Hydrodynamic Cavitation-Assisted Photo-Fenton Pretreatment and Yeast Co-Culture as Strategies to Produce Ethanol and Xylitol from Sugarcane Bagasse

Abstract

This study explored innovative approaches to produce ethanol and xylitol from sugarcane bagasse using a hydrodynamic cavitation-assisted photo-Fenton process as the pretreatment, and yeast co-culture for hydrolysate fermentation. Pretreatment conditions were optimized (20 mg/L of iron sulfate, pH 5.0, and reaction time of 14 min) resulting in glucan and xylan hydrolysis yields of 96% and 89%, respectively. The hydrolysate produced under these conditions was fermented using a co-culture of Saccharomyces cerevisiae IR2 (an ethanol-producing strain) and Candida tropicalis UFMGBX12 (a xylitol-producing strain). Optimal co-culture conditions consisted of using an inoculum concentration of 1.5 g/L for each yeast strain. After 36 h of fermentation, ethanol and xylitol concentrations reached 20 g/L and 13 g/L, respectively. These results demonstrate the potential of combining hydrodynamic cavitation-assisted photo-Fenton pretreatment with co-culture fermentation to simultaneously produce ethanol and xylitol. This strategy presents a promising approach for enhancing the efficiency of lignocellulosic biorefineries.

1. Introduction

In recent decades, growing concerns over environmental issues, energy security, and economic sustainability have driven research into alternative fuels derived from renewable sources. The production of biofuels in biorefineries and the presence of green compounds on the market present promising alternatives to gasoline and other petroleum-based products [1]. In this context, the study of viable technologies for the sustainable production of bioproducts from renewable sources is essential to support the transition toward a more sustainable society [2].

The development of biorefineries for lignocellulosic materials offers a promising approach to enhance the production of second-generation (2G) ethanol. In countries like Brazil, sugarcane bagasse is an abundant by-product of the sugar and ethanol industry [3]. However, efficient utilization of this material requires the hydrolysis of its carbohydrate fractions to release fermentable sugars, which can then be converted into ethanol or other bioproducts through fermentation.

Lignocellulosic biomass is composed of a complex structure of cellulose, hemicellulose, and lignin [4], which contributes to its recalcitrance and makes fractionation and conversion into valuable products challenging. An effective pretreatment step is essential to disrupt this structure, enhancing enzyme accessibility in the subsequent step of the hydrolysis of polysaccharides into monomers. Various pretreatment methods have been developed, including physical, chemical, biological, and physicochemical processes. However, these methods often require large amounts of water, chemicals, and energy, operating under harsh conditions and generating inhibitory compounds that can be difficult to remove, causing inhibition in subsequent fermentation steps and increasing costs [4].

Hydrodynamic cavitation (HC) is an emerging pretreatment technology that has shown promising results for sugarcane bagasse. HC is a phenomenon involving the formation, growth, and subsequent collapse of vapor or gas bubbles within a liquid due to pressure variations. Initially, the pressure is reduced below the vapor pressure of the fluid by devices such as orifice plates, Venturi tubes, or rotary systems, resulting in the formation of small cavitation bubbles. As the liquid then enters a region of higher pressure, these bubbles collapse violently, producing intense microjets and localized shock waves. This collapse can generate extreme local conditions, releasing a large amount of energy. HC-assisted pretreatment of lignocellulosic biomass can be effectively combined with other methods, such as alkaline and oxidative processes, to enhance biomass digestibility. For example, Hilares et al. [5] reported carbohydrate hydrolysis yields exceeding 90% when using sugarcane bagasse pretreated by an HC process in a medium containing 1% peroxide and 3M of NaOH over 10 min. Recently, Prado et al. [6] investigated HC pretreatment in acid medium (pH 5.0) with the addition of 76 mL/min of ozone and 0.67% peroxide, achieving a hydrolysis yield of approx. 92% in the subsequent enzymatic step. The same study also conducted a preliminary evaluation of iron-catalyzed (Fenton) HC pretreatment, which further improved the hydrolysis of sugarcane bagasse carbohydrate fractions. These findings highlight the need for further research to better understand the key influencing variables and optimize the process for maximum efficiency.

HC facilitates pretreatment through both physical and chemical mechanisms [7], with the latter primarily driven by the generation of hydroxyl radicals (HO•). These highly reactive species promote the oxidative degradation of the biomass structure, particularly aiding in lignin breakdown and reducing lignocellulosic recalcitrance. The efficiency of HC can be further enhanced by combining it with Fenton or other oxidative pretreatment methods, which intensify HO• radical production, leading to improved biomass deconstruction and conversion efficiency.

After pretreatment, the solid material that is rich in carbohydrates is subjected to enzymatic hydrolysis and fermentation. During hydrolysis, a glucose-rich broth is produced, which can be converted into ethanol using the same yeast strains traditionally employed in first-generation (1G) ethanol production [8]. The hemicellulosic fraction also contains significant amounts of sugars (mainly pentoses like xylose), which can also be used to produce ethanol or other valuable products such as xylitol [9]. Xylitol is a five-carbon polyalcohol with high sweetening power and diverse applications in the food, dental, and pharmaceutical industries [10,11]. It is recognized as one of the top 10 top bioproducts for development in biorefineries [12]. Although xylitol is currently produced by chemical synthesis, the fermentative route presents important advantages including the use of milder pressure and temperature conditions and reduced substrate purification requirements, making the process more sustainable [9,13].

Some microorganisms can produce both ethanol and xylitol [14,15,16,17], and the simultaneous production of these biomolecules has been explored in some studies [14,15,16]. Cortivo et al. [14], for example, used a recombinant S. cerevisiae strain to co-produce both biomolecules. In another study, Cunha-Pereira et al. [15] used soybean hull hydrolysate for ethanol and xylitol production by Candida guilliermondii BL13, achieving an ethanol productivity of 1.4 g L⁻¹ h⁻¹ and reaching a maximum yield of 0.41 g g⁻¹ (80.4% of the theoretical value), with small quantities of xylitol produced. More recently, Prado et al. [6] investigated a sequential strategy to produce ethanol and xylitol from sugarcane bagasse pretreated by an HC-assisted method. After enzymatic hydrolysis, which generated a glucose- and xylose-rich broth, ethanol was produced using a non-engineered S. cerevisiae strain (which does not ferment xylose). Following ethanol removal by distillation, Candida tropicalis, a yeast naturally capable of fermenting xylose, was used to convert the remaining sugars into xylitol.

Although some microorganisms can co-produce ethanol and xylitol, their industrial performance is often hindered by metabolic limitations. Co-culturing specialized strains, such as S. cerevisiae for hexose fermentation and Candida tropicalis for pentose conversion, enhances adaptation to mixed sugar substrates and improves overall yield. Co-culture systems have been explored for the concurrent production of ethanol and xylitol [18]; however, no studies have previously investigated co-culture fermentation using the S. cerevisiae IR2. This strain exhibits flocculating behavior, which can facilitate downstream processing steps such as cell recovery and reuse after fermentation [6].

In this study, sugarcane bagasse was used for the simultaneous production of ethanol and xylitol through co-culture fermentation with S. cerevisiae IR2 and C. tropicalis UFMGBX12. The biomass was pretreated by an HC-assisted photo-Fenton method, which was optimized with the aid of experimental design tools. After pretreatment, the material underwent enzymatic hydrolysis, and the resulting hydrolysate was used as fermentation medium for the co-culture experiments.

2. Results and Discussion

2.1. Enzymatic Hydrolysis in Erlenmeyer Flasks

Sugarcane bagasse was pretreated under different conditions using an HC-assisted photocatalytic process. The resulting material was evaluated regarding its composition and the enzymatic hydrolysis yields of its cellulosic and hemicellulosic fractions. The untreated biomass consisted of 40% glucan, 26% hemicellulose (24% xylan, 1% arabinan, and 1% acetyl), and 25% lignin, along with 1% ash and 3% extractives.

Table 1. Analysis of variance: ANOVA. Pretreated material composition, removal of components, and enzymatic hydrolysis yield obtained for sugarcane bagasse pretreated by HC-assisted photo-Fenton method according to 2² face-centered design.

Run	Conditions (Codified Values)		Solid Recovery * (%)	Pretreated Material Composition (%)			Removal of Components (%)			Hydrolysis Yield (%)
	Iron(II) Sulfate (mg/L)	Pretreatment Time (min)		Glucan	Xylan	Lignin (%)	Glucan (%)	Xylan (%)	Lignin (%)	Glucan (%)	Xylan (%)
1	10 (−1)	5 (−1)	70	51.08	21.08	16.33	10.6	38.51	54.27	70.03	42.00
2	20 (+1)	5 (−1)	64	57.42	22.32	12.21	8.1	40.48	68.74	70.57	68.04
3	10 (−1)	20 (+1)	71	51.82	21.82	17.01	8.0	35.45	51.69	70.38	61.70
4	20 (+1)	20 (+1)	71	53.01	17.01	20.60	5.9	49.68	41.50	92.50	83.70
5	10 (−1)	12.5 (0)	64	55.46	22.76	20.41	11.3	39.31	47.75	95.98	52.00
6	20 (+1)	12.5 (0)	64	55.56	21.06	20.65	11.1	43.84	47.14	90.27	90.30
7	15 (0)	5 (−1)	72	50.7	22.7	21.19	8.7	31.90	38.97	79.79	60.50
8	15 (0)	20 (+1)	64	51.4	22.4	20.44	17.8	40.27	47.67	80.59	72.50
9	15 (0)	12.5 (0)	71	51.3	22.7	18.69	8.9	32.85	46.92	90.51	72.00
10	15 (0)	12.5 (0)	71	51.2	22.40	15.38	9.1	33.74	56.32	90.20	69.00
11	15 (0)	12.5 (0)	71	51.22	22.40	15.01	9.1	33.70	57.37	90.30	70.00

* Solid recovery is the ratio of the total dry mass after pretreatment to the total dry mass before pretreatment.

presents the results of the statistical design used to evaluate the effects of iron sulfate concentration (10 to 20 mg/L) and pretreatment time (5 to 20 min) on glucan and xylan yields after enzymatic hydrolysis. The table also includes the residual biomass composition and the removal of components for each condition. As shown, the pretreatment altered the composition of sugarcane bagasse, yielding materials with high cellulose and low lignin contents. The pretreatment primarily removed lignin (38–70%), followed by xylan (32–49%), while glucan had a maximum removal of approx. 18%.

The effects of HC on the pretreatment of lignocellulosic biomass have been studied and documented [5,19]. Research indicates that the chemical and physical impacts of cavitation provide several advantages, including milder conditions for oxidative degradation and significant lignin reduction [5]. During the HC process, a substantial amount of energy is released into the medium, leading to the production of highly reactive oxidative radicals. Moreover, the integration of oxidizing agents such as ozone, iron (II) sulfate, and UV light in an acidic medium further enhances the process efficiency. This improvement is attributed to the high oxidative potential generated by the photo-Fenton reaction. The combination of HC and these oxidizing agents promote the breakdown of lignin molecules and facilitate hemicellulose removal [7,20]. In addition to chemical effects, HC also produces mechanical impacts such as high-speed microjets and shockwaves resulting from the violent collapse of cavitation bubbles. These mechanical forces contribute to the disruption of the lignocellulosic structure, enhancing the accessibility of cellulose and hemicellulose and thereby improving their conversion into fermentable sugars during enzymatic hydrolysis. This enhancement occurs because HC increases the specific surface area of the biomass and expands total pore and micropore volumes [19,21].

As shown in Table 1, the compositional changes achieved during pretreatment resulted in varying glucan and xylan hydrolysis yields during the subsequent enzymatic process. The highest yields were 96% for glucan (achieved with 10 mg/L iron(II) sulfate and 12.5 min) and 90% for xylan (obtained with 20 mg/L iron(II) sulfate and 12.5 min). Empirical models (

Table 2. Empirical models for glucan and xylan enzymatic hydrolysis yield, according to the results obtained in the experimental design. A: iron II sulfate (mg/L); B: pretreatment time (min).

Response Variable	Empirical Model
Glucan enzymatic hydrolysis yield (X1%)	X1 = 64.26 − 1.24∙A + 4.64∙B + 0.144∙A∙B − 0.25∙B2
Xylan enzymatic hydrolysis yield (X2%)	X2 = −2.11 + 2.88∙A + 3.68∙B − 0.11∙B2

) were developed to describe the behavior of the dependent variables, glucan and xylan hydrolysis yields. The models were refined by removing non-significant terms (p > 0.1), except where their inclusion was necessary to maintain model hierarchy. ANOVA results (

Table 3. Analysis of variance (ANOVA) and coefficient of determination (R²) for the fitted models for hydrolysis yield (glucan and xylan) as a function of the studied variables.

Glucan Enzymatic Hydrolysis Yield (%)						Xylan Enzymatic Hydrolysis Yield (%)
Source	Sum of Square	Degrees of Freedom	Mean Square	F	p	Source	Sum of Squares	Degrees of Freedom	Mean Square	F	p
Model	798.53	4	199.63	7.19	0.0179	Model	1711.84	3	570.61	40.94	<0.0001
Iron II sulfate (A)	47.88	1	47.88	1.72	0.2372	Iron II sulfate (A)	1242.43	1	1242.43	89.14	<0.0001
Time (B)	88.78	1	88.78	3.20	0.1241	Time (B)	373.83	1	373.83	26.82	0.00613
AB	116.42	1	116.42	4.19	0.0866
B2	545.44	1	545.44	19.63	0.0044	B2	95.58	1	95.58	6.86	0.0345
Residual	166.69	6	27.78			Residual	97.57	7	13.94
Lack of fit	166.64	4	41.66	1664.17	0.006	Lack of fit	92.90	5	18.58	7.96	0.1153
Pure Error	0.050	2	0.025			Pure Error	4.67	2	2.33
R2	0.83					R2	0.95

) confirmed that both models were significant (p < 0.05) for predicting glucan and xylan hydrolysis yields, although presenting a significant lack of fit for glucan hydrolysis yield. As noted by Kraber et al. [22], a significant lack of fit does not necessarily render a model unusable. If model improvement is not feasible, validation through confirmation experiments can ensure the reliability of the results. With R² values of 83% for glucan and 95% for xylan hydrolysis yields, the models were considered suitable for generating response surfaces (Figure 1) and optimizing the process. As shown below, the models successfully predicted the experimental results of optimized conditions.

As shown in Figure 1A,B, and supported by the empirical models in Table 2, enzymatic hydrolysis of the carbohydrate fractions in the pretreated material is influenced quadratically by the concentration of iron sulfate. This suggests the existence of an optimal concentration within the studied range of values that maximizes enzymatic hydrolysis yield. The observed quadratic behavior is likely due to the oxidative environment created during UV light exposure. While studies confirm that acidic catalytic pretreatment alters biomass structure, enhancing hydrolysis yields, excessively oxidative conditions can lead to the formation of inhibitory compounds, which may hinder both enzymatic hydrolysis and subsequent fermentation processes.

The addition of catalytic iron (II) sulfate has a beneficial effect on glucan and xylan hydrolysis yields by inducing structural and compositional modifications in the biomass. Through its catalytic action, iron sulfate enhances the oxidative power of the process, accelerating the generation of free radicals, such as hydroxyl radicals (·OH), which play a crucial role in breaking down recalcitrant biomass components [7,23]. The application of UV light further amplifies this catalytic effect by generating highly reactive chemical species. For instance, Yang et al. [24] demonstrated the effectiveness of oxidative pretreatment using ultraviolet light for sisal waste. In their study, a 500 W UV light source (200 nm) was applied while varying peroxide concentration (0.02–0.8 g/g biomass) and pH (8–12) over a 6 h process at 60 °C. Their results showed that the optimal conditions (pH 12 and 0.8 g/g peroxide) yielded a glucan hydrolysis efficiency of 91.6% during the saccharification stage, showcasing the potential of such pretreatments.

Optimized results from a related study [25] demonstrated that increasing UV light exposure during pretreatment significantly enhanced lignin degradation, achieving a 21% higher reduction compared to untreated samples and a glucan hydrolysis yield exceeding 80%. However, this specific approach had not previously been reported for HC-assisted processes. The results obtained in our study highlight the potential of this method, achieving high hydrolysis yields in relatively short reaction times. To further evaluate the impact of UV light, a control experiment was carried out in triplicate under the same conditions of runs 9, 10, and 11 in Table 1, but without UV light exposure. In the control, glucan and xylan hydrolysis yields were 85.2 ± 2.1% and 77.1 ± 1.1%, respectively. These values were lower than the average hydrolysis yields observed in runs 9, 10, and 11, which reached 90.3 ± 0.2% for glucan and 82.0 ± 3.1% for xylan.

The empirical models presented in Table 2 were used to identify the optimal conditions for maximizing sugar hydrolysis yields, which consisted of using 20 mg/L of iron (II) sulfate and 14 min of pretreatment. Under these conditions, the predicted enzymatic hydrolysis yields for glucan and xylan, with a 95% confidence interval, were 95.67 ± 10.93% and 92.20 ± 14.41%, respectively. Experimental validation, performed in triplicate, closely aligned with these predictions, yielding glucan and xylan hydrolysis rates of 96.1 ± 2.1% and 89.2 ± 2.4%, respectively. This strong agreement between predicted and experimental results underscores the robustness and reliability of the models.

It is worth mentioning that the pretreatment process developed here not only delivered high hydrolysis yields but also produced a hydrolysate that is suitable for bioproduct production, supporting its integration into biorefinery frameworks. Also, it can be considered a short-duration method, requiring only 14 min under optimized conditions. This is particularly noteworthy given that the process is conducted under mild conditions (60 °C, with most of the system operating at atmospheric pressure). When compared to other conventional and emerging pretreatment methods reported for sugarcane bagasse, our approach stands out for its reduced processing time. For example, alkaline pretreatment was reported as requiring 60 min [26], deep eutectic solvents pretreatment was performed for 6 h [27], and dilute acid pretreatment was carried out for 20 min [28].

2.2. Enzymatic Hydrolysis in the Column Reactor

The biomass pretreated under optimized conditions had the following composition (dry weight basis): 45.24% cellulose, 27.45% hemicellulose (25.45% xylan, 1% arabinan, and 1% acetyl groups), and 20.60% lignin. The enzymatic hydrolysis was then performed in a column reactor, yielding up to 77 g/L of glucose and 56 g/L of xylose after 48 h, corresponding to hydrolysis yields of 83% for glucan and 80% for xylan.

As observed, the hydrolysis yields obtained in the column reactor were lower than those obtained in Erlenmeyer flasks, where glucan and xylan yields reached 96% and 89%, respectively. This difference is likely due to product inhibition in the column reactor [29], as the solid loading was higher in this system than in the flasks. However, the column reactor offered the advantage of producing a hydrolysate with a higher sugar concentration in a short reaction time, demonstrating its potential for more efficient sugar production. The obtained hydrolysate was subsequently used to formulate the medium for co-culture fermentations.

2.3. Ethanol and Xylitol Production from Co-Culture Fermentation

Table 4. Experimental matrix of the 2² face-centered design performed to evaluate the influence of the concentration of C. tropicalis and S. cerevisiae inoculated in the co-culture process on the production of ethanol and xylitol.

Run	Inoculum, g/L (Codified Values)		Products, g/L
	C. tropicalis	S. cerevisiae	Ethanol	Xylitol
1	0.5 (−1)	0.5 (−1)	4.20	6.30
2	1.5 (+1)	0.5 (−1)	6.80	10.28
3	0.5 (−1)	1.5 (+1)	16.70	6.00
4	1.5 (+1)	1.5 (+1)	20.37	10.29
5	0.5 (−1)	1.0 (0)	5.20	7.40
6	1.5 (+1)	1.0 (0)	7.10	10.30
7	1.0 (0)	0.5 (−1)	6.05	8.24
8	1.0 (0)	1.5 (+1)	19.85	7.02
9	1.0 (0)	1.0 (0)	16.90	11.20
10	1.0 (0)	1.0 (0)	16.50	10.70
11	1.0 (0)	1.0 (0)	16.70	11.21

presents the results of the experiments conducted to evaluate the effects of the variables S. cerevisiae concentration (0.5 to 1.5 g/L) and C. tropicalis concentration (0.5 to 1.5 g/L) inoculated in the fermentation process on ethanol and xylitol production. The table includes the statistical experimental matrix, with ethanol and xylitol production as the response variables. As can be seen, when both S. cerevisiae and C. tropicalis were added at concentrations of 0.5 g/L each, ethanol production reached 4 g/L, while xylitol production was 6 g/L. The highest production of ethanol (20 g/L) and xylitol (11 g/L) was observed in Experiment 4, where the highest concentrations of both microorganisms were inoculated into the reactor. These results demonstrate the significant impact of the inoculum concentration on the production of ethanol and xylitol in co-culture systems.

Empirical models (

Table 5. Empirical models for the response variables of the production of ethanol and the production of xylitol, according to the results obtained in the experimental design. The initial concentration of (A) C. tropicalis (g/L) and (B) S. cerevisiae (g/L).

Response Variable	Empirical Model
Ethanol concentration (X1, g/L)	X1 = −21.37 + 43.83∙A + 13.29∙B − 20.55∙A2
Xylitol (X2, g/L)	X2 = −1.61 + 3.72A + 16.60∙B − 8.55∙B2

) were developed to describe the experimental behavior of the dependent variables, ethanol and xylitol production. The quadratic models were refined by removing non-significant terms (p > 0.1), except where their inclusion was necessary to maintain model hierarchy. ANOVA results for the quadratic models are presented in

Table 6. Analysis of variance (ANOVA) and coefficient of determination (R²) for the fitted models for ethanol and xylitol concentration as a function of the studied variables.

Glucan Enzymatic Hydrolysis Yield (%)						Xylan Enzymatic Hydrolysis Yield (%)
Source	Sum of Square	Degrees of Freedom	Mean Square	F	p	Source	Sum of Squares	Degrees of Freedom	Square Mean Square	F	p
Model	348.07	3	116.02	12.72	0.0032	Model	33.65	3	11.22	10.81	0.0051
C. tropicalis (A)	11.12	1	11.12	1.22	0.3059	C. tropicalis (A)	20.79	1	20.79	20.04	0.0029
S. cerevisiae (B)	264.94	1	264.94	29.05	0.0010	S. cerevisiae (B)	0.38	1	0.38	0.37	0.5642
A2	72.01	1	72.01	7.89	0.0262
						B2	12.47	1	12.47	12.02	0.0839
Residual	63.85	7	9.12			Residual	7.26	7	1.04
Lack of fit	63.77	5	12.75	318.84	0.0031	Lack of fit	7.10	5	4.46	17.04	0.0564
Pure Error	0.080	2	0.040			Pure Error	0.17	2	0.083

. Although the ethanol production model exhibited an R² value of 85%, it showed a significant lack of fit (p < 0.1). Similarly, the model for xylitol production achieved an R² of 82% but also displayed a significant lack of fit (p < 0.1). As explained before, despite the significant lack of fit, these models were used to generate response surfaces and optimize the process, with a confirmation of prediction performed for the optimum values.

The coefficients of the empirical models (Table 5) and the correspondent response surfaces obtained for both ethanol and xylitol (Figure 2) indicated that, for ethanol production, S. cerevisiae inoculum concentration had a linear positive effect and C. tropicalis inoculum concentration presented a quadratic behavior, with an optimum value near the center point. A similar behavior can be observed for xylitol production, but with a linear positive effect of C. tropicalis inoculum concentration and a quadratic behavior of S. cerevisiae inoculum concentration.

As shown in Figure 3, within 12 h of the process, glucose consumption had already reached about 90%, with xylose consumption occurring over the course of 36 h of cultivation. The ethanol concentration achieved after 36 h of cultivation was 20 g/L, corresponding to a volumetric productivity (Qp) of 0.56 g/L/h, while the xylitol volumetric productivity was 0.36 g/L/h. Although a more detailed study would be needed to accurately determine the substrate-to-product yield values (Yp/s), assuming a fast glucose metabolism by S. cerevisiae to produce ethanol, and exclusive xylose consumption by C. tropicalis, it is possible to estimate the Yp/s for ethanol based on glucose consumption and for xylitol based on xylose consumption. Under these assumptions, the Yp/s for ethanol was 0.27 g/g, and for xylitol was 0.37 g/g. Both Yp/s values were lower than the theoretical values of 0.51 g/g for ethanol and 0.917 g/g for xylitol (this last was based on [17]).

The co-cultivation fermentation process offers a significant advantage by enabling the simultaneous and optimized production of two value-added bioproducts—ethanol and xylitol. In our study, this process was completed in just 36 h, demonstrating a substantial improvement compared to previous reports, such as Antunes et al. [30], where 96 h were required to produce these compounds. This highlights the efficiency and relevance of the co-cultivation strategy employed in our work.

Control experiments were performed using the yeasts separately (1.5 g/L of inoculum concentration) cultivated under the same conditions. In this case, S. cerevisiae produced a higher concentration of ethanol, 25.27 ± 0.18 g/L, with no xylitol production, while C. tropicalis produced a lower concentration of xylitol, 9.53 ± 0.24 g/L, with no ethanol production. Thus, considering the low Yp/s values obtained in co-culture and the results of the control experiments with the yeasts grown separately, some antagonism between the yeasts for ethanol production can occur, with a slight benefit for xylitol production. To enhance the process, strategies such as concomitant ethanol removal during cultivation and non-simultaneous inoculation of the yeasts should be explored in future work. Additionally, since the evaluated S. cerevisiae strain is flocculent, a possible entrapment of C. tropicalis in their pellets could be considered, offering opportunities for yeast reuse or continuous operation in co-culture.

3. Materials and Methods

3.1. Biomass Pretreatment and Enzymatic Hydrolysis in Erlenmeyer Flasks

Sugarcane bagasse in natura was characterized [31] and presented 45.24% cellulose, 26.45% hemicellulose (24.45% xylan, 1% arabinan, 1% acetyl groups), 20.60% lignin, 1.5% ashes, and 2.4% extractives. This material was pretreated using an HC system [5] consisting of centrifugal pumps (1.5 HP) and a cavitation zone, where the cavitation phenomenon was generated by two orifice plates (16 holes, each with 1 mm diameter). Pressure gauges are used to measure the pressure upstream of the orifice plates, which can be adjusted by varying the pump rotation speed through a frequency inverter. The total volume of the cavitation system was 2.5 L.

The photo-Fenton pretreatment assisted by HC was performed in acid medium (pH 5.0, adjusted using 10 mol/L sulfuric acid solution), at temperature 60 °C, with a pressure between the pump and the orifice plate of 3 bar, with atmospheric pressure in the other parts of the system, with an ozone inlet of 10 mL/min. A 2² face-centered design with triplicates at the center point was carried out considering iron sulfate concentration (10–20 mg/L) and pretreatment time (5–20 min) as the input variables. All experiments were carried out under UV light incidence. For this, a UV light source emitting at 280 nm with a frequency of 1018 Hz was placed 5 cm above the liquid level in the cavitation reactor. The response variables were the removal of glucan, xylan, and lignin during pretreatment, as well as the hydrolysis yield (glucan and xylan) in a subsequent enzymatic hydrolysis reaction performed in Erlenmeyer flasks. The hydrolysis was performed as follows: pretreated sugarcane bagasse was added to a 50 mmol/L citrate buffer solution (pH 4.8) in a 125 mL Erlenmeyer flask, with 5% solid loading and a working volume of 50 mL. Commercial cellulase enzyme blend Cellic^® CTec2 (Novozymes Latin America Ltda, Nova York, NY, USA) was added at 20 FPU/g of dry pretreated sugarcane bagasse. The reaction was performed at 50 °C and 200 rpm for 24 h.

The biomass composition after pretreatment was also determined [31].

3.2. Enzymatic Hydrolysis in a Column Reactor

In this step, the pretreated sugarcane bagasse was subjected to enzymatic hydrolysis in a 200 mL column reactor, as described elsewhere [32]. The initial solid loading was set at 20% (40 g of pretreated material) in a 50 mM citrate buffer solution at a pH of 4.8. The enzymatic hydrolysis was carried out using Cellic^® CTec2 (Novozymes Latin America Ltd., Araucária, PR, Brazil), a commercial cellulase enzyme blend, at a loading of 20 FPU/g of dry pretreated sugarcane bagasse. The reaction was conducted at 50 °C for 48 h, with liquid recirculation in the reactor at a flow rate of 12 mL/min in an ascending flow direction. The concentrations of glucose and xylose released during the hydrolysis were analyzed by high-performance liquid chromatography (HPLC), as described in Section 3.4.

3.3. Production of Ethanol and Xylitol in Co-Culture Process

3.3.1. Inoculum Preparation

Saccharomyces cerevisiae IR2 and Candida tropicalis UFMGBX12 were from the strain stock available in the Laboratory of Bioprocesses and Sustainable Products (LBIOS) of the Engineering School of Lorena-University of Sao Paulo (EEL-USP), Brazil. The strains were stored at 4 °C in Malt Extract Agar medium, with periodic subculturing.

The inoculum was prepared by transferring a loopful of a slant storage culture to 250 mL Erlenmeyer flasks containing 100 mL of medium, and the cultivation was carried out in a BIO CB SSB rotary movement incubator (ERETZBIO, São Paulo, SP, Brazil) at 150 rpm for 24 h at 30 °C. For S. cerevisiae IR2, the medium consisted of 50 g/L glucose, 10 g/L bacteriological peptone, and 10 g/L yeast extract [33]. For C. tropicalis UFMGBX12, the medium contained 50 g/L xylose, 20 g/L yeast exact, 2 g/L ammonium sulfate, and 0.1 g/L calcium chloride dihydrate, with the initial pH adjusted to 5.5 [34]. After 24 h, the cells were recovered by centrifugation (3000× g for 15 min), and washed and resuspended in distilled water to obtain a high-density cell suspension, which was used as inoculum to obtain initial cell concentrations of 0.5–1.5 g/L for S. cerevisiae and for C. tropicalis in the co-culture process.

For S. cerevisiae, before inoculum preparation, as a flocculant strain was used, 3 Erlenmeyer flasks were previously prepared as the above described procedure. After cultivation, the content of each of these flasks was separately centrifuged at 3000× g for 15 min, and the cells were washed and dried in an oven to determine their dry mass. The cell concentration in the inoculum flasks could then be estimated, and these data were used to calculate the volume of cell suspension to be inoculated into the fermentation flasks.

3.3.2. Co-Culture Fermentation

For co-cultivation of S. cerevisiae IR2 and C. tropicalis, 125 mL Erlenmeyer flasks containing 75 mL of medium were used. The medium consisted of hydrolysate with 77 g/L of glucose and 35 g/L of xylose, supplemented with 1 g/L bacteriological peptone and 5 g/L yeast extract. The process was carried out in a rotary movement incubator at 150 rpm and 30 °C. A 2² face-centered design with triplicates at the center point was performed, with initial concentrations of S. cerevisiae and C. tropicalis varying between 0.5 and 1.5 g/L for both yeasts. The response variables were ethanol and xylitol concentrations obtained after 48 h of cultivation.

3.4. Analytical Methods

The moisture content of the sugarcane bagasse was determined using an infrared balance (Mark M163, BEL Engineering, Piracicaba, SP, Brazil).

Sugars, ethanol and xylitol concentrations were analyzed by HPLC using an Agilent Technology 1200 series chromatograph (Agilent Technologies 1200, New York, NY, USA). Prior to analysis, the samples were filtered through Sep Pak C18 filters. The chromatographic conditions were as follows: BIO-RAD AMINEX HPX-87H (300 × 7.8 mm) column, maintained at 45 °C; injection volume of 20 μL; RID 6A refractive index detector; 0.01N sulfuric acid aqueous solution as the mobile phase; and a flow rate of 0.6 mL/min.

For the experiments with S. cerevisiae, after cultivation, the entire volume of the flask was centrifuged at 3000× g for 20 min. The cells were then washed twice with distilled water, and their concentration was determined based on the dry mass obtained after drying the cells in an oven at 105 °C. For C. tropicalis UFMGBX12, the yeast concentration was measured by optical density at 600 nm in a spectrophotometer. The optical density values were converted to grams per liter of cells using a pre-established calibration curve.

3.5. Statistical Analysis

Design-Expert v.6 software (Stat-Ease, Inc., Minneapolis, MN, USA) was used to identify statistically significant parameters and adjust the results to empiric models. Statistica software (Tibco Software Inc. V.14, San Ramon, CA, USA) was used to generate the response surfaces.

4. Conclusions

The proposed method using photo-Fenton HC-assisted pretreatment of sugarcane bagasse yielded promising results. Key variables influencing the pretreatment were evaluated, and the optimized conditions were found to be 20 mg/L of iron sulfate and 14 min of process time. Under these conditions, glucan and xylan hydrolysis yields reached 96% and 89%, respectively. The hydrolysate was then used for ethanol and xylitol production via a co-culture process. A process enabling the simultaneous production of both ethanol and xylitol with a co-culture of flocculent S. cerevisiae IR2 and C. tropicalis was explored for the first time. After 36 h of fermentation under optimized conditions, the process achieved 20 ± 0.5 g/L of ethanol and 13 ± 0.4 g/L of xylitol. The optimized conditions consisted of using an inoculum containing 1.5 g/L of S. cerevisiae IR2 inoculum and 1.5 g/L of C. tropicalis. These outcomes show significant promise for application in lignocellulosic biorefineries, particularly when using sugarcane bagasse as a feedstock.