions to prevent external contamination.

The experimental runs required aeration. Therefore, atmospheric air was introduced by bubbling at a constant flow rate of 0.45 L/min for 8 hours daily using a Neuberger N810FT.18 pump (KNF), considering that cell growth increased with aeration periods greater than 6 h [16, 17]. Once the tests were completed, each experimental combination was evaluated in terms of dry biomass concentration, reductions in COD and five-day biochemical oxygen demand (BOD₅), and decreases in the concentrations of K, Ca and Mg.

Determination of Physicochemical Parameters

SThe behavior of the culture was evaluated by determining the daily cell count of aliquots taken from the culture and the cell density was calculated using a Neubauer blood chamber. The optical density was measured at 680 nm in a Genesys 20 spectrophotometer [18]. Total chlorophyll was estimated from the quantification of chlorophyll a and b, according to the procedure described by Barajas Solano, et al. [19], applying the expressions proposed by [20] (Equations 1, 2 and 3).

Dry biomass was quantified by filtering and drying the samples to a constant weight according to Nayak, et al. [20]. In the culture medium, the COD, BOD₅ and the concentration of minerals (K, Ca and Mg) were determined by atomic absorption spectrophotometry, following the procedures described by [21-23]. The purification efficiency was calculated according to Equation 4.

Experimental Design

In the experimental design matrix, the levels of the operating variables were established as indicated in Table 1. The evaluated variables were the type of substrate (X1), culture time (X2), aeration (X3), illumination time (X4) and substrate concentration (X5). These variables were evaluated according to the methods of [24-27]. The response variables included the production of dry biomass (g/L), the removal of COD and BOD₅ (%) and the removal of minerals (K, Ca and Mg) (%).

The analysis of the factors that influence growth and removal was performed using an adapted fractional Plackett-Burman design, which allowed the simultaneous study of different variables, reducing the number of tests (Table 2). In total, eight experiments were carried out, considering five real variables and two fictitious variables.

The selection of influential factors was based on the magnitude of the main effects estimated in the Plackett-Burman design, considering those with greater absolute values as relevant, which is a criterion used in screening designs for the identification of significant variables [28]. Additionally, the consistency of the effects between the response variables and the trends observed in the experimental results was considered, especially in cases with similar values, such as K, Ca and Mg.

Results and Discussion

Growth of Chlorella vulgaris

The parameters evaluated for vinasse from ethanol distillation and the digestate from biogas production are presented in Table 3. The distillery vinasse had a lower pH and higher concentrations of COD, BOD₅, potassium, calcium and magnesium. In contrast, the digestate had a higher pH and lower values for the other parameters, which allowed us to clearly differentiate the two byproducts as culture media for the growth of Chlorella vulgaris.

The cell density of Chlorella vulgaris reached between 5.0×10⁷ and 8.0×10⁷ cells/mL in distillery vinasse in Experiments E.1, E.2 and E.3, and the cells reached the exponential phase before Day 10 (Figure 2). This result is attributed to the greater availability of nutrients in this medium, which led to rapid and prolonged growth. In contrast, in assays performed for Chlorella vulgaris in digestate (E.4 to E.8), the cell density did not exceed 2.0×10⁷ cells/mL, confirming nutritional limitations [29]. The reduction in biomass concentration in E.1 and E.3 after the stationary phase is related to nutrient depletion and the accumulation of inhibitory metabolites, which is in agreement with findings reported by Candido and Lombardi[30].

Comparative analysis revealed that distillery vinasse is a more favorable substrate for the growth of Chlorella vulgaris because of its higher carbon and mineral contents, whereas the digestate was less efficient because of the reduced availability of compounds during anaerobic digestion [8]. These findings are in agreement with those proposed by [31], who reported the potential of ethanol vinasse as a culture medium, although the results differed from those of Quintero Dallos [32], who reported better growth of Chlorella vulgaris in digestate. These variations can be explained by differences in the chemical composition of the wastes.

The optical density at 680 nm was greatest in cultures grown in distillery vinasse (E.1, E.2 and E.3), reaching 2.5 around Day 12, which indicated a relatively high cell density and good nutrient utilization capacity (Figure 3). When digestate was used, the increases in cell density were lower and it did not exceed 1.5, confirming a lower availability of assimilable organic compounds. This finding is consistent with that reported by Kuo, et al. [18], who reported a direct relationship between optical density and biomass in cultures of Chlorella vulgaris in effluents of livestock origin, and with that of Chi, et al. [33], who reported that cell accumulation depends on the nutritional quality of the medium used.

The total chlorophyll concentration was greatest in the cultures grown in distillery vinasse (E.1, E.2 and E.3), with concentrations between 23 and 28 mg/mL during Days 6-12 (Figure 4). These results revealed an increase in photosynthetic activity and biomass accumulation. When digestate was used, the chlorophyll concentrations were lower and did not exceed 15 mg/mL, confirming limitations in the availability of nutrients for the synthesis of pigments. This behavior is similar to that described by Barajas Solano, et al. [19], who reported that the concentration of chlorophyll is directly related to the growth of Chlorella vulgaris in culture systems. In addition, Nayak, et al. [20] reported that the variation in chlorophyll a and b depends on the nutritional balance of the medium, which explains the differences observed between the two substrates.

Distillery vinasse favored the highest growth of Chlorella vulgaris, with productivities of 4.34 and 3.99 g/L in Experiments 1 and 3, respectively, whereas in the digestate, the values did not exceed 1 g/L (Table 4). These values should be interpreted as apparent dry biomass because the quantification was performed by filtration, a procedure in which inorganic residues present in the culture medium may also be retained. Consequently, the dry mass obtained can overestimate the real organic fraction of the biomass. For a more accurate estimate, determining the ash content by calcination and subtracting that fraction from the dry biomass value is recommended. In parallel, the removal of contaminants reached higher percentages in distillery vinasse, indicating that the reduction in COD was greater than 40% compared with less than 20% in the digestate.

This behavior is attributed to the greater availability of carbon and nutrients in the distillery vinasse, which are favorable conditions for cell growth and purification. The initial composition of carbon and phosphorus in the effluent determines the efficiency of the process, with removal rates of up to 84% for COD and 57% for phosphorus in microalgae systems under optimized conditions [34]. Similarly, Zhou, et al. [35] reported that the ability of Chlorella vulgaris to remove nitrogen and phosphorus depends on carbon supplementation, and the organism reached removal efficiencies greater than 80% when glucose was added to digested residual medium. A comparison of the results of the experiments performed with vinasse revealed that aeration modified the response of the system. In particular, the behavior differed between Experiment 5 and other treatments with vinasse. This variation is associated with a shorter culture time and the absence of aeration, conditions that limit biomass production and removal efficiency of contaminants.

The coefficients of the Plackett-Burman design, where the type of vinasse (E1) and aeration (E3) had positive effects on dry biomass content, whereas Ef1 had a negative effect are shown in Table 5. With respect to the removal of COD and BOD₅, the variables time (E2) and vinasse concentration (E5) had the greatest effects, which indicated their direct influence on the purification processes. With respect to potassium, time had a favorable effect, whereas light (E4) and some interactions were unfavorable. For calcium and magnesium, minor effects associated with time and combinations of factors were identified. These findings reveal that the availability of nutrients and oxygen determines the performance of Chlorella vulgaris [36]. A study by Melikoglu [37] reported greater than 90% removal of nitrogen and phosphorus in systems with aqueous phases of treated sludge.

Regarding the standard error, which is determined from the coefficients of the fictitious variables, and the significance of the coefficients, which is determined by comparing the calculated and tabulated values obtained from the Student’s (t) test, the significant variables were identified for each parameter at a significance level [not significant (NS)] of 95 and 80% probability, respectively (Table 6). Few variables were significant, indicating the simplicity of the system and the strong dependence of Chlorella vulgaris on specific conditions.

The results revealed that a maximum of two significant variables affected the final values, and only time influenced the removal of calcium. This led to the implementation of a 2² full factorial design, which is recommended in the literature [38], to analyze the interactions between the factors identified in Table 6. The removal of magnesium was discarded because of its low significance. The optimal values and the models obtained are shown in Table 7. Aeration and the type of vinasse were identified as determining factors for dry biomass content (4.42 g/L), whereas the vinasse concentration and time were associated with the removal of COD (61.5%) and BOD₅ (36.9%). In the case of potassium, a maximum removal of 85.3% was reached, with light and time as the influencing variables, indicating the sensitivity of the process to culture conditions. These findings support the usefulness of the 2² factorial design to optimize both productivity and depuration, in agreement with recent reports that highlight the ability of Chlorella vulgaris to integrate biomass production and nutrient removal in agroindustrial systems [37].

The correlation between the physicochemical parameters of the vinasse and the growth of Chlorella vulgaris reveals underlying mechanisms that explain the observed differences. The acidic pH of distillery vinasse (4.67) favored the solubility of essential minerals such as potassium, calcium and magnesium, a condition that facilitated their assimilation and resulted in higher values of cell density, chlorophyll content and biomass concentration. In contrast, the pH of digestate closest to neutrality (6.07) was accompanied by lower mineral concentrations, limiting growth and reducing productivity. Jui, et al. [11] reported that the interaction between pH and nutrient availability regulates photosynthetic efficiency and biomass accumulation in microalgae through its direct effect on ionic absorption and enzymatic stability. These results indicate that pH modulation together with mineral supplementation improves purification and productivity in systems based on residual vinasse.

The removal efficiencies observed in the experiments performed with vinasse indicated a high capacity of Chlorella vulgaris to reduce the organic load and the contents of some of the mineral ions present in the effluent. The values of 0.61505 for COD and 0.3690 for BOD₅ indicated a higher purification compared with 36% for the biodegradable compounds, whereas the highest fraction removed was that of K (0.85325), indicating the efficient use of this nutrient in metabolism (Table 8). In contrast, Ca (0.2836) and Mg (0.4476) were partially removed, possibly because of surface adsorption and chemical precipitation rather than biological incorporation. These results are in agreement with those described by Zhou and Broodbank [39] and Yu, et al. [40], who reported the ability of green microalgae to remove organic matter and fix divalent cations to functional groups on their cell walls.

Lower removal fractions were obtained in experiments performed with digestate, with values of 0.1726 for COD and 0.1035 for BOD₅, indicating limited purification of organic compounds, whereas potassium (0.0109), calcium (0.0854) and magnesium (0.1006) exhibited little mineral assimilation (Table 8). This behavior is attributed to a lower availability of usable carbon, the presence of inhibitors, and high turbidity that reduces the photosynthetic efficiency. Studies by Wu, et al. [41] and Liu, et al. [42] reported that light intensity, the proportion of nutrients and the ionic balance strongly influence the productivity and purification capacity of Chlorella vulgaris. Thus, the results confirm that vinasse is a more favorable medium than digestate for promoting microalgal growth and the removal of organic and mineral contaminants, which is consistent with the observations of Bartoli, et al. [43] on the dependence of biological yield on the physicochemical conditions of the crop.

Conclusions

For cultures grown in distillery vinasse, higher values of cell density, chlorophyll content and biomass concentration were reached, accompanied by significant reductions in COD, BOD₅ and minerals, whereas for cultures grown in the digestate, limitations associated with lower nutrient availability were observed. These results allowed us to identify the type of substrate, along with variables such as time and concentration, as decisive factors in the performance of the culture. In addition, the application of experimental designs such as Plackett-Burman and full factorial 2² was effective for establishing interactions between variables and defining conditions that simultaneously increase biomass production and clearance. In this context, the use of microalgae in agroindustrial effluent treatment systems is projected to be a sustainable strategy with potential application in biorefineries.

References

Notes

* Research paper

Author notes

^aCorresponding author. E-mail: rd.vinocungap@uea.edu.ec

Additional information

How to cite this article: R D Vinocunga-Pillajo, A C de Armas Martínez, E González Suárez, V V Kafarov, E Guardado Yordi, A Pérez Martínez, “Growth of Chlorella vulgaris in and removal of organic and mineral contaminants from vinasse, a byproduct of ethanol production” Ing. Univ. vol. 30, 2026. https://doi.org/10.11144/Javeriana.iued30.gcvr

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