Abstract
Introduction:
Vinasse is an organic and nutrient-rich byproduct of the ethanol industry with potential for microalgae cultivation. Although its use is generally limited by the need for dilution, appropriate vinasse pretreatment can improve its suitability as a culture medium, enabling microalgae to thrive. This study investigated the feasibility of cultivating microalgae in 100% previously treated vinasse and evaluated the use of vinasse-grown cultures as inoculum for successive cultivation cycles.
Methods:
Nine microalgal species were evaluated in an initial screening test to determine their tolerance to full-strength treated vinasse. Three species, Chlorolobion braunii, Tetradesmus sp., and Haematococcus pluvialis, were selected for further testing in 6-well plates with 12 mL cultures. Growth rate, cell volume, and the effect of inoculum origin (vinasse vs. synthetic culture medium), were evaluated. Additionally, vinasse was characterized by nuclear magnetic resonance spectroscopy (NMR) before and after algal growth.
Results & Discussion:
C. braunii had the best performance in full-strength treated vinasse, achieving the highest growth rate (1.3 d−1), compared to 0.66 d−1 for Tetradesmus sp. and 0.68 d−1 for H. pluvialis. Notably, C. braunii was the only species that showed a higher growth rate in vinasse than in its control, suggesting mixotrophic growth. Mixed effect modeling showed that cell volume increased 1.6 times in vinasse-grown C. braunii (208 μm3) and 6.3 times in Tetradesmus sp. (250 μm3) in comparison to their controls, but H. pluvialis cell volume was maintained (5575 μm3). Compared to the respective controls, when the inoculum was obtained from vinasse, C. braunii presented 3.2 times lower growth rate (0.4 d−1) and larger cells, but no variation in growth response was observed for Tetradesmus sp. cultures (0.67 d−1 growth rate; cell volume 13.2 times larger), while H. pluvialis formed small cysts. NMR results showed that acetate and butyrate were consumed by the microbial consortium. Additionally, H. pluvialis cultures in vinasse released cis-aconitate, a compound that can be related to physiological adjustments in a stressing condition.
Conclusion:
This study advances the circular economy concept in the ethanol industry by showing that full-strength treated vinasse can serve as a viable culture medium for microalgae, given appropriate species selection.
Graphical Abstract
1 Introduction
The global population has surpassed 8 billion people, increasing the demand for food, energy, and industrial production while intensifying pressure on natural resources and the environment. The Sixth Assessment Report of the Intergovernmental Panel on Climate Change (IPCC) highlights that mitigating global warming requires not only reducing dependence on fossil fuels but also adopting more sustainable resource management practices. Consequently, innovative strategies for waste valorization are prominent (). At the same time, population growth and industrial expansion have substantially increased wastewater generation, making its efficient treatment and reuse essential to prevent the release of excessive nutrients and contaminants into terrestrial and aquatic ecosystems (). Therefore, developing sustainable strategies to recover resources from industrial wastes has become an increasingly important component of the circular bioeconomy.
Sugarcane vinasse is an agro-industrial waste that stands out due to its large production and nutritive potential. It is generated at volumes of approximately 13 L per liter of ethanol (). The characteristics of vinasse depend on the feedstock and processing conditions for the bioethanol generation (). Although vinasse is commonly applied to sugarcane fields through fertigation, due to its high organic load and inorganic nutrient concentrations (), it poses risks to agricultural soils. However, if accumulated in the soil, it may lead to salinization (), acidification (), and modification of soil microbiome () and structure (), requiring a rotational strategy for soil preservation. Additionally, the mobility of nutrients increases the risk of leaching into groundwater and runoff into surface water bodies, leading to eutrophication and other forms of aquatic pollution ().
Brazil is one of the world’s largest producers of sugarcane ethanol, generating approximately 26 billion L during the 2025/2026 harvest, which resulted in nearly 340 billion L of vinasse (). Given its volume, strategies relying exclusively on soil irrigation are insufficient to fully exploit the by-product. Consequently, the sugarcane industry has explored opportunities to implement the biorefinery concept, integrating technologies to convert its by-products into renewable energy, chemicals, and value-added products (; , ). Within this framework, developing complementary approaches for vinasse valorization represents an important opportunity to improve resource recovery and contribute to a circular production system.
Microalgae attract attention because of their microbial attributes and chemical composition, which are related to physiological plasticity that enables them to grow in different conditions and accumulate biomolecules of industrial interest and diverse applications (; ). As photosynthetic organisms, microalgae assimilate CO2 and other inorganic nutrients, but some of them can also use dissolved organic compounds, thus performing mixotrophic growth. Such ability makes vinasse a useful substrate for their growth while bioremediation and biomass generation are obtained (). However, not all algae are mixotrophic, and only some strains support the conditions imposed by vinasse (; ; ). Therefore, screening for microalgae with attributes such as resilience, high growth rates, and mixotrophy can reveal robust candidate species for remediation of wastewater (; ) and of interest to the agro-industrial sector ().
Industrial by-products frequently contain indigenous microorganisms, and vinasse is no exception, given its organic and nutrient-rich composition. It has been shown that algae can profit from the presence of bacteria, which furnish CO2 for photosynthesis (; ). Therefore, growing microalgae in non-sterile vinasse can be considered a consortium, where bacteria, yeast, and algae coexist and may interact (). With proper precautions, the resulting biomass may be exploited for different uses as biofuels, pigments, animal feed, and other bioproducts (; ). Therefore, microalgae cultivation represents a promising strategy within integrated biorefinery systems, where wastewater remediation and resource recovery can be achieved simultaneously.
Several studies have shown that microalgae can grow in sugarcane vinasse, but most have relied on diluted vinasse or used it as a supplement to synthetic culture media. However, successful cultivation of microalgae in vinasse has generally been reported at relatively low vinasse concentrations, typically ranging from 5% to 50%. It depends on the microalgal species, on the vinasse itself, and if any pretreatment has been applied, including vinasse centrifugation, filtration, clarification, pasteurization, biodigestion, sterilization, or combinations thereof (; ; ; ; ; ; ; ; ; ; ; ).
and have improved vinasse quality through physicochemical pretreatments with a specific focus on microalgae growth. Therefore, the patent INPI BR 102015024100-3 resulted in a vinasse that was used in concentrations as high as 80% for the growth of Chlorella vulgaris. Existing patented technologies are predominantly based on physicochemical and biological treatments aimed at improving vinasse management but not necessarily focusing on microalgae growth. In general, the improvements on vinasse include membrane filtration and concentration (), carbon fixation and turbidity reduction (), pH adjustment (), potassium recovery (), biological treatment using kefir microorganisms (), and the use of vinasse in enzymatic hydrolysis processes (). Patents involving microalgae cultivation in vinasse have mainly focused on biomass production using diluted vinasse, nutrient supplementation, and/or CO2 supply (; ), whereas other inventions address downstream processing of microalgal biomass rather than vinasse treatment itself (). Collectively, these patented technologies illustrate the growing effort to integrate sugarcane vinasse into biorefinery processes while highlighting the need for more efficient strategies to maximize the reuse of this agro-industrial effluent.
Despite the advances, cultivation in undiluted treated vinasse remains poorly explored for most microalgal species, particularly regarding successive cultivation cycles. In microalgae cultivation, the quality and physiological condition of the inoculum are critical determinants of culture performance. From an industrial perspective, the ability to propagate successive generations directly in treated vinasse would simplify inoculum production, facilitate process automation, and reduce labor and operational costs. Nevertheless, most studies have employed inocula propagated in synthetic culture media before transfer to vinasse (; ; ; ; ; ). Consequently, the feasibility of using vinasse-grown cultures as inoculum for subsequent cultivation cycles remains largely unexplored.
An important knowledge gap concerns the organic composition and fate of the organic compounds present in vinasse to microalgae. Vinasse contains a complex mixture of organic compounds, including lactic and acetic acids, residual sugars (e.g., glycerol and fructose), and other soluble metabolites (). Several of these compounds may serve as carbon sources stimulating mixotrophy in microalgae (), but which modifications occur in the vinasse as a result of algae cultivation is still unknown. Nuclear magnetic resonance (NMR) spectroscopy provides a non-destructive and highly reproducible means for characterizing complex organic mixtures, making it particularly suitable for investigating potential changes in vinasse.
Based on key knowledge gaps regarding microalgal cultivation in vinasse, this research addresses the following questions: Can microalgae be cultivated in 100% vinasse? How does microalgal growth affect vinasse composition? Can vinasse support inoculum preparation for successive cultures? To address these questions, previously treated vinasse was used to grow the Chlorophyta Chlorolobion braunii, Tetradesmus sp., and Haematococcus pluvialis, and specific growth rate, cell volume, changes in vinasse composition by NMR spectroscopy, and the potential of vinasse-grown cultures to serve as inoculum for successive cultivation cycles were evaluated. The outcomes of this study are a contribution to the development of strategies for vinasse valorization within integrated sugarcane biorefineries.
2 Materials and methods
2.1 Vinasse, microalgae, and culture conditions
The vinasse used in the present research was donated by Usina Santa Lucia (Araras, São Paulo, Brazil). After collection, it was stored in 1-L-capacity plastic bottles and kept in a freezer (−22 °C) until use. Before being used, they were defrosted and treated according to . It refers to a method of vinasse treatment and uses that can be applied to either biodigested or conventional vinasses, with a special focus on microalgae growth. In the present research, the vinasse was percolated sequentially through three different materials used in the same proportion. The filtering materials consisted of coarse sand (particulate material retention), smectite clay, and activated carbon. The final filtrate exhibited a pH of 7 and low turbidity, suitable for microalgae culturing. It consists of a low-cost medium for generating microalgal biomass. The proposed technique enabled the use of the residue (vinasse) while simultaneously generating a product of commercial interest, microalgae (). Before use, no sterilization was performed on the treated vinasse, and the microalgae were inoculated directly into the treated vinasse. After the treatment, microscopic observation showed that bacteria and yeast were still present in the effluent, but they were not monitored in the present research.
The algal strains Botryococcus braunii (Kützing) (CCMA477), Botryococcus protuberans (West and G.S.West) (CCMA480), Botryococcus terribilis (Komárek and Marvan) (CCMA481), Chlorolobion braunii (Komárek) CCMA377, and Tetradesmus sp. (CCMA500) were generously donated by Prof. Dr. Inessa Lacativa Bagatini, curator of the Freshwater Microalgae Culture Collection at Federal University of São Carlos (CCMA). The strains C. vulgaris (Beijerinck) (LBA05), Haematococcus pluvialis (Flotow) (LBA11), and Scenedesmus obliquus (Kützing) (LBA10) were obtained at the Algae Biotechnology Laboratory (UFSCar), and the strain Chlorella sorokiniana (Shihira and R.W.Krauss) (IBVF211-32) was from the Institute of Plant Biochemistry and Photosynthesis, University of Sevilla (Spain). They were selected based on previous descriptions of mixotrophic capacity and ability to produce biomolecules of interest (; ; ). The nine microalgae were used in a screening test, after which three strains were selected to proceed to the experiment.
Stock cultures were kept in BG11 synthetic culture medium () under controlled environmental conditions of light intensity (190 µmol photons m−2 s−1), 12:12-h light/dark cycle, and temperature (25 °C ± 1 °C). The light intensity was measured inside cultures using a photosynthetically active radiation (PAR) sensor connected to a light meter LI 250A (LI-COR, USA). The stock cultures were kept in exponential growth by frequent inoculation into sterile BG11 culture medium that was autoclaved at 121 °C, 1 atm, for 20 min. Cultures were monitored daily by absorbance at 684 nm and cell counts under an optical microscope (BEL Photonics BIO3 Research, China) using a Fuchs-Rosenthal chamber.
2.2 Experimental design
First, an algae screening test was performed to select vinasse concentration and microalgae species to follow for the next experiment. The selection was based on growth performance and the highest vinasse concentration that would support growth, with the goal of 100% vinasse. The screening test consisted of cultures with nine algal strains in BG11 (control), and 50%, 60%, 70%, 80%, 90%, and 100% treated vinasse distributed in 96-well microplates (280-µL volume). Vinasse concentrations were obtained by diluting the treated vinasse with deionized water. Microalgae inocula (105 cells/mL) were obtained from sterile BG11 culture medium. The strains were selected for further testing in 6-well plates with 12-mL cultures. The total volume inoculated in the 96-well screening test was 8% for all algae, except C. braunii, Tetradesmus sp. and H. pluvialis, whose volume was 5% for both 96- and 6-well plates.
The microplates were incubated under the same conditions as described for the stock cultures and were monitored daily for absorbance at 570, 684, and 750 nm using an Epoch™ BioTek Microplate Spectrophotometer (USA). Absorbances measured at several vinasse concentrations without microalgae were used as blanks, which were subtracted from the culture’s absorbance measured daily. Three experimental replicates were performed. From this test, growth rates based on absorbance 684 nm were obtained, and a heat map was plotted (Supplementary Figure 1, Supplementary Material). Based on these results, C. braunii, Tetradesmus sp., and H. pluvialis were selected to proceed.
The incubation time for the screening test (96-well plates) and experiment (6-well plates) was 5 days and based on the growth curve of the algae, after which cells enter the stationary growth phase. Maximum culture evaporation was 7% of the total volume. The evaporation volume was quantified in well plates filled with water instead of vinasse. The water mass was determined on a 10−6-g precision balance (Mettler Toledo XPE26, Switzerland), and the difference between the initial (day 1) and final values (day 5) was assumed to be evaporation. All plates were maintained covered with their original lids and kept under the controlled conditions described above. Light intensity was measured inside the wells using a PAR sensor to ensure 190 µmol photons m-² s-¹ irradiance independently of vinasse concentration. Irradiance was adjusted using dimmers connected to the LED light source.
Cultures of C. braunii, Tetradesmus sp., and H. pluvialis were performed in 6-well microplates and 100% treated vinasse (12 mL) in three experimental replicates to analyze the potential of vinasse to support successive algal generations, count and measure the algal cells, and carry out NMR analysis, in addition to absorbance measurements. Treatments consisted of control cultures in BG11 whose inoculum was obtained from cells growing in BG11, cultures in vinasse whose inoculum was obtained from cells in BG11 (here referred to as V1), and cultures in vinasse whose inoculum was obtained from cells growing in 100% treated vinasse (here referred to as V2). Independent of the inoculum origin, the cells to be inoculated were in exponential growth phase. Daily, cell counts were done on a Fuchs-Rosenthal chamber under an optical microscope, and absorbance was measured at 570, 684, and 750 nm using an Epoch™ BioTek Microplate Spectrophotometer (USA). On the last experimental day, an aliquot of the cultures was obtained for cell biovolume estimation. The remaining culture was centrifuged (15,000 rpm, 5 min) and then underwent NMR analysis.
2.3 Cell volume and growth rates
Cell volume was estimated after cell measurements using an optical microscope (Olympus BX41TF, Japan) with an attached camera (Olympus U-SPT, Japan) and scale (TCapture software). Culture samples were obtained on the 5th day, preserved in 4% formaldehyde, and kept refrigerated at 4 °C for subsequent cell volume analysis. Measurements were performed using the imaging software ImageJ, following the methodology described in . For each replicate, 30 cells were measured. A cylinder shape with two cones was used as the cell shape for C. braunii, a prolate spheroid for Tetradesmus sp., and a sphere for H. pluvialis.
Specific growth rates were calculated by plotting the natural logarithm of cell density (cell/mL) as a function of experimental time (days) and adjusting a linear regression for the exponential growth phase. From the linear regression equation, the angular coefficient is here considered as a representation of the maximum growth rate. It should be mentioned that bacteria and yeast may have influenced growth rate metrics in the screening test, since the 96-well microplates were monitored solely by absorption at 684 nm, even though this absorption wavelength applies to the chlorophyll peak. This observation does not apply to the 6-well plates, since these cultures were monitored by cell counts under a microscope and no bacteria or yeast was recorded.
2.4 NMR analyses
To evaluate possible modifications in the organic content of vinasse induced by microalgae, vinasse was characterized by NMR spectroscopy analyses. Its raw and treated forms before and after microalgae cultivation were analyzed. For this analysis, 450 µL of vinasse was transferred to Eppendorf® tubes and mixed with 50 µL of a D2O solution containing 0.5 mM of sodium 3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionate (TMSP-d4). This solution was used as a chemical shift reference, along with a phosphate buffer adjusted to pH 7.42. After homogenization, the mixtures were transferred to 5-mm NMR tubes for spectral acquisition. One (1D) and two-dimensional (2D) NMR spectra were recorded on a Bruker® Avance III spectrometer operating at 400.23 MHz for ¹H detection, equipped with a broadband inverse (BBI) probe. The 1D spectra were acquired using the zgpr pulse sequence for presaturation of the water signal at 4.70 ppm. Spectral processing was carried out using TopSpin® software (version 3.7.0, Bruker®) following the manufacturer’s standard parameters. For data analysis, the compounds were identified with the support of 2D NMR spectra. For quantitative data analysis, the areas of the least overlapped signals in the ¹H spectrum corresponding to each metabolite of interest were integrated in relation to the TMSP-d4 signal, arbitrarily adjusted to 1.0. For data analysis, the compounds were identified with the support of 2D NMR spectra.
2.5 Data analysis
Graphical representations and statistical analysis were done using GraphPad Prism 10. Statistical analysis included two-way ANOVA followed by Tukey’s multiple comparison tests for comparing growth rates (Supplementary Figure 2—residual analysis shown in the Supplementary Material). Cell volume was analyzed using a mixed-effects model where replicates were modeled as random effects. A natural logarithm transformation was used on cell volume to ensure model assumptions were satisfied, which is shown in Supplementary Figure 2. The graphical abstract was created with BioRender.com.
3 Results
The results of the screening assay (96-well microplates) are shown in the Supplementary Material as a heat map of the growth rates for the nine algal species grown in 50%–100% treated vinasse concentrations. It can be observed that C. braunii and Tetradesmus sp. performed the best in vinasse 90%–100%. Although H. pluvialis did not grow as well, due to its commercial interest and better performance in vinasse in comparison to its control in this screening test, it was selected. Therefore, C. braunii, Tetradesmus sp., and H. pluvialis were selected for the subsequent experiment and evaluated in 100% treated vinasse, as well as in successive inoculations into vinasse (V1 to V2 treatments).
Figure 1 shows the growth curves obtained for the control and cultures in 100% treated vinasse inoculated with cells from BG11 (V1) and from vinasse (V2). In general, maximum cell density was similar for both the control and V1 (~106 cells/mL−1), but approximately 60% lower for V2. Microscopic observations revealed that H. pluvialis formed cysts in V2, a characteristic response of this species to environmental stress.
Figure 1
Figure 2 reports growth rates for treatments and control. Among the treatments in vinasse, C. braunii grown in V1 presented the highest growth rate (1.3 day−1), nearly twice that obtained for the other microalgae and its control (0.7 day−1). However, the performance of this species in V2 (0.4 day−1) was not as good, decreasing by 1.6 times in comparison to V1. Tetradesmus sp. and H. pluvialis growth rates in either vinasse treatment were almost half of their control value—1.3 day−1 for Tetradesmus sp. and 1.5 day−1 for H. pluvialis. Nevertheless, the growth rates of Tetradesmus sp. were similar in both V1 (0.66 day−1) and V2 (0.67 day−1) treatments, while H. pluvialis encysted in V2.
Figure 2
The results of microalgae cell volumes are reported in Figures 3, 4. Figure 3 shows that cell volume in C. braunii varied from 53 µm3 in the control to 1,073 µm3 in V2, Tetradesmus sp. from 13 µm3 in the control to 855 µm3 in V2, and H. pluvialis from 905 µm3 (V2) to 33,510 µm3 (V1). In the control, the volume of H. pluvialis varied from 2,144 to 11,494 µm3. In V2, Tetradesmus sp. presented a cell volume almost 14 times larger than its control and 1.7 times larger in relation to V1 (250 µm3). According to Figure 4, except for H. pluvialis, cell volume was dependent on the inoculum origin, with larger cells in V2 than in V1.
Figure 3
Figure 4
The NMR results are reported in Figure 5, Table 1. Figure 5 shows the 1D spectra, and Table 1 shows the relative abundance of major metabolites in vinasse before and after algal growth. The NMR-1H spectrum of raw vinasse exhibited signals of butyrate, isobutyrate, ethanol, acetate, succinate, isocitric acid, methanol, and 1, 2-propranediol (Figure 5). Figure 5 shows that, in general, after algal growth, the vinasse differed in comparison to the raw vinasse, including the reduction of some signals, the disappearance of others, and the appearance of a new metabolite. The signal at 3.45 ppm can be assigned to the metabolite trans-aconitate, present in the vinasse where H. pluvialis was grown. From the data presented in Table 1, which were assessed through ¹H NMR signal integration, variations in signal intensities were observed, suggesting differential metabolite consumption or production by each algal species when grown in the effluent.
Figure 5
Table 1
| Compounds | Treated vinasse | C. braunii | Tetradesmus sp. | H. pluvialis |
|---|---|---|---|---|
| Ethanol | 0.0475 | 0.0407 ↓ | 0.0533 ↑ | 0.0482 ↑ |
| Acetate | 0.6754 | 0.0089 ↓ | 0.0135 ↓ | 0.0104 ↓ |
| Butyrate | 0.2699 | 0.0856 ↓ | 0.0579 ↓ | ND |
| 1, 3-Propanediol | 0.1535 | 0.0713 ↓ | 0.0332 ↓ | ND |
| Succinate | 0.0957 | ND | ND | ND |
| Methanol | 0.0684 | 0.0591 ↓ | 0.0713 ↑ | 0.0823 ↑ |
| Trans aconitate | ND | ND | ND | 0.0092 ↑ |
| Isocitric acid | 0.0554 | ND | ND | 0.0514 ↓ |
Relative integral values (arbitrary units) of ¹H NMR signals corresponding to key compounds detected in treated vinasse and in vinasse where C. braunii, Tetradesmus sp., and H. pluvialis were cultivated.
Integrals were normalized using the TMSP-d4 signal as reference (set to 1.0).
ND, not detected; ↓, indicates a decrease; ↑, indicates an increase in signal area relative to treated vinasse.
4 Discussion
4.1 Microalgal growth rate
The present study showed that full-strength-treated sugarcane vinasse was suitable for growing selected microalgal species, supporting its potential as an alternative culture medium even though the responses differed among species. The successful growth of C. braunii, with a higher growth rate in vinasse compared to the control, is an indication of mixotrophy and suggests that dissolved organic carbon in the vinasse contributed to cellular metabolism. Photosynthetic determinations can be conducted to support mixotrophy in C. braunii under vinasse growth. A similar behavior has been reported for C. vulgaris cultivated in treated and diluted 40% vinasse, in which reduced photosynthetic activity did not limit cell growth, supporting the role of mixotrophy (). According to , supplementing algae cultures with organic substrates as a supplementary energy source makes irradiance less restrictive and enables algal growth in darker media. Therefore, the vinasse pretreatment was a key step for microalgae cultivation in full-strength-treated sugarcane vinasse.
The present results confirmed those of , who reported on Chlamydomonas biconvexa and Micractinium sp. grown in full-strength-treated vinasse (centrifuged and clarified). Although the authors did not report growth rate values, they obtained biomass output higher in vinasse than the respective controls. Interestingly, investigated the growth of C. vulgaris in vinasse and used the same method for vinasse treatment as the one of the present study, except that vinasse pasteurization was used at the end of the treatment. Similar to the present results, they obtained microalgae growth in 80% vinasse, which is similar to the control, but unfortunately, the authors just reported optical density, with no growth rates.
The results of , ), who applied only physical pretreatment to vinasse, contrasting with the physical and chemical processes used in the present study, are a demonstration of the importance of chemical treatment in enhancing the suitability of vinasse for microalgae growth. Additionally, , ) showed the importance of cell acclimation to achieve better growth rates. Compared to the controls, which were performed in BG11, the authors showed that C. vulgaris and Scenedesmus sp. previously acclimated to vinasse had growth rates nearly 1.5 times higher than those that were not acclimated. However, the concentrations of vinasse above 15% led to cell death. We rationalize that microalgae acclimation can be a strategy for improving its growth in vinasse, which could further enhance the response of successive generations maintained in the effluent.
In the present research, the similarity of growth rates observed for Tetradesmus sp. regardless of the inoculum source, whether from vinasse or synthetic medium, highlighted this organism as a robust candidate for industrial applications, even though its growth rate was half of the control. The different behavior observed for C. braunii (lower growth) and H. pluvialis (encystment) deserves further investigation, but as suggested from the results of , ), cell acclimation can be an alternative. Additionally, it must be observed that the scalability of the proposed systems (96- and 6-well microplates) has still to be verified. It is known that laboratory conditions greatly differ from those in the natural environment usually present in large-scale systems, such as dead zones and variation in light intensity. These aspects were controlled for the present experiments.
Among the species tested in the present research, H. pluvialis was the most sensitive, with a marked reduction in growth rate in vinasse in comparison to the control. Considering that H. pluvialis is a known mixotrophic alga (), one possible explanation is that the organic carbon profile in the treated vinasse did not favor a balanced mixotrophic metabolism. Additionally, the presence of toxic substances may have contributed to its inhibition. Literature has shown that different carbon sources trigger distinct metabolic responses in H. pluvialis, with glucose favoring heterotrophic growth and acetate promoting mixotrophy (). Further studies evaluating the relationship between specific carbon sources and H. pluvialis physiology are required to optimize its growth in vinasse.
The success of growing microalgae (C. braunii and Tetradesmus sp.) in 100% treated vinasse (V1) can be due to a combination of factors that include vinasse treatment described in and the microalgae species. The treatment successfully converted dark vinasse into a light, translucent effluent, facilitating light penetration while preserving essential inorganic () and organic nutrients (this work). Additionally, the treatment adjusted the vinasse pH from 4.0 to approximately 7.0, providing an ideal range for the freshwater green microalgae used in this study. Unlike most patented vinasse treatments, which target general waste management, the method used in this study focuses specifically on enhancing microalgae growth. Consequently, each stage of the vinasse treatment process was designed and evaluated based on microalgae cultivation parameters and growth rates.
4.2 Microalgal cell volume
It is known that microalgae cell morphology, including its volume, is affected by environmental stressors such as pH () and chemical pollutants such as glyphosate (). One of the consequences of increasing cell volume is a decrease in surface area to volume ratio, thus decreasing nutrients (and toxicants) in the cells. showed that cell size increase can occur as a consequence of exposure of microalgae to toxic compounds and that this directly affects cell division rate. This is similar to the cell morphology effect observed in the present research, in which larger cells had a lower division rate, reflected in lower growth rates.
The increased cell volumes of C. braunii and Tetradesmus sp. in vinasse may have had the contribution of exposure time to the toxic compounds, since among the larger cells, those of the greatest size were detected in the V2 treatment. The prompt encystment of H. pluvialis in vinasse (V2) and the lack of its volume increase obtained in the present study differ from the results of , ). However, the authors used diluted vinasse (5%), meaning a lower toxicant concentration. The morphological response observed in the present research as cell size increased can, therefore, be related to the chemical nature, complexity of the substrate, and exposure time. Vinasse contains a heterogeneous mixture of organic molecules, minerals, and fermentation by-products (), which may promote distinct metabolic pathways and impact cell morphology.
4.3 NMR in vinasse
As shown by the NMR analysis in this research, the decrease in the organic acids acetate and butyrate in vinasse after microalgae growth can be considered a demonstration of their use as organic nutrients by the microbial consortium. According to , ), acetate is a compound normally used to induce mixotrophic metabolism in microalgae, but butyrate is less generalized and its use is species-dependent. investigated the growth of 10 microalgae in acetate and butyrate and confirmed the lower tolerance and induction of stress in 9 of the 10 microalgae strains to butyrate. point out that to take advantage of mixotrophy, an equilibrium between heterotrophic metabolism and carbon fixation by photoautotrophy should be considered. Therefore, the clarification of vinasse accomplished as a result of its treatment in the present research, which supports light penetration into the cultures, is an important factor for mixotrophy.
The possibility that bacteria and yeasts present in the vinasse may have produced/consumed some of the compounds detected by NMR cannot be ruled out. According to , certain bacteria can produce butyrate, whereas the yeast, Saccharomyces cerevisiae, neither produces nor consumes such an organic compound. The authors report on a bacteria–yeast consortium for the synthesis of butyrate whereby the yeast produces ethanol and acetate, which are further metabolized by bacteria. In a study of genome-resolved metagenomics of sugarcane vinasse bacteria, showed that the predominant bacteria in vinasse were Lactobacillus, Megasphaera, and Mitsuokella (phylum Firmicutes, 35%–97% of the sample reads), Arcobacter and Alcaligenes (phylum Proteobacteria, 0%–40%), Dysgonomonas (phylum Bacteroidetes, 0%–53%), and Bifidobacterium (phylum Actinobacteria, 0%–18%). Considering these candidate bacterial genera to be present in vinasse, acetate producers can be assigned to Bifidobacterium, Dysgonomonas, Mitsuokella, and Lactobacillus, while acetate consumers can be assigned to Megasphaera, Arcobacter, and Alcaligenes. Among them, Alcaligenes consumes butyrate, while Lactobacillus and Megasphaera produce butyrate. However, only Alcaligenes is aerobic, while the others are either anaerobic/facultative anaerobic or aerotolerant. In conclusion, the experimental conditions of the present research may have favored mixotrophy in C. braunii, which exhibited a growth rate 1.8-fold higher in vinasse compared to its control. Conversely, vinasse did not enhance population growth in Tetradesmus sp. nor in H. pluvialis, both of which showed lower growth rates than their respective controls.
The NMR analysis performed in vinasse after microalgae growth revealed cis-aconitate exclusively in H. pluvialis cultures, indicating a species-specific response. Although aconitic acid is typically present in sugarcane vinasse (), it was removed during treatment (this research). Its conjugate base, cis-aconitate, was not detected in the raw or treated vinasse, nor in the vinasse where C. braunii and Tetradesmus sp. were grown. Literature data show that several physiological aspects related to the TCA cycle and stress acclimation can be related to the excretion of cis-aconitate in H. pluvialis. showed that high light intensity, high salinity, or nitrogen starvation can lead to excretion of cis-aconitate in H. pluvialis, preventing mitochondrial imbalance. argued that such physiological responses have an important role in balancing the redox state of the cell. Therefore, the presence of cis-aconitate in the vinasse medium, coupled with a 60% reduction in growth rate, confirms that H. pluvialis cells were under stress. Citric acid, the first intermediate in the citric acid cycle, was identified in both raw and treated vinasse. Its depletion after the cultivation of C. braunii and Tetradesmus sp., in contrast with its presence in the H. pluvialis medium, suggests that the organic acid was assimilated by the former two species but not by the latter. This is consistent with mixotrophic metabolism, but the persistence of citric acid in H. pluvialis medium may reflect a metabolic difficulty at the aconitase step, leading to reduced conversion of citrate into downstream intermediates. Recent studies in engineered cyanobacteria have shown that partial suppression of aconitase activity increased citrate accumulation and excretion as overflow metabolism (). The present findings highlight the species-specific nature of metabolic modulation in vinasse media. While C. braunii and Tetradesmus sp. assimilated available organic compounds, H. pluvialis exhibited metabolic signatures consistent with stress adaptation, emphasizing the distinct physiological adjustments required by this species under vinasse exposure.
5 Conclusion
Full-strength-treated sugarcane vinasse successfully supported microalgal growth, but performance varies significantly by species. C. braunii can be categorized as the most successful organism in the effluent, followed by Tetradesmus sp. and then H. pluvialis. With a growth rate higher than the control, only C. braunii performed mixotrophically in the treated vinasse, but it was Tetradesmus sp., cultivated in vinasse, that was unaffected by the inoculum source, growing at a similar rate whether inoculated from vinasse or synthetic medium. Cell volume of the Chlorophyta C. braunii and Tetradesmus sp. in vinasse increased, with larger cells present at longer exposure times, as detected by the successive cell inoculation in vinasse. By contrast, vinasse-grown H. pluvialis cells were similar or smaller compared to the control. H. pluvialis was the only organism that produced cis-aconitate in vinasse, which, when combined with its decreased growth rate, indicates that cells were experiencing stress. Therefore, this research suggests that pretreating vinasse is a crucial step for successful microalgae cultivation, alongside careful species selection. Future work should focus on scaling up culture volumes to evaluate biomass and biomolecule productivity.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contributions
VN: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing. VF: Formal analysis, Visualization, Writing – review & editing. TV: Resources, Supervision, Writing – review & editing. RP: Formal analysis, Writing – review & editing. LZ: Funding acquisition, Project administration, Supervision, Writing – review & editing. AL: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. Vitória G. Navarrete was supported by a CAPES PhD scholarship. This study was also supported by the São Paulo Research Foundation (FAPESP), Brazil (Grant No. 2018/07988-5), and by the National Council for Scientific and Technological Development (CNPq), Brazil (Grant No. 308003/2023-3). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments
RP is acknowledged for his voluntary participation in this study, which was conducted entirely during his personal time and is unrelated to his professional responsibilities at Stanford University. The authors thank Dr. Natalia L. de Oliveira for statistical support and Prof. Dr. Josmar Pagliuso for valuable suggestions regarding vinasse treatment operation and equipment design.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was used in the creation of this manuscript. ChatGPT (GPT-5.6) was used to assist with English refinement.
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Correction note
This article has been corrected with minor changes. These changes do not impact the scientific content of the article.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/finmi.2026.1917607/full#supplementary-material
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Summary
Keywords
Chlorolobion braunii, industrial effluent, mixotrophy, sugarcane industry, Tetradesmus sp., wastewater reuse
Citation
Navarrete VG, Ferreira VS, Venâncio T, Pulgrossi RC, Branco LHZ and Lombardi AT (2026) Growing microalgae in treated vinasse: effects on cell morphology and growth rate. Front. Ind. Microbiol. 4:1917607. doi: 10.3389/finmi.2026.1917607
Received
23 June 2026
Revised
31 July 2026
Accepted
04 August 2026
Published
15 September 2026
Corrected
28 September 2026
Volume
4 - 2026
Edited by
Igor Carvalho Fontes Sampaio, Universidade Estadual de Santa Cruz, Brazil
Reviewed by
Aman Singh Rajpoot, PDPM Indian Institute of Information Technology, Design and Manufacturing, India
Adriana Ramírez-Romero, Institute of Agrifood Research and Technology (IRTA), Spain
Updates
Copyright
© 2026 Navarrete, Ferreira, Venâncio, Pulgrossi, Branco and Lombardi.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Vitória G. Navarrete, vitoria.navarrete@unesp.br
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.