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Article

Consortium of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae Yeasts for Vinasse Fermentation of Agave americana L. Liquor for Biomass Production and Reduction in Chemical Oxygen Demand

by
Roberto Robles Calderón
1,*,
Francisco Alcántara Boza
2,
Elmer Benmites-Alfaro
3,
Oscar Tinoco Gómez
4 and
Jaqueline Chirre Flores
5
1
Faculty of Chemistry and Chemical Engineering, Professional School of Chemical Engineering, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
2
Faculty of Geological, Mining, Metallurgical and Geographical Engineering, Professional School of Geographical Engineering, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
3
School of Environmental, Universidad César Vallejo, Trujillo 13001, Peru
4
Faculty of Industrial Engineering, Professional School of Industrial Engineering, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
5
Faculty of Environmental Engineering, Professional School of Sanitary Engineering, Universidad Nacional de Ingeniería, Rímac 150101, Peru
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(5), 281; https://doi.org/10.3390/fermentation11050281
Submission received: 25 March 2025 / Revised: 21 April 2025 / Accepted: 23 April 2025 / Published: 14 May 2025
(This article belongs to the Special Issue Food Wastes: Feedstock for Value-Added Products: 5th Edition)
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Abstract

The vinasse from Agave americana L. (blue cabuya) liquor has a high concentration of organic matter, nutrients with high chemical oxygen demand (COD), and low pH, properties that give it the potential to generate adverse impact on the environment if improperly disposed of. In other countries, studies have been conducted on yeast strain combinations in vinasses to produce biomass and reduce organic load, but there are no studies of the aforementioned yeast consortium in blue cabuya liqueur vinasses to produce biomass and reduce COD. Given this problem, the objective of the research was to reduce this adverse environmental impact through aerobic fermentation of this vinasse with the yeast consortium Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae (D 47-Lalvin). As a result, biomass production and COD reduction were achieved. The study evaluated temperature variables of 28 °C, 30 °C, and 32 °C, and pH values of 3, 4, and 5 under conditions of consortium and nutrient diammonium phosphate (DAP) concentrations of 1.32 g/L and 1.5 g/L, respectively, in a bioreactor with automatic control of temperature, time, stirring speed of 100 RPM, and air flow of 1 VVM. The result was a biomass yield of 93.4% and a COD reduction of 33.3%. It is concluded that the aerobic fermentation process of blue cabuya liquor vinasse with the yeast consortium employed produces a high biomass yield, which can be used for its protein value as an animal feed supplement and, due to its low COD value, as an agricultural fertilizer.

1. Introduction

Taxonomically, Agave tequilana Weber is classified as a member of the Rigidae group within the genus Agave of the Agavaceae family [1]. Several varieties of A. tequilana Weber have been described, including “blue”, “blue striped”, “moraleño”, “sahuayo”, “bermejo”, “zopilote”, and “Creole”, the preferred variety has been A. tequilana Weber var. “azul” due to its relatively shorter life cycle and capacity to accumulate high levels of fructans (inulins), which can be utilized for the production of tequila [2].
In Peru, due to the diversity of ecosystems, the blue cabuya (Agave americana L.) grows wild on large tracts of land in rural areas of the departments of Cajamarca, Huancavelica, Huánuco, and Ayacucho. Despite its significant availability, it is not exploited industrially, and this raw material is wasted. In the department of Huancavelica, residents use cabuya juice for the artisanal processing of liquor and chancaca [3].
Tequila production generates vinasse; this by-product is generated in significant quantities. For each liter of tequila, 10 L to 12 L of vinasse are produced [4]. The discharge of this residue into water bodies or soil has a negative impact on the ecosystem. As a result, its treatment is necessary before discharge due to its low pH of 3.4 to 4.5, its high phosphate content (100 to 700 mg/L), its biochemical oxygen demand (BOD) (35,000 to 60,000 mg/L) [4], and its high outlet temperature from the distilleries of 70–80 °C.
In 2018, tequila production in Mexico was 309 million liters, generating 3090–3708 million liters of tequila vinasse [5], which, without proper treatment, is equivalent to the annual biochemical oxygen demand (BOD) pollution produced by 8.4 million people [4]. Large tequila companies with the financial resources are implementing vinasse treatment systems, but small companies that do not have these financial resources discharge it directly into water bodies (rivers, streams, lakes, reservoirs) and municipal sewage systems or directly into the soil as fertilizer without receiving proper treatment [4].
This by-product, “vinasse”, has a high organic load and contaminants, posing environmental risks if not properly treated before disposal [6,7]. In this context, the treatment of vinasse is important to avoid soil degradation and fertility due to its acidic nature and the presence of toxic heavy metals. Vinasse can contaminate water due to high COD and Biochemical Oxygen Demand (BOD), cause eutrophication and acidification of water bodies, and harm aquatic life [8]. Other related impacts are its contribution to greenhouse gas emissions by releasing methane [7].
Poor management of vinasse can cause serious contamination when discharged into aquatic bodies, resulting in the death of fish and negative impacts on other aquatic organisms such as crustaceans and reptiles [9,10]; this occurs due to the high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) of vinasse, which contributes to the depletion of oxygen in the water [11]. Contamination also spreads to groundwater when vinasse is applied to the soil for very long periods of time [12]. In agricultural soil it can improve its quality due to the high content of organic matter and nutrients, but in excess it can produce salinization and changes in pH levels [13]. Another danger is the presence of traces of heavy metals that vinasse may contain and, when accumulated, can be incorporated into the food chain [14].
The importance of vinasse treatment is to take advantage of this residual effluent to recover the organic matter present and convert it into usable biomass that can be used in animal feed by having important nutritional components, such as potassium, nitrogen, and organic matter that, applied as a natural fertilizer, can improve soil fertility [15,16]. For all this, the treatment of vinasse and taking advantage of its potential within the framework of a circular economy is essential, adopting approaches such as aerobic digestion using a yeast consortium [16]. Other approaches are its use in fertigation and biorefinery, so that vinasse, from being a pollutant, becomes a valuable resource promoting sustainability and economic benefit [17].
Scientific literature indicates that yeasts have beneficial properties for industrial applications, such as K. marxianus, which has a rapid growth rate, is thermotolerant, and has a wide range of sugars, which gives it the potential for use in biotechnological processes [18,19], in the same way the yeast Candida utilis has the ability to generate extracellular metabolites such as citric acid, ethanol, and xylitol, with application in the food, cosmetics, and pharmaceutical industries [20,21]. Other Candida species, such as Candida Glabrat and Candida parapsilisius, produce significant biomass with a high protein power when fermenting vinasse of approximately 46.85% and reduce the COD by more than 50% [22,23]. Saccharomyces cerevisiae strains are also effective growth media for up to 40% vinasse [24].
Researchers [25] analyzed individual the biological and physicochemical treatment methods and combined the technologies for their use on distillery vinasse. Contamination of distillery vinasse depends on the quality of the substrates and the unit operations used for alcohol production, which means that vinasse characteristics may differ between distilleries [26,27].
Treatment of vinasse is conducted using the aerobic microbial method [28], using activated sludge, with a microorganism concentration of 13.578 g/L, and an air flow of 5 L/min, for 2 h to 10 h. The objective of the biological treatment is to remove or reduce the concentration of organic and inorganic compounds [29]. The addition of oxygen is important to treat these contaminants so that their concentration is reduced or even completely eliminated [30,31].
Researchers [24] biologically treated vinasse with yeast for the production of single-cell protein for feed supplementation. Two strains of Saccharomyces cerevisiae (CCMA0187 and CCMA 0188) and one strain each of Candida glabrata (CCMA 0193) and Candida parapsilosis (CCMA 0544) showed the highest biomass production with 306, 312, 388, and 306 mg/L, respectively. The applied biological treatment was promising, with a 55.8 and 46.9% decrease in BOD and COD, respectively. These results confirmed the potential use of yeast in the treatment of vinasse while producing protein biomass for use in other applications, such as animal feed.
The objective of the research was to determine the biomass yield and COD reduction in the treatment of blue cabuya liquor vinasse through the aerobic fermentation process treated with the yeast consortium C. utilis, K. marxianus and S. cerevisiae, at a concentration of 1.32 g/L, at temperatures of 28 °C, 30 °C and 32 °C, at pH of 3, 4 and 5; at a DAP nutrient concentration of 1.5 g/L, with an airflow of 1 VVM; in a bioreactor with automatic temperature and time control, and a stirring speed of 100 RPM.

2. Materials and Methods

The vinasse by-product of the distillation of alcohol from the juice of the Agave americana L. was obtained in the community of La Merced, (Huancavelica, Perú) where the inhabitants process the juice of the Agave americana L. to obtain liquor and other products. The Agave americana (blue cabuya) used in the process must be in a state of complete maturity and obtained before the floral scape comes out, as the plant consumes its sugars for the floral scape to come out—this way, the extracted juice will conserve the sugars [32] (see Figure 1).
Hydrolysis of the juice of blue cabuya (Agave americana L.)
The blue cabuya juice had 12.8 °Brix; the sweetness was due to the presence of sucrose, fructose, glucose and a high content of fructans, mainly inulin, so it was necessary to hydrolyze the fructans to release the fermentable sugars, which was carried out in a 20 L capacity autoclave Taisite (Taisite Lab sciences, new York, USA, vertical pressure steam sterilizer) at temperature conditions of 110 °C for 15 min and pH of 2.5 [33]. The blue cabuya juice had an average pH of 4.5 ± 0.2. To bring the pH to the hydrolysis conditions, 1.2 mL of H2SO4 was added to 1 L of cabuya juice; 9.4 M, so as not to dilute the concentration of sugars in the sample. After the hydrolysis, the juice with 14.4 degrees Brix was obtained.
Fermentation and distillation of blue cabuya juice to obtain vinasse
Once the hydrolysis of the fructans present in the blue cabuya juice was completed, the alcoholic fermentation process was carried out in batches of 20 L of juice in a 27 L capacity fermenter (Lafayette, IN, USA), at ambient temperature conditions of 22 °C, pH 4 (the high acidity of the juice due to the hydrolysis conditions was regulated with CaCO3), DAP nutrient concentration of 1.5 g/L and Saccharomyces cerevisiae D 47–LALVIN (Montreal, QC, Canada) yeast concentration of 1.2 g/L.
The process involved distilling 4600 mL of must from the alcoholic fermentation in a 5 L glass tower still with distillation rings, yielding 709 mL of 55 °GL distillate (1 L of liquor/6.5 L of vinasse) and 4116 mL of vinasse. The distillate is shown in Figure 2.
Measuring biomass production
The concentration of the yeast consortium during the aerobic fermentation of Agave americana L. liquor vinasse was determined as follows: 10 mL of the must was centrifuged at 4000 rpm for 15 min; the sediment was placed in a crucible and 10 mL of 0.85% w/v alkaline NaCl solution was added. The solution was dehydrated in an oven at 80 °C for 6 h until a constant weight was reached. The amount of biomass was determined by the weight difference between the crucible with dry biomass and the crucible without sample [3].
Determination of COD
The chemical oxygen demand (COD) and oxygen consumption in the aerobic fermentation process of Agave americana L. liquor vinasse was determined with the RS-xx-N01-HHT Portable Water Quality Quick Test Recorder User (Jinan, China).
Determination of CO2
The production of CO2 in the aerobic fermentation process of the Agave americana L. (blue cabuya) liquor vinasse was measured with the TES-1370 NDIR CO2 analyzer equipment (Brookfield, WI, USA).
Inoculum activation
The yeast’s Saccharomyces cerevisiae varieties Ellipsoideus, D 47–LALVIN, C. utilis, and K. marxianus were in a dehydrated state, so it was necessary to rehydrate and activate them. The process was as follows: to 3 L of blue Agave americana L. liquor vinasse, the nutrient DAP was added at a concentration of 2 g/L, then the solution was sterilized at 110 °C for 3 min and transferred to a 5 L flask, in which the aeration system was installed. The solution was cooled to the incubation temperature of 26 °C, the yeast consortium was added, and sterile air was supplied at a flow of 1 vvm for 24 h, until reaching a concentration of 1.3 g of biomass/L. It is estimated that 1.0 g of cells ≅ 1.4 × 107 cells, then the concentration of consortium used is in the range of number of cells/mL to inoculate the vinasse [34].
Aerobic fermentation process of Agave americana L. liqueur vinasse
For each experimental test, 10 L of cabuya liquor vinasse were used, which was carried out in a 12 L bioreactor (Lima, Perú), with the following characteristics: automatic temperature control, stirring speed of 100 RPM with a 1/4 HP motor, with a sterile air supply with a volumetric flow of 1 VVM. The experimental part of the research had a 3 × 3 arrangement, resulting in 9 trials (see Table 1), which was carried out by manipulating and controlling the variables pH and temperature and analyzing the incidence on the variables biomass yield and COD, and with two repetitions. Temperatures of 28 °C, 30 °C and 32 °C and pH levels of 3, 4 and 5 were shown. The treatment was performed over a period of 28 h, and each sample for analysis was taken every 4 h, to determine the production of biomass and CO2, consumption of O2 and decrease in COD of the blue cabuya liquor vinasse by aerobic digestion using the yeast consortium Saccharomyce cerevisiae, Candida utilis and Kluymeromyces marxianus, Figure 3 shows the bioreactor used in the aerobic fermentation process of the blue cabuya liquor vinasse.
Yeasts are the microorganisms responsible for the fermentation process. Several factors affect their ability to reproduce in the fermentation media. Therefore, it is important to provide these microorganisms with the appropriate conditions for their reproduction, such as temperature and pH. The growth rate of yeast is related to temperature; the optimal temperature range is 26 to 32 °C. The initial pH of the wort is also a factor that influences yeast growth. The optimal pH for growth can vary from 4.0 to 6.0, depending on temperature, nutrients, and other factors.

3. Results

3.1. Biomass Production

After conducting the nine trials according to the experimental design, Table 2 shows the biomass production obtained at different temperature conditions (28 °C, 30 °C, and 32 °C) and pH conditions of (3, 4 and 5), with the consortium to concentration of 1.32 g/L, a DAP nutrient concentration of 1.5 g/L, and an air flow of 1 VVM, using undiluted vinasse samples (100%).
It is observed that in Trial T6 (Table 2), under temperature conditions of 30 °C and a pH of 5, consortium concentration of 1.32 g/L and a DAP nutrient concentration of 1.5 g/L, the highest biomass yield was achieved with 2.556 g/L (93.4%). Trial T3 also showed notable results with 2.134 g/L, The T3 and T9 tests at equal pH also showed remarkable results with 2.134 g/L and 2.116 g/L, but much lower than the T6 test, demonstrating that temperature is an important factor in yeast metabolism and the performance of the fermentation process. Additionally, it was determined that the longest treatment time (28 h) resulted in the highest biomass production. Therefore, the research will continue to determine the optimal time for obtaining the highest biomass yield. Table 2 shows the biomass production obtained at different temperature and pH conditions.
Figure 4 shows the biomass production of the 9 experimental tests of the fermentation process of the blue cabuya liquor vinasse.

Analysis of Biomass Yield Results

Table 3 shows the statistical treatment (IBM SPSS statistics, version 26) of biomass production in the experimental trials conducted during the aerobic fermentation process of the blue cabuya liquor vinasse.
Table 3 shows the inter-subjects effects.
The corrected model value of 0.000 indicates a significant difference between the treatments.
There are significant differences between time and trial.
Ho: Time does not influence biomass production.
Ha: Time influences biomass production.
The value 0.000 compared to alpha 0.05 → reject Ho. It is concluded that time influences biomass production, so Ha is accepted.
Figure 5 of the estimated marginal means shows that trial 6 exhibits the highest biomass production in the aerobic fermentation process of blue cabuya liquor vinasse.

3.2. Reduction in Chemical Oxygen Demand (COD)

Table 4 shows the COD reduction in the nine experimental trials conducted under different experimental conditions: temperature (28 °C, 30 °C, and 32 °C), pH (3.4 and 5), yeast consortium concentration of 1.32 g/L, and DAP nutrient concentration of 1.5 g/L and with airflow of 1 VVM, for undiluted vinasse (100%).
It was observed that the greatest reduction in COD occurred in trial T6, at 30 °C and pH 5, where a value of 179.7 mg/L (33.3%) was achieved. The trials will continue to determine the optimal treatment time, as longer treatment durations tend to result in greater COD reductions.
Figure 6 indicates the COD reduction in the nine experimental trials of the aerobic fermentation process of the blue cabuya liquor vinasse.

Analysis of the Results of COD Reduction in the Aerobic Fermentation Process of Blue Cabuya Vinasse

Table 5 shows the statistical treatment of COD reduction in the experimental trials conducted during the aerobic fermentation process of the blue cabuya liquor vinasse.
Table 5 shows the inter-subjects effects.
The corrected model value of 0.000 indicates a significant difference between the treatments.
There are significant differences between time and trial.
Ho: Time does not influence COD reduction.
Ha: Time influences COD reduction.
The value 0.000 compared to alpha 0.05 → reject Ho. It is concluded that time influences COD reduction, so Ha is accepted.
Figure 7 of estimated marginal means shows that trial 6 exhibits the highest COD reduction in the aerobic fermentation process of the blue cabuya liquor vinasse.

3.3. Biomass and CO2 Production, COD Reduction and O2 Consumption Trial T6

Figure 8 shows the evolution of biomass and CO2 production, COD reduction and O2 consumption, for trial T6, over 28 h of the fermentation process.

4. Discussion

To obtain 1 litter of Agave americana L. (blue Cabuya) liquor at 55%, 6.25 L of vinasse were generated. Similarly, in a study in Mexico, for every liter of tequila at 55%, between 7 and 10 L of vinasse were produced, this difference could be due to the quality of the juice obtained from the agave in terms of its physicochemical characteristics, the method of extraction and efficiency of the distillation process and maturity of the Agave americana L. [35].
Continuing the comparison of physicochemical properties between the vinasse obtained in the research (Perú) and in a study conducted in Mexico, a country where agave is industrialized for tequila production, the value of Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) are highlighted. The COD values in the vinasse from the research ranged from 269.3 to 286.2 mg/L and the BOD values ranged from 38.56 to 43.65 mg/L, In contrast, in Mexico, the COD values were between 40,000 mg/L to 80,000 mg/L and the BOD values ranged from 25,000 to 50,000 mg/L [4]. As observed, the values in Mexico were much higher, which was likely due to the industrial extraction of the juice, where hydrolysis methods and milling were applied leading to juice with high COD and BOD values. In contrast, the juice used in the research was obtained at an artisanal level by collecting the juice that accumulates in the hole drilled in the top of the head, resulting in juice with low organic load values reflected in the COD and BOD.
Regarding biomass production, in this research, when treating Agave americana L. (blue Cabuya) vinasse with the yeast consortium C. utilis, K. marxianus and S. cerevisiae D47 (Lalvin), a yield of 2.56 g/L was obtained (equivalent to 93.4% yield), and the COD reduction in the vinasse reached 33.3%. In comparison, another study reported a biomass production of 18 g/L while achieving a 33.3% reduction in COD [17]. The difference in biomass production is likely due to the efficiency of the yeast consortium used, as Rhodotorula mucilaginosa has a better capacity for biomass production and synthesis of valuable bioactive substances such as carotenoids and lipids. It has also been found that this yeast effectively reduces COD by up to 80.8% while producing significant biomass (15.7 g/L) with a high lipid content (31.5%) and protein content (48.12%) [36]. It is also notable that S. cerevisiae of significantly reduces both COD and BOD with the added benefit of a high content of essential amino acids [37]. Kluyveromyces marxianus has a high growth rate, is thermotolerant, and has a broad spectrum of sugar assimilation, making it a promising candidate for industrial applications. It has been used for contaminant removal and biomass production [17,38].
The pH is a critical factor that affects the fermentation of vinasse by yeasts, as well as cellular growth and metabolite production. Yeasts also differ in their ability to develop at certain pH levels; Saccharomyces cerevisiae can grow at pH levels between 3 and 6; it has been found that at pH 3 less ethanol is produced compared to pH 4.5 which is optimal for ethanol production [32]. Similarly, Candida parapsilosis, like S. cerevisiae, shows variations in biomass production at different pH levels. Studies have shown that it is one of the most promising yeast protein production from vinasse due to its high biomass productivity [39]. It should also be noted that at a low pH, organic acids generation increases, which can reduce ethanol production and yeast growth. The adaptation time to low pH levels should be considered as it could affect fermentation efficiency [40].
The best results for biomass production in this study were obtained at a temperature of 30 °C. Studies indicate that the optimal temperature for yeast biomass production is related to the specific strain of yeast and the conditions of the fermentation process. For Saccharomyces cerevisiae, the highest biomass production occurs at 25 °C [41], but other studies mention that temperatures between 30 °C and 37 °C also yield good results [42].
A notable outcome of this research was the production of microbial biomass from the vinasse, with characteristics suitable for use as an animal feed supplement due to its high content of single-cell proteins. Alternatively, it can be used as a fertilizer in agricultural activities [39]. Furthermore, the reduction in COD from the vinasse contributes to the sustainability of the process. This research provides significant results that indicate the potential utilization of vinasse in an environmentally sustainable way, contributing to the management of waste or byproducts from the Agave americana L. industry.

5. Conclusions

It is concluded that with the process employed, for every liter of 55% blue cabuya liquor, 6.25 L of vinasse were generated, while in Mexican studies, between 7 L and 10 L of vinasse are produced per liter of 55% tequila.
The COD values in the vinasse from the research ranged from 269.3 to 286.2 mg/L, and the BOD values ranged from 38.56 to 43.65 mg/L. In contrast, in Mexico, the COD values ranged from 40,000 mg/L to 80,000 mg/L, and the BOD values ranged from 25,000 to 50,000 mg/L [4]. As observed, the COD and BOD values in Mexico are high, and this difference is attributed to the fact that in Mexico, the juice is obtained by hydrolyzing the head and milling it, resulting in juice with a high organic load and small fibers, which increases the COD and BOD values. On the other hand, the juice used in this research had low COD and BOD values because it was collected from the liquid that accumulates in the hole made in the top of the head.
From the utilization of the vinasse after the distillation process of the Agave americana L. juice, 2.556 g/L (93.4%) of biomass was obtained by using a yeast consortium consisting of Saccharomyces cerevisiae variety Ellipsoideus, D 47–LALVIN—Canada, C. utilis, and K. marxianus. This biomass can be used as an animal feed supplement due to its significant protein content or as an agricultural fertilizer.
Additionally, during the same process, the COD was reduced to a value of 179.7 mg/L (33.3%), a concentration lower than the maximum permissible limit set by Peruvian regulations, which is 200 mg/L. Therefore, it is determined that the yeast consortium used is viable for decontaminating vinasse, reducing its environmental impact when disposed of in ecosystems.

Author Contributions

For the formulation of this scientific article, the authors’ contributions were as follows: conceptualization, R.R.C.; methodology, R.R.C.; validation, F.A.B.; formal analysis, R.R.C. and E.B.-A.; investigation, R.R.C. and J.C.F.; resources, R.R.C.; data curation, R.R.C.; writing—original draft preparation, R.R.C. and E.B.-A.; writing, R.R.C.; supervision, O.T.G.; project administration, R.R.C.; funding acquisition, R.R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We express our gratitude to the National University of San Marcos for providing us with the facilities to use the laboratory equipment of the Faculty of Chemistry.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 11 00281 g001
Figure 1. Agave americana fields.
Figure 1. Agave americana fields.
Fermentation 11 00281 g001
Fermentation 11 00281 g002
Figure 2. Distillation of Agave americana L.
Figure 2. Distillation of Agave americana L.
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Fermentation 11 00281 g003
Figure 3. Bioreactor used in the fermentation process.
Figure 3. Bioreactor used in the fermentation process.
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Fermentation 11 00281 g004
Figure 4. Biomass production in the nine experimental tests of the aerobic fermentation process of the blue cabuya liquor vinasse.
Figure 4. Biomass production in the nine experimental tests of the aerobic fermentation process of the blue cabuya liquor vinasse.
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Fermentation 11 00281 g005
Figure 5. Estimated marginal means of biomass production from the 9 experimental tests of the aerobic fermentation process.
Figure 5. Estimated marginal means of biomass production from the 9 experimental tests of the aerobic fermentation process.
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Fermentation 11 00281 g006
Figure 6. COD reduction in the nine experimental tests of the fermentation process of the blue cabuya liquor vinasse.
Figure 6. COD reduction in the nine experimental tests of the fermentation process of the blue cabuya liquor vinasse.
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Fermentation 11 00281 g007
Figure 7. Estimated marginal means of COD reduction in the nine experimental tests at different temperature and pH conditions.
Figure 7. Estimated marginal means of COD reduction in the nine experimental tests at different temperature and pH conditions.
Fermentation 11 00281 g007
Fermentation 11 00281 g008
Figure 8. Biomass and CO2 production, COD reduction, and O2 consumption in trial T6 of the aerobic fermentation process of the blue cabuya liquor vinasse.
Figure 8. Biomass and CO2 production, COD reduction, and O2 consumption in trial T6 of the aerobic fermentation process of the blue cabuya liquor vinasse.
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Table 1. Experimental setup for vinasse treatment.
Table 1. Experimental setup for vinasse treatment.
VinasseTemperaturepHTrial
% °C
100283.0T1
4.0T2
5.0T3
303.0T4
4.0T5
5.0T6
323.0T7
4.0T8
5.0T9
Table 2. Biomass production of the nine experimental tests at different temperatures and pH conditions.
Table 2. Biomass production of the nine experimental tests at different temperatures and pH conditions.
Time
(h)		Biomass (g/L)
T1T2T3T4T5T6T7T8T9
01.341.321.341.2691.3151.3231.4121.3521.36
41.4651.4221.5121.3941.4111.4671.5481.4311.477
81.5411.5821.7461.4851.4851.6751.5871.5771.756
121.6011.6281.9081.6381.6252.1281.6551.6721.952
161.6751.72520521.6611.6952.481.8221.7712.011
201.6821.7112.1051.6681.6852.4611.8361.7782.132
241.6871.7692.1131.671.7422.5161.8431.7992.187
281.7051.7582.1341.6731.7352.5561.8411.8122.200
Table 3. Statistical treatment of the biomass yield from the nine experimental trials conducted under different temperature and pH conditions.
Table 3. Statistical treatment of the biomass yield from the nine experimental trials conducted under different temperature and pH conditions.
Tests of Inter-Subject Effects
Dependent Variable: Biomass
OrigenType IIIglSquareFSig.
Sum of SquaresRoot Mean
Corrected model5.378 a150.35921.1150.000
Intersection213.6421213.64212,820.6450.000
Time3.35670.47928.7670.000
Trial2.02280.25315.1700.000
Mistake0.933560.017
Total219.95372
Total corrected6.31171
a. R squared = 0.845 (Adjusted R squared = 0.804).
Table 4. COD reduction in the nine experimental trials at different temperatures and pH.
Table 4. COD reduction in the nine experimental trials at different temperatures and pH.
Time
(h)		COD (mg/L)
T1T2T3T4T5T6T7T8T9
0276.9286.2282.36279.7281.3269.3269.7274.7269.7
4251.2274.6272.3274.1275.2255.6264.3264.2257.4
8234.3271.9261.4263.9271.7233.1258.4259.7251.0
12235.1270.2257.4260.8265.6220.4251.7257.8248.1
16232.3268.6245.4258.5261.4215.5249.8254.3241.3
20231.3261.5241.3257.4260.4201.3248.7251.2231.3
24224.3262.3239.4256.2259.2186.1247.2249.7228.0
28222.0261.5237.58255.4257.6179.7245.5248.7227.0
Table 5. Statistical treatment of COD reduction in the 9 experimental trials performed at different temperatures and pH levels.
Table 5. Statistical treatment of COD reduction in the 9 experimental trials performed at different temperatures and pH levels.
Tests of Inter-Subject Effects
Dependent Variable: COD
OrigenType III Sum
of Squares		glSquare Root MediaFSig.
Corrected model 27,280.506 a 15 1818.700 27.039 0.000
Intersection 4,575,413.334 1 4,575,413.334 68,023.247 0.000
Time 11,662.813 7 1666.116 24.770 0.000
Trial 15,617.694 8 1952.212 29.024 0.000
Mistake 3766.700 56 67.262
Total 4,606,460.540 72
Total corrected 31,047.206 71
a. R squared = 0.879 (Adjusted R squared = 0.846).
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MDPI and ACS Style

Calderón, R.R.; Boza, F.A.; Benmites-Alfaro, E.; Gómez, O.T.; Flores, J.C. Consortium of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae Yeasts for Vinasse Fermentation of Agave americana L. Liquor for Biomass Production and Reduction in Chemical Oxygen Demand. Fermentation 2025, 11, 281. https://doi.org/10.3390/fermentation11050281

AMA Style

Calderón RR, Boza FA, Benmites-Alfaro E, Gómez OT, Flores JC. Consortium of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae Yeasts for Vinasse Fermentation of Agave americana L. Liquor for Biomass Production and Reduction in Chemical Oxygen Demand. Fermentation. 2025; 11(5):281. https://doi.org/10.3390/fermentation11050281

Chicago/Turabian Style

Calderón, Roberto Robles, Francisco Alcántara Boza, Elmer Benmites-Alfaro, Oscar Tinoco Gómez, and Jaqueline Chirre Flores. 2025. "Consortium of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae Yeasts for Vinasse Fermentation of Agave americana L. Liquor for Biomass Production and Reduction in Chemical Oxygen Demand" Fermentation 11, no. 5: 281. https://doi.org/10.3390/fermentation11050281

APA Style

Calderón, R. R., Boza, F. A., Benmites-Alfaro, E., Gómez, O. T., & Flores, J. C. (2025). Consortium of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae Yeasts for Vinasse Fermentation of Agave americana L. Liquor for Biomass Production and Reduction in Chemical Oxygen Demand. Fermentation, 11(5), 281. https://doi.org/10.3390/fermentation11050281

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