Impact on the Rheological Properties and Amino Acid Compositions of the Industrial Evaporation of Waste Vinasse in the Production of Nutritional Supplements for Livestock

Abstract

Vinasse a byproduct of ethanol manufacturing, is a challenge for ethanol producers which possesses a high organic content that presents a considerable environmental threat. This complicates its management and treatment utilizing standard technologies like anaerobic digestion. This residue contains a substantial quantity of dead and lysed yeast cells, which can function as a protein source for livestock’s nutritional needs. The application of multi-effect evaporation enhances the characteristics of this residue by increasing protein concentration, reducing volume, and minimizing water content. This study examines the impact of the five-effect evaporation procedure on vinasse waste, focusing on its rheological properties and the concentrations of proteins, amino acids, RNA, and DNA. This study aims to assess the thermal impacts linked to the evaporation process. The findings of the one-way statistical analysis demonstrate that the five evaporation effects are relevant in the utilization of waste as feed for livestock. The substance has a viscosity of 0.933 Pa s, comprising 6.3 g/100 g of crude protein, 4.08 g/100 g of amino acids, 0.1158 g/L of DNA, and 0.1031 g/L of RNA.

1. Introduction

In recent years, there has been a substantial global population expansion, necessitating the mass production of diverse products to satisfy numerous demands. This vast production generates many forms of garbage that adversely affect and disrupt numerous ecosystems. Vinasse, a byproduct of distillation, is one of these leftovers. Owing to its physicochemical characteristics and absence of prior treatment before disposal, it represents a significant contaminant for aquifers and soil [1]. The physicochemical properties of vinasses are contingent upon the raw material utilized for ethanol production, including barley, wheat, corn, or sugarcane [2]. Typically, they predominantly comprise organic matter, which includes glycerol, lactic acid, ethanol, and acetic acid [3], as well as dissolved and suspended solids [4]. Due to its substantial generation (ranging from 9 to 14 L per liter of ethanol produced) and significant contamination potential [2], it is imperative to identify and cultivate alternative applications for vinasse to mitigate the adverse environmental effects of its excessive discharge.

Vinasse, characterized by its substantial organic load, nutrient composition, and mineral content including potassium [5], is employed in fertigation [6]. Furthermore, if this effluent is subjected to anaerobic digestion, it can yield biogas, electricity, and biofuels [7]. The distillation process results in residual biomass from the Saccharomyces cerevisiae yeast utilized in fermentation [8], which can be employed as a supplement in animal feed due to its protein content [4].

The application of vinasse as a fodder supplement commences with its concentration. Various techniques are available for this objective, including microfiltration-nanofiltration, flocculation-sedimentation-filtration, and evaporation [9]. The concentration process utilizing a multiple-effect evaporator system effectively decreases water content while preserving the solid percentage [1]. The evaporation method for concentrating vinasse is the most used, as it allows for the integration of distillation and cogeneration facilities within the sugar and ethanol production process [10]. The quantity of effects employed is contingent upon the volume of steam accessible for the process; a larger volume necessitates fewer effects. [11] The concentration of vinasse yields advantages including water collection and its subsequent reuse in the process [12], a decrease in transportation expenses [13], and the generation of a supplementary product for animal feed. This approach, coupled with the cremation of vinasse, addresses the pollution issue associated with this effluent.

The physicochemical characteristics of vinasse and the impact of temperature on yeast lysis are closely related, as this lysis releases cellular materials (proteins, polysaccharides, nucleic acids, and lipids) [14], which in turn affects the rheological properties of the fluid. Understanding this can enhance the final disposal methods of vinasse in the alcohol industry [15].

Rheology serves as a monitoring tool in industrial operations, particularly in the rheological characterization of algal-derived hydrocolloids utilized in the food industry [16]. It aids in the analysis of algal polysaccharides [17], elucidates the agarose gelation process [18], and provides insights into microstructure [19], deformation, and flow of processed foods across various stages, including raw materials, intermediate products, and finished goods. This knowledge facilitates the minimization of textural defects, optimization of processes, enhancement of quality control for raw materials, and development of new products. Industrial processes utilize inputs rich in organic matter, including yeasts, carbohydrates, lipids, and proteins. The latter induces structural changes that characterize the rheological behavior of a compound [20].

This study aims to assess the physicochemical alterations of vinasse throughout the evaporation process stages and to model its rheological behavior in relation to modifications in chemical structure, particularly concerning the variations in protein and amino acid content during the quintuple effect vacuum evaporation process.

2. Materials and Methods

2.1. Collection of Samples and Analysis of Physicochemical Properties

A vinasse sample was collected from a must column in an ethyl alcohol production process, following the methodology outlined in Mexican regulation [21], from an industry situated in the central region of Veracruz. The sample was maintained at room temperature for two days. Upon collection, the preliminary characteristics presented in

Table 1. Parameters, regulation, and equipment utilized in the preliminary physicochemical assessment of the sample.

Parameter	Regulation	Equipment
pH	NMX-AA-008-SCFI-2016 [22]	Hanna® potentiometer HI2002-01 Model, Mexico City, Mexico
Conductivity	NMX-AA-093-SCFI-2018 [23]	Conductronic® potentiometer PC45 Model, Mexico City, Mexico
Total COD	NMX-AA-030-SCFI-2012 [24]	ThermoScientific® spectrophotometer UV-VIS Genesys 10S Model, Mexico City, Mexico
Dissolved oxygen and saturation	NMX-AA-012-SCFI-2001 [25]	Hatch® multiparameter HQ40D model, Mexico City, Mexico
Density	NOM-CH-50-1984 [26]	Ohaus® analytical balance Scout model, Mexico City, Mexico
Turbidity	NMX-AA-038-SCFI-2001 [27]	Hatch® turbidimeter 2100Q model, Mexico City, Mexico
Reducing sugars	NMX-F-312-NORMEX-2016 [28]	Thermofisher Multiskan EX® microplate reader, Mexico City, Mexico
Viscosity	NMX-AA-135-SCFI-2007 [29]	Brookfield® viscometer DV2T model, Mexico City, Mexico
Temperature	NMX-AA-007-SCFI-2013 [30]	Brannan® thermometer, Mexico City, Mexico
TS and TVS	NMX-AA-034-SCFI-2015 [31]	Felisa® stove 4840 model, Mexico City, Mexico

COD: Chemical Oxygen Demand, TS: Total Solids, TVS: Total Volatile Solids.

were assessed.

2.2. Rheological Analysis of the Quintuple-Effect Evaporator System

A multiple effect evaporation system with five effects was employed to concentrate the vinasse, accompanied by an in situ rheological analysis. A sample from each effect was collected and analyzed using the Brookfield^® DV2T viscometer, which features a ULA LV-1 needle and an adapter with a temperature control jacket, linked to a recirculating water thermostatic bath. Sixteen milliliters of each sample were utilized for this analysis. The experiments were performed at the exit temperature corresponding to each effect (as indicated in

Table 2. Operating temperatures of each evaporator.

Evaporator 1	Evaporator 2	Evaporator 3	Evaporator 4	Evaporator 5
80 °C	80 °C	78 °C	34 °C	67.7 °C

) and additionally at room temperature.

The hydraulic configuration of the five-effect evaporation system is depicted in Figure 1, where Line 1 represents the discharge from evaporator 1 and is fed into evaporator 2. Line 2 represents the discharge from evaporator 2 and is fed into evaporator 4. From evaporator 4, discharge 3 exits and fed into evaporator 3, line 4 enters evaporator 5. Finally, outlet line is the channel via which the evaporated product is discharged. All the vapor discharges are consolidated into a single line of steam outlet.

2.3. Analysis of Proteins, Amino Acids, and Nucleic Acids

Protein determination for each effect was conducted using the Bradford method, with a dilution of 1:1000 and PBS 1X as the diluent. The sample was allowed to mature for 60 min and then quantified using the Thermo Fisher Multiskan EX^® microplate reader. The identical dilution was employed for assessing DNA and RNA content, measured using the Eppendorf^® spectrophotometer, with the selection of either the dsDNA or RNA option based on the molecule being quantified. Amino acid determination was conducted utilizing gas chromatography-mass spectrometry, specifically the GCMS-QP2010 Plus model (high-power oven), equipped with a ZB-AAA 10 mm × 0.25 mm (I.D.) column. Gas chromatography operating conditions include an injection temperature of 280 °C, a column temperature starting at 110 °C with a ramp of −30 °C/min to a final temperature of −30 °C, using helium as the carrier gas. Flow control is maintained by pressure at 15 kPa, and the injection method employed is split with a ratio of 1:15. Operating conditions for the mass spectrometer include an interface temperature of 280 °C, an ion source temperature of 200 °C, a scan range of m/z 45–450, and an analysis interval of 0.15 s.

2.4. Statistical Techniques

The experimental results from the evaporation process were recorded in Microsoft Excel^® 2021. Afterwards, a one-way analysis of variance (ANOVA) was carried out using Minitab^® 19 Statistical Software to analyze the influence of each evaporation stage on the response variables (pH, total solids, cell count, proteins, DNA, and RNA). Group disparities were evaluated utilizing Tukey’s mean comparison at a 95% confidence interval. Furthermore, their correlation coefficient (R²) for the rheological parameters was assessed.

3. Results and Discussion

3.1. Physicochemical Characterization of Vinasse

The physicochemical characterization results of the vinasse are presented in

Table 3. Results of the physicochemical characterization of vinasse.

Analysis	Value	Units
pH	4.73	NA
Conductivity	3.8	µS
COD	181.6	g O2
Dissolved O2	0.0619	g/L
Density	1.0385	g/mL
Turbidity	469	NTU
Reductor sugars	104.605	g/L
Viscosity	2.01	cP
Total Solids	10.35	% w/w

. The acidity arises from the initial acidification of the must with H₂SO₄ to enhance fermentation [32]. Ibarra-Camacho et al. [32] reported conductivity results between 15 and 17 µS, suggesting that the sample analyzed has a low concentration of soluble salts [32]. The reference values for COD, as reported by Zúñiga-Cerón et al. [33], range from 10.2 to 43 g/L, which are lower than the values obtained for this sample. The production of ethanol from molasses results in vinasse with elevated organic matter content [32], leading to a high COD. The concentration of dissolved O₂ is influenced by the temperature of the vinasse leaving the process and the reduction in photosynthetic activity caused by organic compounds, leading to a decrease in oxygen levels [34].

The density approximates the value reported by Aristizábal [35], which is 1.04 g/mL. The measurement is influenced by the temperature at which it is conducted. The turbidity value is below the 999 NTU reported by González et al. [36]. The variation arises from melanoidins, caramels, and high molecular weight compounds produced by the Maillard reaction [37], which impart the vinasse its distinctive color. The levels of reducing sugars are lower than those reported by Parsaee et al. [34], which measured 911.7 g/L. This discrepancy may be attributed to factors such as the type, maturity, and variety of sugarcane, in addition to the fermentation and distillation processes, as well as the reagents used in the must [34]. The viscosity of vinasse is influenced by its water content and the temperature during analysis [38]. The total solids value of the sample analyzed is lower than the 22.98% reported by Dávila-Rincón et al. [39]. The observed difference is ascribed to the impact of factors including the planting system, soil type, sugarcane fertilization, and the fermentation and distillation conditions involved in the process on the composition of the vinasse [40].

3.2. Rheological Characterization of the Evaporation Stages

Figure 2 illustrates the rheological behavior associated with the inlet vinasse and each evaporator. The samples were identified as Non-Newtonian fluids, indicating a non-linear relationship between shear stress and shear rate, with viscosity variations dependent on the applied shear range [41].

A comparison of three rheological models (Herschel–Bulkley, Bingham, and Ostwald–Waele) was conducted to identify the model that best represents the fluid’s behavior. The rheological model exhibiting the highest correlation coefficient, nearest to one, was chosen.

Table 4. Comparison of rheological models.

Evaporator	Temperature (°C)	Bingham			Ostwald–de Waele			Herschel–Bulkley
		${\mathit{\tau }}_0$	${\mathit{\mu }}_0$	${\mathit{R}}^2$	$\mathit{k}$	$\mathit{n}$	${\mathit{R}}^2$	${\mathit{\tau }}_0$	$\mathit{k}$	$\mathit{n}$	${\mathit{R}}^2$
1	80	0.0223	0.0019	0.9713	0.0106	0.5798	0.8134	0.1258	0.0001	1.5138	0.999
2	80	0.0265	0.002	0.9634	0.0119	0.5701	0.7856	0.0161	0.0001	1.6055	0.99
3	78	0.0091	0.0028	0.9974	0.014	0.6576	0.9334	0.0091	0.0028	1.0001	0.9974
4	34	0.006	0.0038	0.9993	0.0158	0.6682	0.9407	0.0089	0.0033	1.0269	0.9994
5	67.7	1.088	0.0267	0.9081	0.0933	0.6452	0.9802	0.0372	0.1375	0.5196	0.9712

presents a comparative analysis of these models. The stages of the evaporation process influence the rheological properties of the evaporated vinasse.

Evaporators 1, 2, and 4 were analyzed using the Herschel–Bulkley model, demonstrating correlation coefficients exceeding 0.999. In evaporator 3, the Bingham or Herschel model is applicable, given their comparable fit of 0.9974, whereas evaporator 5 was fitted to the Ostwald–de Waele model. Variations in rheological models are expected, as the evaporation process advances. The viscosity of the vinasse evaporate increases with each effect it undergoes, noting that the sequence of effects is non-linear and follows a specific arrangement (1, 2, 4, 3, and 5). Observations indicate that, as the process evolves, both the apparent viscosity of the sample and the yield stress (${\tau }_0$) decrease. The Herschel model effectively captured the initial effects, as it necessitates a yield stress for fluid movement, supported by the correlation index result [42]. Conversely, the Ostwald model was suitable for the final effect, as it demonstrates a decrease in viscosity with increasing applied stress [42]. The final product’s flow index, being less than 1, indicates that the fluid demonstrates pseudoplastic behavior [43]. The pronounced non-linearity indicates the fluid’s non-Newtonian characteristics, which correlate with the chemical composition discussed in the subsequent section.

3.3. Physicochemical and Chemical Structure Changes in the Vinasse Evaporation Process

Essential amino acids are carbon chains that cannot be synthesized by the organism [44] yet are crucial for animal nutrition. These are termed Nutritionally Essential Amino Acids (NEAA) and include cysteine, histidine, isoleucine, leucine, lysine, phenylalanine, methionine, threonine, tryptophan, valine, and tyrosine [45]. The concentrations of essential and non-essential amino acids found in the vinasse, across each effect, as well as in the evaporated vinasse extract, are summarized in

Table 5. The impact of the evaporation system on the amino acid profile.

AMINOGRAM ANALYSIS
AMINO ACID	VINASSE	E1	E2	E3	E4	E5	UNITS	METHOD
Ala	0.3	0.02	0.04	0.10	0.1	0.22	g/100 g	GC-MS
Gly	0.02	0.03	0.03	0.05	0.05	0.1
Val	0.01	0.01	0.02	0.05	0.04	0.14
Leu	0.01	0.01	0.02	0.04	0.03	0.14
Ile	0.009	0.009	0.02	0.03	0.03	0.09
Thr	0.01	0.01	0.02	0.03	0.03	0.07
Ser	0.02	0.02	0.02	0.03	0.03	0.09
Pro	0.01	0.01	0.02	0.03	0.02	0.07
Asp	0.38	0.42	0.44	0.86	0.89	1.29
Met	0.009	0.009	0.009	0.009	0.009	0.16
Glu	0.24	0.22	0.4	0.77	0.8	1.24
Phe	0.009	0.009	0.009	0.009	0.009	0.08
Lys	0.01	0.01	0.02	0.04	0.03	0.12
His	0.02	0.02	0.02	0.02	0.02	0.08
Tyr	0.009	0.009	0.009	0.009	0.009	0.05
Cys	0.02	0.02	0.02	0.02	0.02	0.14
Amino acids	1.086	0.836	1.117	2.097	2.117	4.08
Crude Protein	1.3	1.7	2.0	3.8	3.4	6.3		Volumetric essay

. The protein and amino acid content increases as the process advances, attributed to the removal of water from the vinasse.

Furthermore, the evaporate exhibits a suitable nutritional profile, comprising 16 amino acids, including 8 non-essential amino acids (NEAA) [46]. Figure 3 illustrates the relationship between vinasse evaporation and the concentration of essential amino acids, indicating an increase in these biomolecules as evaporation occurs.

The obtained extract lacks two essential amino acids: tryptophan and arginine. Gao et al. [47] indicates that the first amino acid contributes to colitis relief by regulating gastrointestinal secretion and permeability. Additionally, arginine plays a role in reducing obesity, enhancing cardiovascular function, promoting lean tissue development, and increasing insulin sensitivity [48]. These biomolecules are thus significant in animal nutrition.

The concentration of proteins and amino acids, measured in g/100 g against total solids by effect, was illustrated in Figure 4 to elucidate their behavior throughout the process. The evaporated extract exhibits a higher concentration of proteins and amino acids than the vinasse. The production of vinasse evaporates, utilized as an animal feed supplement, necessitates the inclusion of proteins, which enhance nutrient digestibility by promoting optimal ruminal fermentation [49].

Determining a reference amount for the nutritional requirements of amino acids and proteins in animal diets is influenced by various factors, including species-specific physiological demands, sex, and the animal’s life stage, among others [50]. A comparison of the NEAA requirements for broiler consumption, as established by various authors, is presented alongside the contents of evaporated vinasse in Table 6. Generally, the evaporated vinasse-based feed supplement contains the non-essential amino acids (NEAA) identified by various authors as necessary for adequate animal nutrition. Although the values of this supplement are lower than those established, it is important to note that the primary source of amino acids should originate from the feed provided to the animals [51].

The three-dimensional structure of proteins exhibits low thermal stability, characterized by a melting point ranging from 40 to 60 °C [52]. Subjecting proteins to temperatures exceeding those previously mentioned results in modifications to their native structure [53]. However, the energy available is insufficient to disrupt the peptide bonds [54], thus preserving the amino acid sequence [53]. Moderate temperatures induce reversible denaturation [54], whereas temperatures exceeding 80 °C result in irreversible denaturation and the formation of protein aggregates [55]. The initial effects experienced by the sample occur at 80 °C, leading to the conclusion that proteins undergo structural unfolding, transitioning from tertiary to secondary structure, ultimately resulting in a linear chain of amino acids [54]. Consequently, the quantity of these molecules increases as the process advances.

Table 6. Comparison of amino acid content in evaporated vinasse vs. minimum requirements established by various authors for chickens, expressed as a percentage.

Amino Acid	Evaporated Vinasse	Baker et al. (1994) [53]	Wu et al. (2014) [54]
Ala	0.22		0.9
Gly	0.1	0.6	1
Val	0.14	0.69	0.78
Leu	0.14	0.98	1.52
Ile	0.09	0.6	0.7
Thr	0.07	0.6	0.61
Ser	0.09		0.8
Pro	0.07	0.4	1.31
Asp	1.29		1.03
Met	0.16	0.325	0.38
Glu	1.24	12	1.45
Phe	0.08	0.5	0.53
Lys	0.12	0.9	0.82
His	0.08	0.32	0.41
Tyr	0.05	0.45	0.41
Cys	0.14	0.325	0.29
Total amino acids	4.08	19.8

Table 6. Comparison of amino acid content in evaporated vinasse vs. minimum requirements established by various authors for chickens, expressed as a percentage.

Amino Acid	Evaporated Vinasse	Baker et al. (1994) [53]	Wu et al. (2014) [54]
Ala	0.22		0.9
Gly	0.1	0.6	1
Val	0.14	0.69	0.78
Leu	0.14	0.98	1.52
Ile	0.09	0.6	0.7
Thr	0.07	0.6	0.61
Ser	0.09		0.8
Pro	0.07	0.4	1.31
Asp	1.29		1.03
Met	0.16	0.325	0.38
Glu	1.24	12	1.45
Phe	0.08	0.5	0.53
Lys	0.12	0.9	0.82
His	0.08	0.32	0.41
Tyr	0.05	0.45	0.41
Cys	0.14	0.325	0.29
Total amino acids	4.08	19.8

Temperature affects non-covalent bonds, and during protein unfolding, hydrophobic groups (such as thiol groups) interact with the medium, thereby preventing the dissolution of molecules in water, which leads to the gradual appearance of aggregates during the process [56] and increases the viscosity of the fluid [57]. This is why it is important to evaluate the rheological behavior of the sample, as this analysis will reflect the simplicity or complexity of the chemical composition of the fluid in question [58].

The content of nucleic acids (DNA and RNA) in the vinasse and each of the effects are detailed in

Table 7. The nucleic acid concentration at the discharge of each evaporative stage.

Evaporator	DNA (g/L)	RNA (g/L)
Vinasse	0.0751	0.0238
1	0.0373	0.0306
2	0.0466	0.0368
3	0.0866	0.0690
4	0.0923	0.0732
5	0.1158	0.1031

. Initially, the vinasse presents a significant amount of DNA, but when subjected to the first thermal treatment at 80 °C, this content decreases drastically, as it is close to the denaturation temperature of DNA, which is 85 °C [59].

Elevated temperatures lead to the denaturation and fragmentation of DNA and RNA chains [60]. As the process unfolds, the concentration of this molecule escalates until it attains its peak level, a phenomenon attributed to the evaporation of water [61]. This mirrors the behavior of the RNA molecule, which similarly begins at a low concentration and rises as the process continues. The alterations are visually represented in Figure 5.

While no studies have been conducted in humans, nucleic acids are metabolized in the body into uric acid, and excessive intake may result in clinical disorders such as gout, pre-eclampsia, chronic kidney disease, and hypertension [62]. Therefore, the consumption of molasses-based dietary supplements in humans is not advised. The intake of nucleic acids contributes to the development of the immune system and intestinal mucosa in animals; subsequently alleviating stress associated with enteric diseases prevalent among them. Their brief life cycle facilitates the absorption of these substances without inducing metabolic issues [63]. The rheological properties resulting from elevated levels of proteins, amino acids, and nucleic acids yield a desirable consistency for the final product in the market.

4. Conclusions

The physicochemical characteristics assessed in the vinasse, including a total COD of 181.6 g COD/L, a low dissolved oxygen concentration of 0.0619 g/L, and a high turbidity of 469 NTU, indicate its significant pollution potential; thus, the evaporation of vinasse emerges as a feasible reuse option. The vinasse concentration process utilized in this study exhibited an increase in solid content, significantly impacting the viscosity of the final product.

Therefore, it is essential to monitor this variable during plant operation to prevent blockages and malfunctions in the piping system. Evaporation resulted in the denaturation of proteins in the vinasse, leading to the release of essential amino acids, including phenylalanine, histidine, valine, leucine, methionine, isoleucine, and threonine, along with alanine, aspartic acid, glycine, serine, proline, glutamic acid, tyrosine, and cysteine. However, the extract was noted to lack tryptophan and arginine.

The concentration of amino acids, proteins, and nucleic acids renders this product a viable option for use as an animal feed supplement, while also mitigating the risk of aquifer contamination due to improper disposal practices.