Treatment of Sugarcane Vinasse Using Heterogeneous Photocatalysis with Zinc Oxide Nanoparticles

Abstract

Vinasse is the main by-product of the ethanol industry; for each liter of ethanol, 13 to 18 L of vinasse is generated. Vinasse is composed of 93% water and 7% organic and inorganic solids and has an acidic pH and a high concentration of macro- and micronutrients used by plants, which is the reason for its widespread application in soil fertigation. However, over time, excessive direct discharge of vinasse into the soil causes damage, such as salinization and groundwater contamination. In this study, we used heterogeneous photocatalysis with zinc oxide nanoparticles (ZnO-NPs) to reduce chemical oxygen demand (COD) and biochemical oxygen demand (BOD) and as an antimicrobial treatment. ZnO-NPs were synthesized by the precipitation of zinc sulfate heptahydrate and sodium hydroxide, resulting in nanoparticles with a size of 21.6 ± 0.3 nm and an energy bandgap of 2.6 eV. Microscopic examinations revealed that Saccharomyces cerevisiae microorganisms are present in vinasse and that the minimum inhibitory concentration for the ZnO-NPs is 1.56 g/L. Photocatalysis with 40 mg/L of ZnO-NPs for 4 h of exposure to sunlight resulted in COD and BOD reduction efficacies of 17.1% and 71.7%, respectively. This study demonstrates the viability of using ZnO-NPs in vinasse treatment, contributing to sustainable applications and reducing the environmental impacts of fertigation.

1. Introduction

Vinasse is an effluent produced in agro-industrial activities and is generated in the sugarcane ethanol production process. Sugarcane vinasse is composed of micronutrients used by plants, namely, potassium (2.53–2100 mg·L⁻¹), calcium (354.8 mg·L⁻¹), and magnesium (169.9 mg·L⁻¹) [1], as well as dissolved organic carbon (4000–14,000 mg·L⁻¹) [2,3]. Vinasse has a low pH (3–4.5) [4], a high chemical oxygen demand (COD) (>150,000 mg O₂/L) [5], and a 30–70% biochemical oxygen demand (BOD) [6]. In ethanol production, for each liter of ethanol produced, 13–18 L of vinasse is generated, amounting to a volume of 2.38 × 10⁷/m³ per year [7,8,9]. Vinasse is used in fertigation; however, indiscriminate disposal into the soil and continued exposure to vinasse have environmental impacts, such as saturation with potassium, sulfates, and metal ions; salinization; nutrient leaching; and the permanent acidification of soils and water resources [8,10,11,12]. Studies show that residues and by-products from sugar and ethanol production potentially impact water resources, soils, and the atmosphere [4,13,14]. The degree of impact depends on the concentrations released into the environment, the duration of vinasse management, and the resilience of natural systems. Therefore, technologies for its treatment are needed to prevent side-effects of vinasse use. Conventional treatment methods have already been applied in vinasse management, such as filtration, adsorption, biological oxidation, coagulation–flocculation, and pH adjustment [15,16]. Effluent treatment is complex, in general, and, except for anaerobic digestion, which reaches COD reduction rates higher than 80% [17], greater efficacy can be achieved by combining processes [6], such as advanced oxidative processes (AOPs) mediated by semiconductors, ozonation, Fenton and photo-Fenton reactions, electrochemical remediation, and other processes based on ultraviolet (UV) light irradiation [16,18,19,20]. However, combined processes are more complex and have higher costs.

In photocatalytic reactions, the electron in the semiconductor absorbs a photon with greater energy than the bandgap. The electron is excited from the valence band to the conduction band, producing an electron pair, creating a hole (h+) in the valence band, where oxidation and/or reduction of the adsorption substrates begins. In aqueous solutions, the hydroxyl ion (OH·) reacts with the catalyst surface and generates an oxidizing agent for effluent treatment [21].

The main semiconductors used in heterogeneous photocatalysis are catalysts in the form of metal oxides, such as zinc oxide (ZnO), titanium dioxide (TiO₂), tin dioxide (SnO₂), sulfates, selenium, and telluride minerals, such as cadmium sulfide (CdS), zinc sulfide (ZnS), and cadmium selenide (CdSe) [22,23,24]. The photocatalytic properties of zinc oxide nanoparticles (ZnO-NPs) have been extensively studied due to their chemical stability, high thermal conductivity, energy bandgap compatible with UV-Vis, and low production costs compared to other metallic oxides [25,26]. In recent years, heterogeneous photocatalysis using ZnO-NPs as semiconductors have been used in the decontamination of food waste, as well as pharmaceutical, textile industry, and domestic effluents. Kee et al. [16] studied the efficacy of COD reduction and discoloration of anaerobically digested vinasse with photocatalytic degradation under adjusted conditions. Samples were processed using a 0.45 µm membrane filter and pH levels modified by sodium hydroxide (NaOH) and sulfuric acid (H₂SO₄) to achieve photocatalytic degradation in an alkaline condition (pH 10). When the catalyst dosage of ZnO-NPs increased from 0.5 to 2.0 g/L, the efficacy of decolorization and COD reduction also increased in the solution by 99.29% and 83.40%, respectively.

This work provides an approach for sugarcane vinasse treatment without the need for complex additional infrastructures and energy expenditures. We used heterogeneous photocatalysis mediated by zinc oxide nanoparticles with sunlight irradiation. The work was divided into three parts: (i) the synthesis and characterization of zinc oxide nanoparticles (ZnO-NPs); (ii) sugarcane vinasse treatment by heterogeneous photocatalysis, comparing different energy sources; and (iii) microbiological studies of vinasse and antimicrobial effects of ZnO-NPs.

2. Materials and Methods

2.1. ZnO-NP Synthesis and Characterization

The synthesis of zinc oxide nanoparticles (ZnO-NPs) was performed by precipitation. Solutions of 0.1 M zinc sulfate heptahydrate (ZnSO₄.7H₂O) and 0.4 M sodium hydroxide (NaOH) were used in the proportions of 1:4 M. The ZnSO₄.7H₂O solution was added (at a rate of 1 mL/min) to the NaOH solution, which was kept under constant stirring (800 rpm) for 30 min at 21 °C. The white product obtained was washed with deionized water, filtered with 3 µm filter paper under negative pressure, and heated to 60 °C for 1 h until a constant mass was reached. The chemical reaction is described by Equation (1).

$${\mathrm{ZnSO}}_4·7{\mathrm{H}}_2\mathrm{O} + 2\mathrm{NaOH} \stackrel{\Delta \mathrm{t}}{\to } \mathrm{ZnO} + {\mathrm{Na}}_2{\mathrm{SO}}_4 + 8{\mathrm{H}}_2\mathrm{O}$$

(1)

ZnO-NPs were characterized using X-ray diffraction (XRD) with a Shimadzu–XRD 6000, λ = 1.54984 Å. The ZnO-NPs samples were spread on a carbon tape for the morphological analyses performed via field emission scanning electron microscopy (FEG-SEM), using JEOL-JSM-7500F, PC-SEM software v.2.1.0.3., and transmission electron microscopy (TEM) with a JEOL-JEM-100 CXII microscope. The samples were dropped on the surface of a copper TEM grid and dried before the analysis. Size distributions from TEM images were acquired with ImageJ software [27]. The UV-Vis absorption spectra for colloidal dispersions were recorded with an Ultrospec 2100 Pro spectrophotometer (Amersham Pharmacia).

2.2. Analytical Methods and Performance Assessment

Sugarcane vinasse from Saccharum spp. was collected at a sugarcane mill in the municipality of Osvaldo Cruz, São Paulo State, Brazil. Chemical oxygen demand (COD) was determined by the closed reflux colorimetric 5220D method [28] and biochemical oxygen demand (BOD) was determined by the 5-day 5210B and 5210D respirometric methods [29] before and after vinasse treatment with photocatalysis. Moreover, potential of hydrogen (pH) measurement was performed using a Quimis pHmeter-Q400MT at 21 °C.

The percentage of reduction in COD values after treatment was calculated according to Equation (2), where CODi stands for the average initial COD and CODf represents the average of final values. All experiments were performed in triplicate.

$$\% \mathrm{COD} \mathrm{reduction} \frac{\left(CODi-CODf\right)}{CODi}\times 100$$

(2)

The percentage of BOD reduction was calculated using Equation (3), where BODi is the initial biochemical oxygen demand and BODf is the final biochemical oxygen demand measured at the end of 5 days [11,29].

$$\% \mathrm{BOD} \mathrm{reduction} \frac{\left(BODi-BODf\right)}{BODi}\times 100$$

(3)

One-way ANOVA (analysis of variance) and the Tukey test were used to determine the statistical significance of differences between COD values after 4 h of treatment. Differences were considered statistically significant when p < 0.05.

2.3. Photocatalysis of Vinasse Using ZnO-NPs

The heterogeneous photocatalysis experiments on vinasse were performed with two concentrations of ZnO-NPs (40 mg/L and 1560 mg/L) and using three radiation sources: UV-C (Osram Puritec germicidal, 18 W), white halogen light (Osram Haloline, 80 W), and sunlight. Solar exposure data at the moment of the experiment were collected in situ from the Unoeste Meteorological Station [30]. The UV-C lamp power was similar to that in other reports in the literature [31].

Table 1. Description of vinasse treatments through photocatalysis.

Sample	ZnO-NPs (mg/L)	Radiation Source
Control	0	n/a
T1	0	Sunlight
T2	40	Sunlight
T3	1560	Sunlight
T4	0	UV-C
T5	40	UV-C
T6	1560	UV-C
T7	0	White halogen light
T8	40	White halogen light
T9	1560	White halogen light

Note: n/a: not applied.

presents a description of each treatment. All processes were monitored every 1 h until the completion of a 4 h period.

The photocatalysis experiment under UV-C and white halogen light was performed with a volume of 250 mL of vinasse and ZnO-NPs, with an exposure surface area of 113.04 cm², according to Table 1, using a photocatalytic reactor (Figure 1a). The solution was kept under constant agitation (300 rpm) and a constant temperature during the experiment.

The photocatalysis under sunlight exposition was made using 250 mL vinasse and ZnO-NPs, with the same exposure surface area (113.04 cm²) (Table 1). The samples were exposed directly to sunlight for 4 h, between 11h00 and 15h00 [30], at the following coordinates: latitude: −22.116009, S22°6′57.63168″; longitude: −51.450153, W51°27′0.55116″ (Figure 1b). In this experiment, the samples (vinasse and ZnO-NPs) remained at rest.

For COD monitoring, aliquots of 1 mL were extracted from the total solution every hour until the 4 h period was completed. COD values were measured without sedimented material. For BOD measurement, the entire volume (vinasse and nanoparticles) was homogenized and used in the experiment.

2.4. Susceptibility of Microorganisms to ZnO-NPs

The Gram method for staining was used to investigate which microorganisms were mostly present in the vinasse [32]. The antimicrobial activity of ZnO-NPs against microorganisms in vinasse was quantitatively measured by the minimum inhibitory concentration (MIC) assay using Eucast/BrCast 7.3.2 [33].

For the microdilution of ZnO-NPs, 12 tubes were used containing a negative control (C−) with 50 mg of ZnO-NPs and 1 mL of dimethyl sulfoxide (DMSO) and a positive control (C+) with 2 mL of in natura vinasse. The remaining 10 tubes contained serial microdilutions of ZnO-NPs (Figure 2a). For the serial microdilutions, firstly, a volume of 1 mL of sterile distilled water was placed in each tube. Subsequently, 1 mL of ZnO-NP solution from C− was transferred to the first tube, resulting in a solution with a half concentration of ZnO-NPs. The same procedure was repeated for the other tubes, allowing a serial dilution in the tubes, ranging from 25 mg/mL to 0.049 mg/mL. After this process, 1 mL of vinasse was added to each tube, totaling 2 mL of solution (Figure 2a). Figure 2b shows the aliquots of 100 µL transferred to microplates with 96 wells. The plates with the microdilutions were incubated in an oven for 24 ± 2 h at 35 ± 2 °C [33].

For the microbial growth analysis, 20 µL of 0.01% resazurin solution and 20 µL of sterile distilled water were added to each well. The plates were then incubated again in an oven for 1 h at 35 °C. With this technique, the blue color represents the absence of viable cells, while the pink/red color represents their presence. To aid in the determination, since the vinasse is colored, a plugin of ImageJ software was used [27] which indicates the RGB coordinates of each color. The reddish coloration was found with this procedure, and then the breakpoint was determined as the previous concentration and confirmed with CFU counting. The tests were performed in sextuplicate.

The viability of microorganisms in vinasse after the treatments was tested by the drop plate method and CFU counting [33]. The microdilution samples were removed from the oven after incubation (24 ± 2 h at 35 ± 2 °C) and shaken in a Vortex mixer, variable speed, at 2000 rpm for 15 s for seeding in agar Sabouraud plates. Each plate was divided into five sections, and three aliquots of 30 µL of the corresponding microtube were placed in each section. After preservation for 48 h in an oven at 35 °C, the CFUs were quantified with ImageJ software [27], and the non-parametric Kruskal–Wallis and Student–Newman–Keuls tests were applied. The inhibition efficacy in % (η) is defined in Equation (4) [34]:

$$\mathsf{\eta }=\frac{N1-N2}{N1}\times 100$$

(4)

where η corresponds to the inhibition efficacy (%), N1 is the CFU number of C+, and N2 is the number of colonies formed after ZnO-NP treatment.

3. Results and Discussion

3.1. Synthesis and Characterization of ZnO-NPs

Figure 3a,b show FEG-SEM images of ZnO-NPs synthesized by precipitation. Figure 3c shows a TEM image of the nanoparticles, while Figure 3d presents a histogram of their size distribution.

Figure 4 shows a diffractogram (XRD) of the ZnO-NPs, with the XRD pattern structure with a sharp and well-defined diffraction peak according with the reference catalog (JCPDS n. 36-1451), suggesting the formation of nanoparticles with highly crystalline structures. The average crystalline diameter of the ZnO particles was obtained by the Scherrer equation, Equation (5), for the diffraction peak at (101).

$$d_{XRD=\frac{0.9\lambda }{\beta Cos\theta }}$$

(5)

where λ is the wavelength (nm) of the X-ray, θ is the angle of the Bragg diffraction at the (101) plane, and β is the full width at half-maximum (FWHM) of the diffraction peak at the (101) plane [35].

The size of the ZnO-NPs was 21.5 ± 0.3 nm, which agrees with the value obtained by the TEM-derived results for histogram size (Figure 3d).

The synthesis of ZnO-NPs by precipitation is a method that requires a source of zinc and a precipitating agent. Different morphologies and sizes can be obtained by changing precursors, reaction temperatures, and calcination processes [34]. Sharma et al. [36] obtained similar results. The authors used the same precursors, zinc sulfate heptahydrate and sodium hydroxide, in aqueous solution, with a stirring time of 15 min and microwave treatment for 1 min, producing spherical ZnO-NPs with an average size of 2 to 28 nm. Similarly, Ni et al. [37] produced ZnO-NPs of nanorod morphology with peaks also indexed as the hexagonal wurtzite structure of ZnO (JCPDS n. 36-1451) [38].

These characterizations are important because the antimicrobial properties of nanoparticles can be altered according to their morphologies and average sizes. This can be attributed to the surface areas of particles, which affect antimicrobial efficacy [34]. The effect of particle size on antimicrobial activity was studied by Padmavathy and Vijayaraghavan [39], who studied Escherichia coli in suspensions of ZnO-NPs with sizes ranging from 12 to 45 nm. Via disk-diffusion plating on agar, starting from the radius of the zone of inhibition, the authors observed that a ZnO-NP suspension with 12 nm nanoparticles was more effective than suspensions with larger particles, suggesting that the size of the nanoparticles damaged cell membranes [34].

The bandgap is the energy distance between the valence band and the lowest empty conduction band [40]. Therefore, the bandgap determines the minimum energy required to excite electrons from one band to another, resulting in photoelectrons and holes [41].

Figure 5a shows the UV-visible absorption spectrum of ZnO-NPs dispersed in water, while Figure 5b shows a Tauc plot for bandgap energy.

The broad absorption band centered at 375 nm is the characteristic peak for the hexagonal wurtzite ZnO structure [42]. The optical bandgap was calculated from the absorption spectrum based on the direct Tauc equation, Equation (6) [43]:

(αhυ)² = K(hυ − Eg)

(6)

(2.303 × A × 1240/λ)² = k (1240/λ − Eg)

(7)

where A is the absorbance, λ is the wavelength, hυ is the photon energy, Eg is the energy bandgap, k is a constant, and α is the absorption constant.

The results for the Tauc plot for the bandgap energy were extrapolated in the straight part of the graph by linear fitting to the energy (eV) axis, and the sample bandgap obtained was 2.6 eV.

The theoretical bandgap value for non-doped ZnO-NPs is around 3.37 eV; however, this value can change depending on the type of morphology analyzed and show values around 3.16 eV, 3.18 eV, and 2.72 eV [44]. Rusdi et al. [45] also found variation in energy bandgap values for different morphologies, for example, 3.35 eV for nanotubes, 3.29 eV for nanoroads, and 3.25 eV for spherical morphologies. This variation was associated with spacings in crystal structures, and smaller spacings resulted in higher bandgap values [45].

Compared with the values reported in the literature, the decrease in the bandgap value found in this study may be associated with high numbers of oxygen vacancies. Wang et al. [46] stated that a decrease may lead to a better efficacy of visible light absorption without affecting photocatalytic capacity. In this sense, the absorption of photons with energies equal to or greater than this bandgap leads to electron excitation from the valence band to the conduction band, thus generating holes in the valence band. The holes generate sites of oxidization or reduction of organic compounds, which are effective for effluent treatment [47].

3.2. Photocatalysis of Vinasse Using ZnO-NPs

Figure 6 shows a decrease in COD values after the treatments with photocatalysis during 4 h of exposure with intervals of 1 h.

The vinasse in natura sample without treatment presented a COD value of 41,441.7 ± 144 mg O₂/L, which was used as a parameter to determine the changes resulting from the treatment with ZnO-NPs. The exposure of the vinasse to different light sources without nanoparticles did not influence the COD values (p > 0.05).

After the first hour of exposure (Figure 6b), treatment T3 with sunlight 1560 mg/L resulted in the smallest COD value compared to the other light sources. This result was stable for up to 2 h. After 3 h (Figure 6d), treatment T3 resulted in the same COD value, while treatments T2 (40 mg/L ZnO-NPs and sunlight) and T6 (1560 mg/L ZnO and UV) resulted in COD reductions of 12.47% and 9.05%, respectively. After 4 h of exposure (Figure 6f), the comparison of different light sources showed that treatments T2 and T6 (40 and 1560 mg/L ZnO-NPs, sunlight and UV) showed greater efficacy in reducing COD values (17.09% and 14.08%) and that T2 was more efficient than T6 (p < 0.05). The samples did not present large variations in pH values at 21 °C, these remaining between 3.71 and 3.96. Sunlight has a higher power compared to artificial light, so it allows more transitions of electrons to the excited state. Such transitions result in recombination with the holes, thus promoting greater numbers of reactive oxygen species (ROS), which are responsible for photocatalysis [12]. As for NPS concentration, 40 mg/L presented a better result than 1560 when sunlight was used, which may be related to the optimal concentration. The application of a higher concentration of NPS may have saturated the sample. In addition, when exposed to the sun, greater numbers of nanoparticles can aggregate, and we did not use agitation in this experiment so as to simulate a real treatment application. When excited state transitions and recombination occur, the energy released by these nanoparticles can be self-absorbed around the NPs themselves (self-absorption) and not deposited in solution to provide reactive oxygen species (ROS). The results also indicate that further studies with varying concentrations are necessary to better elucidate the phenomenon.

Kee et al. [16] observed COD reductions for vinasse treated with 250, 500, 750, and 1000 mg/L ZnO-NPs/vinasse over 10 h of monitoring. The increase in the concentration of ZnO-NPs (1000 mg/L) affected the ·OH radicals on the catalyst surface [16]. The highest COD degradation of 42.85% was achieved with the lowest concentration (250 mg/L).

Similar to our results, and using an individual technique in the treatment, Apollo et al. [48] carried out photocatalysis with ultraviolet (UV) light for the treatment of molasses distillery wastewater, obtaining a COD reduction efficacy of <20%. Robles-González et al. [49] conducted vinasse treatment by ozonation and achieved a COD reduction between 4.5 and 11% (with contact times of up to 1.5 h). David, Arivazhagan, and Ibrahim [50] used photocatalysis with aluminum oxide nanoparticles (Al₂O₃) to treat distillery wastewater and observed that photocatalytic treatment efficacy depended mainly on the number of nanoparticles (0.5 to 2.5 g/L) and had better reduction rates at low pH levels (3) and 25 °C, while agitation had less effect on the treatment (50 to 250 rpm). Other studies on vinasse treatment from tequila production observed that the vinasse of blue Agave tequilana had a COD value of 343.30 mg O₂/L. To increase the quality of the treated effluent, different processes were applied, and combined experiments with Fenton reactions showed an efficacy of COD removal from 79 to 89% [5]. Guerreiro et al. [51] reported on the biodegradability efficacy of sugarcane vinasse. Vinasse samples were submitted to thermophilic anaerobic digestion (UASB) in series. For coagulation–flocculation analysis, a jar test apparatus (22–25 °C) was used, and the ferric chloride coagulant was added to vinasse with a pH of ~7. The dissolved iron salts that resulted from the coagulation–flocculation were used as a catalyst in Fenton oxidation. In sequence, BOD, COD, and dissolved iron levels were measured for clarified vinasse. The results showed a reduction of 45.7% for BOD, 69.2% for COD, and 270 mg/L for dissolved iron [51]. Some treatment methods, such as biodigestion and AOPs, require more complex infrastructures to carry out the processes; however, the processes have high efficacies. Studies in the literature have reported the influence of COD–sulfate ratios (12.0, 10.0, and 7.5) on COD removal and methane (CH₄) production from vinasse biodigestion [8]. At a COD–sulfate ratio of 7.5, CH₄ production was 35% lower compared to the 12.0 ratio. The diversion of electrons to sulfidogenesis was negligible at COD–sulfate ratios >25, considering the increase in CH₄ production [8]. The results showed that organic matter degradation was not affected by sulfidogenesis, with COD removal levels higher than 80%, regardless of the initial COD–sulfate ratio [8].

Afterward, BOD analysis was performed to complete the COD results for vinasse in natura (no treatment) and after 4 h of treatment with 40 and 1560 mg/L of ZnO-NPs (

Table 2. BOD (mg/L) and reduction efficacy (%).

Sample	Description	BOD (mg/L)	% Reduction
Control	Vinasse in natura	17,666.7 ± 577	-
T2	40 mg/L (sunlight)	5000.0 ± 0	71.7
T6	1560 mg/L (UV-C)	6000.0 ± 0	66.0

).

After 5 days of analyses, treatment T2 showed the highest efficacy in reducing BOD (71.7%) and COD. Therefore, T2 was the most effective and the least costly treatment with the lowest concentration of nanoparticles (40 mg/L) and sunlight.

3.3. Susceptibility of Microorganisms to ZnO-NPs

The microorganisms identified via vinasse Gram staining were related to unicellular fungi or yeast of Saccharomyces cerevisiae, with an average size of 1 µm and oval and cylindrical morphologies. Yeast strains have phenotypes of high tolerance and resilience and are considered super-resistant microorganisms in the ethanol production process. Saccharomyces cerevisiae and other yeast strains, such as CAT-1 and PE-2, have been developed to tolerate different stress situations during ethanol fermentation, such as osmotic stress, oxidative stress, and high temperatures [52]. Possibly, the yeasts found in the vinasse samples analyzed in our work are organisms that resisted these processes and were active in the effluent.

Minimum inhibitory concentration (MIC) analysis provides quantitative results for antimicrobial effectiveness. MIC is the lowest antimicrobial concentration that inhibits visible microorganism growth in vitro [37]. The MICs for the ZnO-NPs against microorganisms in vinasse were determined through MIC experiments.

Table 3. Minimum inhibitory concentrations (MICs) (ZnO-NPs mg/mL).

Sample	C+	C−	1	2	3	4	5	6	7	8	9	10
ZnO-NP concentration	0	50	25	12.5	6.25	3.125	1.562	0.781	0.390	0.195	0.097	0.049

C+: positive control, vinasse in natura (growth of viable cells); C−: ZnO-NP negative control with DMSO (absence of viable cells).

shows the ZnO-NP concentrations, and Figure 7 presents a plate photograph after resazurin staining.

A gradual increase in inhibition was observed with increasing ZNO-NP concentrations (Table 3 and Figure 7), showing that the microorganisms were sensitive to ZnO-NPs between 25 and 0.781 mg/mL, with the breakpoint at 0.781 mg/mL. Low concentrations of ZnO-NPs showed no activity against microorganisms, and Saccharomyces cerevisiae strains were resistant between 0.390 and 0.049 mg/mL. MIC analysis allows the classification of microorganisms as sensitive or resistant to an antifungal when ranges and breakpoints are obtained [33].

Table 4. Number of colony-forming units (CFUs) according to the concentration of ZnO-NPs used in the vinasse treatment.

ZnO-NPs (mg/mL)	C+	C−	25	12.5	6.25	3.125	1.562	0.781	0.390	0.195	0.097	0.049
CFUs and SD	267	0	0	0	0	0	0	1 ± 0	3 ± 2	10 ± 3	11 ± 4	12 ± 5
Reduction in CFUs (%)	n/a	n/a	100 a	100 a	100 a	100 a	100 a	99.63 b	98.88 b	96.25 c	95.88 c	95.51 c

SD: standard deviation; n/a: not applied. Different letters indicate significant statistical differences (p < 0.05).

lists the number of CFUs in the vinasse plating experiment with microdilutions of ZnO-NPs.

The non-parametric Kruskal–Wallis and the Student–Newman–Keuls tests showed that the vinasse treatment with ZnO-NPs was effective in eliminating/reducing the microbial load of vinasse up to a concentration of 1.562 mg/mL (Table 4). In the treatment with a concentration breakpoint visually determined, 0.781 mg/mL, viable cells were observed (Table 4). Thus, the MIC value is 1.562 mg/mL, which should be used for the treatment of microorganisms with vinasse in natura.

Other studies analyzed the in vitro toxicity of ZnO-NPs in Saccharomyces cerevisiae strains and reported that growth was inhibited by 80% with 250 mg/L [53]. Babele et al. [54] also investigated the toxic effects of ZnO-NPs using 5, 10, 15, and 20 mg·L⁻¹ in Saccharomyces cerevisiae, as well as the underlying mechanisms. Cell walls were damaged, and the accumulation of reactive oxygen species (ROS) led to cell death at 10, 15, and 20 mg·L⁻¹ of ZnO-NPs. Furthermore, exposure to ZnO-NPs caused cellular toxicity due to lipid disequilibrium and proteostasis. Galván-Marquéz et al. [26] assessed the effects of 1.5 mg/mL of ZnO-NPs in Saccharomyces cerevisiae and suggested that vinasse microorganisms showed greater sensitivity due to ZnO-NPs affecting cell wall integrity and/or ROS accumulation.

Other works have addressed promising and innovative techniques in agriculture, such as the application of metal oxide nanoparticles as plant fertilizers. As micronutrients in soils, ZnO-NP ions promote richness and diversity in soil microbial communities, increasing crop growth and yield by stimulating photosynthesis and respiration in plants and suppressing the growth of soil pathogens [55,56]. Therefore, the presence of ZnO-NPs in treated vinasse favors and does not impede its use as a fertilizer, nor is it a barrier to its sustainable reinsertion into the agro-industrial productivity cycle.

4. Conclusions

In this work, treatments with sugarcane vinasse were carried out by heterogeneous photocatalysis with different concentrations of zinc oxide nanoparticles (ZnO-NPs) and radiation sources, including sunlight, UV-C, and white halogen light. Our results showed reductions in COD and BOD and vinasse microorganism populations using a low concentration of nanoparticles (40 mg/L) and sunlight, demonstrating an alternative treatment which does not require complex infrastructures or artificial radiation sources. The addition of ZnO to vinasse added value to this effluent as a fertilizer, enabling its reinsertion into the ethanol production chain. Future studies should consider different concentrations and treatment durations to increase COD reduction efficiency.

5. Patents

Patent resulting from the work reported in the article: Innovation Privilege. Registration number: BR1020210201789; title: “Treatment of sugarcane vinasse with zinc oxide nanoparticles to reduce microbial load, chemical oxygen demand and biochemical oxygen demand”; institution of registration: INPI—Brazilian National Institute of Industrial Property; deposited: 10 July 2021.