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Article

Chemical Analysis and Antioxidant Capacity of the Stages of Lignocellulosic Ethanol Production from Amazonian Fruit Industrial Waste

by
Gabriela Vieira Pantoja
and
Johnatt Allan Rocha de Oliveira
*
Postgraduate Program in Food Science and Technology, Federal University of Pará, Augusto Corrêa Street, 01, Belém 66075-1101, Pará, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2024, 10(10), 496; https://doi.org/10.3390/fermentation10100496
Submission received: 20 August 2024 / Revised: 16 September 2024 / Accepted: 23 September 2024 / Published: 27 September 2024
(This article belongs to the Special Issue Lignocellulosic Biomass Valorization)
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Abstract

:
Abstract: The production of ethanol from wastes resulting from the process of growing Amazonian fruit is a little-explored approach, in which unknown chemical compounds are released with potential for industrial application. This work aimed to produce lignocellulosic ethanol from waste from Amazonian fruit farming and to chemically characterize the stages of the process. The wastes (açaí seeds, mango peel, and peach palm peel) were pretreated with 1% to 5% H2SO4 and 15% solids; the resulting solid fraction was enzymatically hydrolyzed with cellulase at 20 FPU, and the liquid fraction (liqueurs) and enzymatic and fermented hydrolysates produced were chemically characterized. Via HPLC for sugars and fermentation inhibitors, we determined the antioxidant capacities and total phenolic compounds. The liquors from the pretreatment of açaí seeds released the most significant amount of glucose, while in the hydrolyzed solid fractions, the mango peel produced the highest glucose content. Among the fermented liquors, the highest ethanol content was the açaí seed at 15 and 5% (0.183–0.276 g/L). High glucose levels were produced (0.09–25.05 g/L) and provided ethanol levels that can be improved (0.061–10.62 g/L), in addition to liquors and hydrolysates with interesting amounts of phenolic compounds (14.04–131.87 mg EAG/g DM) and high antioxidant capacities (417.78–2774.07 mmol TEAC/g), demonstrating that these wastes can have other applications in addition to ethanol production.

1. Introduction

The growing consumption of fossil fuels and their negative environmental impacts, such as the emission of greenhouse gases and the accumulation of waste, drive the search for alternative and sustainable energy sources. Lignocellulosic ethanol (LE), obtained from lignocellulosic biomass, appears as a promising alternative to fossil fuels, contributing to the diversification of the energy matrix and mitigating climate change [1,2].
LE production involves complex steps that transform lignocellulosic biomass in agricultural and forestry wastes into fermentable sugars and, subsequently, into ethanol. The process generally includes pretreatment, enzymatic hydrolysis, fermentation, and purification [3,4].
Amazonian fruit farming, characterized by the abundance of native species such as açaí, pupunha, and mango, faces significant challenges related to the management of waste from these fruits, which together amount to approximately 1,069,687 tons [5] that are disposed of inappropriately. Without any management, especially the açaí pit, it is characterized as an urban sanitary inconvenience for the large cities in the North of Brazil, as they contribute to adverse environmental impacts, such as soil and water contamination and greenhouse gas emissions [6,7]. Only a minimal fraction of the waste is estimated to be reused for producing bioproducts, energy, or composting [7]. Therefore, developing sustainable technologies for this waste is crucial to mitigate these impacts and promote the regional bioeconomy [8]. Thus, using fruit farming waste as raw material for LE has several benefits. In addition to contributing to the valorization of this waste, which is generally disposed of inappropriately, the production of LE from Amazonian fruit farming waste can generate income for local communities, promote regional development, and contribute to environmental preservation [9]. Pretreatment is a crucial step in the production of LE. It alters the structure of lignocellulosic biomass, facilitating the access of hydrolytic enzymes to the polysaccharides present in cellulose and hemicellulose. Various pretreatment methods, such as physical, chemical, and biological, can be used, each with advantages and disadvantages. Acid pretreatment, using acids such as diluted H2SO4, is considered one of the most economical and effective methods to disrupt the lignocellulosic matrix, especially hemicellulose, and thus efficiently increase enzyme accessibility in the production of hydrolysates [10,11].
After pretreatment, enzymatic hydrolysis converts the polysaccharides in lignocellulosic biomass into fermentable sugars such as glucose and xylose. This step is carried out by specific enzymes, such as cellulases and xylanases, breaking the glycosidic bonds in polysaccharides. The efficiency of enzymatic hydrolysis depends on several factors, such as the biomass composition, the pretreatment characteristics, and the hydrolysis conditions [12,13].
The enzymatic hydrolysis of lignocellulosic biomass results in two main fractions: liquid and solid; the liquid fraction, or hydrolysate, contains fermentable sugars used in fermentation to produce ethanol. The solid fraction, a lignocellulosic residue, comprises non-hydrolyzed lignin, cellulose, and hemicellulose [14,15].
In addition to fermentable sugars, other molecules of industrial interest may be present in the liquid and solid fractions of LE production from Amazonian fruit farming wastes. In the liquid fraction, phenolic compounds, organic acids, and other compounds with antioxidant and antimicrobial potential can be found in addition to fermentable sugars. In the solid fraction, lignin can be valued for producing bioproducts, such as biomaterials, biofuels, and fine chemicals [16].
Evaluating the antioxidant capacity of the liquid and solid fractions of LE production is essential for several reasons. Firstly, the antioxidant compounds present in these fractions may have beneficial health properties, such as protection against chronic diseases and reduction in oxidative stress. Furthermore, these compounds may have applications in the food, pharmaceutical, and cosmetic industries [17,18].
The production of LE from wastes from Amazonian fruit farming has great potential to diversify the energy matrix, generate income for local communities, and contribute to environmental preservation. Studying the chemical composition and antioxidant capacity of the liquid and solid fractions of LE production is essential to identify new compounds of industrial interest and add value to this process [19,20].
Thus, the main objective of this study was to carry out a comprehensive investigation into the phenolic compounds, the chemical characterization and antioxidant capacity of liquid fractions (liqueurs), and the enzymatic and fermented hydrolysates obtained during the production of lignocellulosic ethanol with lignocellulosic wastes obtained from pretreatment with dilute sulfuric acid (H2SO4).

2. Materials and Methods

2.1. Feedstock

The wastes used were obtained from fruit processing companies in the city of Belém, Pará, Brazil (01°27′21″ S Longitude: 48°30′16″ W). They were açaí seeds, mango peel, and peach palm peel. The materials were dried for 48 h at 40 °C in an oven and then crushed and stored according to the methodology of Oliveira et al. [6]. The flowchart in Figure 1 shows a summary of the steps developed.

2.2. Hydrothermal Pretreatment with Diluted H2SO4

The pretreatment stage used the best condition studied by Oliveira et al. [10] using two concentrations of solids (5 and 15% w/v) for pretreatment with a 1% H2SO4 solution. The material was weighed in a load of study solids in 250 mL borosilicate Schott tubes, and pretreatment was carried out at 120 °C in a vertical autoclave for 1 h. After completion of the reaction, the material was removed from the autoclave and filtered. The liquid fraction was collected and the solid fraction was washed with distilled water until reaching neutral pH. Then, it was dried in an oven at 30 °C for 48 h, and then enzymatic hydrolysis was performed.

2.3. Enzymatic Hydrolysis

The enzymatic hydrolysis of the pretreated material (solid fraction) was adapted from Oliveira [20], using the sodium citrate buffer with pH 4.5. Hydrolysis was carried out in Erlenmeyer flasks with a solid load of 3% of material of the volume of buffer solution and an enzyme load of 20 FPU/mL (Cellulase from Aspergillus niger, Sigma, St. Louis, MO, USA). The Erlemmeyer flasks were incubated in a shaker for 72 h at 50 °C and 120 rpm. The hydrolysates were sterilized in a sterilizing membrane model Minikap HF Filter MK2M-512-V6S, 0.22 µm (Spectrum Laboratories, Inc., Miami, FL, USA) to obtain a sterile liquid fraction for subsequent fermentation and analysis.

2.4. Inoculum Preparation

For the fermentation of the hydrolysate and liquor obtained from the solid fraction, Saccharomyces cerevisiae ATCC 26602 was used, and its preparation was carried out in a 1000 mL Erlenmeyer flask containing 500 mL of distilled water and cultivated in YPX medium (5 g/L of yeast extract, 10 g/L peptone, 10 g/L D-xylose) at 30 °C for 24 h, and stirring at 200 rpm. Lastly, the volume was centrifuged for 5 min (at 2000 rpm and 15 °C) and resuspended in distilled water to obtain a suspension with 180 mg of fresh yeast/mL. Fermentation assays were inoculated with 5 mg fresh yeast/mL (approximately 1 mg dry cell weight (DCW)/mL).

2.5. Fermentation of Liqueurs (Liquid Fraction) and Hydrolysates

The hydrolysates of the solid fraction and the liquid fraction (liqueurs) were fermented after pH correction with potassium phosphate was achieved in the desired pH range (pH 4.0 to 4.5), and then filtered through sterilizing membranes of the Minikap model HF Filter MK2M-512-V6S (Spectrum Laboratories, Inc., Miami, FL, USA). Then, we proceeded to fermentation. Both were supplemented with 0.112 g·L−1 of K2HPO4, 0.01 g·L−1 of (NH4)2SO4, 0.1 g·L−1 of yeast extract, 0.1 g·L−1 of peptone and 0.028 g·L−1 of (NH4)2SO4. Fermentation was adapted from Oliveira et al. [6] and was carried out in Erlenmeyer flasks with 20 mL of hydrolysates or liquors. After adding the Saccharomyces cerevisiae ATCC 26602 yeast inoculum, fermentation occurred in a shaker incubator at 120 rpm and 30° C for 72 h. Finally, the fermented material was analyzed.

2.6. Chemical Composition and Chromatographic Analysis

The moisture content was determined by drying, according to the AOAC standard No. 926.12 [21], and the ash content followed the methodology described by NREL “Determination of Ash in Biomass” [22]. The extractive content was determined with water/ethanol solvents following the method of Sluiter et al. [22]. After removing the extractives from the biomass, the material was subjected to acid hydrolysis with 72% H2SO4, based on Sluiter et al.’s method [23], and their cellulose and hemicellulose contents were determined by HPLC, as described in [24]. The lignin content was performed according to the NREL/TP-510-42618 standard [24]. For the determination of protein, method no. 920.152 by AOAC [21] was used for the micro-Kjeldahl technique. The lipid content was determined in a Soxhlet apparatus, following method no. 920.39 by AOAC [21].
The fractions of fermentable sugars, fermentation inhibitors, and ethanol were analyzed via High-Performance Liquid Chromatography (HPLC). For sugars and ethanol, a BIORAD Aminex HPX-87H column was used at 30 °C with the eluent H2SO4 0.01 mol/L, a flow of 0.6 mL/min and a refractive index detector, at 35 °C; the volume of injected sample was 15 L and the total analysis time was set at 30 min. The average ethanol retention time was 21.1 min, and the standards were solutions in the 0.01 to 4% concentration range. For inhibitors (furfural, hdroxymethylfurfural, and acetic acid), a Nova-Pak C18 column was used, at 30 °C, with eluent acetonitrile/water (1:8), a flow of 0.8 mL/min, and a UV-VIS detector, at 27 °C and 280 nm [24].

2.7. Phenolic Compounds

The liquors from the pretreatment, enzymatic hydrolysis, and fermentation stages were analyzed to determine the levels of phenolic compounds released. The concentration of phenolic compounds was determined by the Folin–Ciocalteu colorimetric method [25]. The solutions used were the extractor solution (80% methanol), diluted extractor solution (8% methanol), 10% Folin–Ciocalteu solution, and 7.5% calcium carbonate solution. The reaction mixture was carried out with 50 µL of samples, 800 µL of distilled water, 50 µL of Folin–Cioucateu solution, and 100 µL of aqueous sodium carbonate solution (Na2CO3). After an incubation period of 2 h at room temperature (25 °C) and in the dark, the reaction mixture had its absorbance read on a spectrophotometer (Ultospec 2000, UV visible, Pharmacia Biotech, Piscataway, NJ, USA) at 765 nm. The content of total phenolic compounds was calculated from the gallic acid calibration curve and expressed as milligrams of gallic acid equivalents per gram of dry extract (mgEAG/gES).

2.8. Antioxidant Capacity (DPPH)

The quantification of antioxidant capacity was carried out using the DPPH method [26,27]. Firstly, the methodology for constructing the calibration curve with the Trolox solution was carried out. For the samples, 1:800 dilutions were made with 70% methanol. Afterwards, 50 µL of diluted samples and 150 µL of DPPH solution were added to each microplate’s well. After 40 min, the reading was performed on a plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 520 nm.

3. Results and Discussion

Table 1 presents the results of the chemical characterization of the waste used.
The results of the chemical characterization of the residues of pupunha shell, mango shell, and açaí seeds reveal significant variations in their compositions. The mango shell presented the highest cellulose content (29.11%), while the açaí seeds had the highest proportion of hemicellulose (39.03%). The pupunha palm peel, although with a lower cellulose content (24.03%), presented a more balanced portion between cellulose, hemicellulose (10.45%), and lignin (18.48%). Compared with data in the literature, the mango shell has higher cellulose contents than other lignocellulosic residues, such as sugarcane bagasse, which has approximately 25% cellulose [28]. The high concentration of hemicellulose in açaí seeds is consistent with studies on this residue [29,30] and with other tropical fruit residues, which generally have high levels of this polysaccharide due to its structural function in the plant [31]. Lignin, more abundant in the peach palm shell, is a critical factor in resistance to enzymatic hydrolysis, negatively impacting biomass conversion efficiency into fermentable sugars, as Haldar et al. [32] observed. The high concentration of extractives in ethanol in açaí seeds (13.01%) suggests a significant potential for the extraction of bioactive compounds, corroborating previous studies on the valorization of these residues for high-value-added bioproducts [33]. Table 2 shows the results for the released glucose content, total phenolic compounds, antioxidant capacity, and fermentation inhibitors in the 5% and 15% pretreatment liquors.
The highest levels of phenolic compounds were observed for mango peel liqueurs (48.06 and 69.31 mg EAG/g DM), followed by the values found for peach palm peel (17.54 and 43.54 mg EAG/ g DM) and açaí seed (14.04 and 42.85 mg EAG/g DM). Safdar et al. [34] found values of 67.58 ± 0.21 mg EAG/g DM for mango peel, a value very close to that found in the pretreatment liquor from mango peel, which demonstrates an essential result as phenolics can be very toxic to yeast or fermenting microorganisms, even in minimum quantities.
According to Adeboye et al. [35], such compounds significantly inhibit yeast growth and fermentation and promote a reduction in product yield, increasing fermentation costs. This information is essential as it demonstrates possible challenges for ethanol production from this residue. Nevertheless, it reflects the potential for using this liquor, which is rich in such compounds, for other purposes. The TPC values contributed to the antioxidant activity values, and the peach palm peel was the material that demonstrated the highest values, which ranged from 380.25 to 2684.44 mmol TEAC/g, followed by the results from the mango peel (780. 74 and 983.70 mmol TEAC/g), and finally, the antioxidant capacity for the açaí seed, which ranged from 417.78 to 655.31 mmol TEAC/g. According to De Souza Mesquita [36], peach palm peel is rich in carotenoids, which probably contributes to its antioxidant capacity and may explain its higher value than other liqueurs.
It is possible to verify that during the pretreatment stage for ethanol production, depending on the pretreated residue, it is possible to obtain compounds with great industrial interest for different application areas, which, if in high quantities, can limit the final alcohol production. The quantification of the primary fermentation inhibitors is essential to verify whether ethanol production can be viable with the steps and conditions used and identify and quantify other compounds of interest that can be generated during the second-generation ethanol production steps with the wastes used. Both the pretreatment of lignocellulose and the fermentation process produce acetic acid and can hinder subsequent fermentation stages as well as the use of lignocellulosic waste, with fermentation by S. cerevisiae, in addition to xylose-fermenting microorganisms [37].
Açaí seed liqueurs were those that demonstrated the highest amounts of the inhibitors furfural (0.034 g/L), HMF (0.125 g/L), and acetic acid (3.191 g/L). Oliveira et al. [10] found values of furfural and HMF higher than those observed in the present work, which were 0.22 g/L and 1.182 g/L, respectively. It is possible to state that the pretreatment carried out promoted the production of inhibitors in reduced and smaller quantities than those recommended by Taherzadeh et al. [38], who reported that the growth of Saccharomyces cerevisiae is inhibited by 70% with the presence of 4 g/L of HMF, and by 89% with 4 g/L of furfural. The same authors reported that furfural (2 g/L) and HMF (2 g/L) can simultaneously act with a synergistic effect of the total inhibition of the same microorganism. Table 3 presents the results obtained for the liquid fraction (liquors) from the pretreatment of biomass with diluted sulfuric acid (pretreatment liquors) after fermentation.
The fermented mango peel liquors presented the highest TPC values (19.58 and 62.48 mg EAG/g DMg/L), slightly higher than those observed in the original liqueurs (14.04–71.48 mg EAG/g DM), which represents an increase ranging from 3.13 to 7.83% and promoted by the fermentation of the pretreatment liquors. This can be explained by the fact that during fermentation, S. cerevisiae can release phenolic compounds linked to other molecules in plant cell walls and also promote the release of phenolic compounds linked to sugars or proteins, increasing the total content of these compounds [39,40].
The antioxidant capacity of the fermented liquor was quite revealing, as it showed that for the majority of pretreatment liquors, there was an increase in the antioxidant capacity, which may be the result of a series of biotransformations promoted in the constituent chemicals of these samples during the fermentation, such as the breakdown and separation of sugar fractions from phenolic compounds, with a change in the phenolic profile, in addition to an increase in the quantity released [27,39,41].
Fermented liquors, although limited, had lower levels of fermentation-inhibiting compounds, which may have occurred because S. cerevisiae, in some cases, reduces furfural to furfuryl alcohol, which is less toxic, a process catalyzed by enzymes such as aldehyde reductases and alcohol dehydrogenases present in yeast [42]. According to Hawkins et al. [43], in the case of HMF, the levels found in liquors may have been reduced due to the conversion of HMF to 2,5-bis-hydroxymethylfuran (HMF-alcohol), a less toxic compound, which is also a process mediated by yeast reductase enzymes. The acetic acid content in the fermented liquors was also lower than in the unfermented liquor because acetic acid is a byproduct of acetaldehyde metabolism. Under normal conditions, Saccharomyces cerevisiae converts acetaldehyde into ethanol, so if this conversion is efficient, the amount of acetaldehyde available to form acetic acid is reduced, which would explain such a reduction [44].
The highest ethanol values were found for solid loads of 15%, as they allowed for higher percentages of released sugars. Nevertheless, they were quite limited for mango and peach palm liqueurs, mainly due to the lower values of released sugars and the microorganisms used, as there were more significant amounts of pentoses in these liquors not fermented by S. cerevisiae. However, the values obtained for the açaí seed proved to be promising, as the sum of the glucose and mannose contents released contributed to higher ethanol values than the possible theoretical yields, which were around 90% of the theoretical yield calculated from the glucose mass found in the açaí pretreatment liquor. However, it is essential to highlight that the sugar content in the liqueurs was generally low due to the degradation caused by the acid content, which could compromise the subsequent distillation stages. Table 4 shows the chemical composition of the solid fraction resulting from pretreatment with 1% of H2SO4.
The results indicate an increase in the cellulose fraction in all biomasses as the solid load increases from 5% to 15%. Previous studies on acid pretreatment of biomass indicate that the cellulose fraction tends to increase after treatment, as hemicellulose and lignin are partially solubilized, allowing for greater accessibility to cellulose [45,46].
At a solid load of 5%, the chemical attack demonstrated remarkable efficiency in removing the hemicellulose fraction. As per the literature, H2SO4 pretreatment leads to the partial or complete degradation of hemicellulose, which is then solubilized into monosaccharides like xylose and arabinose [47].
The greater effectiveness of the chemical attack for the lower solid load (5%) allowed for the excellent removal of this component under this condition. Lignin is one of the most challenging components to remove during pretreatment, and its removal is crucial to improving cellulose accessibility in bioconversion processes [48].
The increase in solid load from 5% to 15% during the pretreatment of lignocellulosic biomass with H2SO4 may have impacted hemicellulose solubilization and cellulose retention. At lower solid loads (5%), there is a more significant interaction between the acid and the biomass, resulting in greater hemicellulose solubilization and partial cellulose degradation. This effect is explained by the greater accessibility of the acid to the lignocellulosic matrix, facilitating polymer degradation [49]. At higher solid loads (15%), the mixture’s viscosity increases, reducing the efficiency of hemicellulose solubilization and better preserving the cellulose fraction [50]. Thus, pretreatment with 5% solids may have resulted in more significant cellulose degradation, while treatment with 15% promoted a more excellent retention of this fraction.
Table 5 presents the results of the chemical analyses of the enzymatic hydrolysates of the solid fraction of the acid pretreatment.
Xylose was the second monomer found in the highest quantity in the hydrolysates obtained, being found in the highest concentration in the açaí seed hydrolysate and ranging from 1.42 to 3.02 g/L. The mannose fraction was detected only for the hydrolysates of the peach palm peel and the açaí seed. The latter was the one that demonstrated the highest mannose values, ranging from 4.95 to 9.35 g/L, which reveals a potential material to be used as a source of mannose production and that has a wide variety of food and pharmaceutical applications, although is still little explored for this material.
Hydrolytic enzymes, such as cellulases, can break down the plant cell wall and release chemical compounds of industrial interest, such as phenolic compounds [6]. Thus, during the enzymatic hydrolysis stage carried out during the production of ethanol from the evaluated wastes, it was revealed that interesting values of phenolic compounds were released, mainly from the plant matrix of the açaí seed, with values ranging from 97.11 to 123.57 mg EAG/g DM and which were followed by the values found for the mango peel, which ranged from 52.14 to 63.68 g/L, and finally by the pupunha peel, with values ranging from 27.80 to 42.86 g/L. Regarding antioxidant capacity, the hydrolysates obtained from the peach palm peel demonstrated the highest values, ranging from 1392.98 to 1828.07 mmol TEAC/g, which can be explained mainly by the high amount of carotenoids, compounds known to have this potential [51], and which were followed by the values presented by the açaí seed (976.61 to 1251.46 mmol TEAC/g) and by the mango peel, with values of 807.02 and 1074.29 mmol TEAC/g.
Table 6 shows the levels of sugars, phenolic compounds, and antioxidant activity of the fermented hydrolysates.
Although the rate of mannose fermentation by S. cerevisiae is generally lower than that of glucose, possibly due to the lower affinity of hexokinase for mannose compared to glucose [55], the S. cerevisiae presents several advantages for the conversion of mannose into ethanol, and which include high tolerance to ethanol, robustness, and the ability to grow in different pH and temperature conditions, which make it an ideal microorganism for large-scale biotechnological processes. The highest mannose reduction rate was 67.87 for the hydrolysate obtained with 15% açaí seeds.
For TPC values, it was observed that fermentation with Saccharomyces cerevisiae provided the release of more significant quantities of these compounds; this same behavior was followed by other authors who worked with lignocellulosic biomasses [56,57]. According to Monteil et al. [58], yeast has several enzymes that can degrade or modify these compounds, reducing their inhibition of fermentation. Furthermore, fermentation can change the pH of the medium, influencing the solubility and bioavailability of phenolic compounds. The type of S. cerevisiae strain used can also affect the release of phenolic compounds, since strains with a greater capacity for adsorption and degradation of these compounds may present better performance in the fermentation of lignocellulosic hydrolysates [59].
In our research, we found that fermentation led to an increase in phenolic compounds in all fermented hydrolysates, which had values ranging from 31.11 to 131.87 mg EAG/g DM, which represents increases of 2.40 to 13.77% in relation to the values observed for hydrolysates without fermentation. As observed for phenolic compounds, fermentation promoted an increase in the antioxidant activity of fermented hydrolysates; this increase can be explained not only by the more significant release of phenolic compounds into the medium but also by the possible modification that yeast enzymes can promote in the profile phenolic, after fermentation [27,53].
The highest AC values for fermented products obtained from peach palm waste ranged from 1904.12 ± 56.01 to 1415.47 ± 63.01 mmol TEAC/g for 5 and 15% solids. It was observed that the values of ethanol produced varied from 4.01 to 10.62 g/L, with mango peel and peach palm peel, both at 15% solid load, being the ones that presented better ethanol production, with theoretical yields ranging from 83 to 87%, which represents reasonable rates of use of the released sugars, since several factors can influence the actual yield, such as the efficiency of the fermentation process, cultivation conditions of the fermenting microorganism, and energy efficiency, among others [60]. Furthermore, the ethanol contents obtained are higher than those obtained by Jahid et al. [61] for banana and pineapple peels, which ranged from 2.75 to 6.3 g/L. However, Favaretto et al. [62] obtained higher values than those obtained in the present study, reaching values of 28.02 g/L for banana peel. These values must depend on several factors related to the intrinsic characteristics of the chosen residue and the process conditions. It is important to emphasize that even if the ethanol levels produced can be considered low, the other compounds explored, characterized as chemicals of industrial interest, such as inhibitors with antimicrobial potential and phenolic compounds with antioxidant properties and their various industrial applications, can justify the exploration of these residues, as carried out in the present work.

4. Conclusions

Fruit industry residues from the Amazon, such as açaí seeds and mango peels, have proven valuable for producing lignocellulosic ethanol and can potentially extract bioactive compounds. An analysis of the process steps revealed that the liquid fractions of the residues presented high levels of glucose and phenolic compounds, with açaí residues producing the highest ethanol yields. Fermentation of the hydrolysates demonstrated that, despite the presence of inhibitors, such as furfural and acetic acid, the concentrations obtained were not high enough to compromise the ethanol yield significantly, demonstrating the viability of the processes used, especially for mango residues considering the amount of residue available and ethanol produced. Furthermore, the antioxidant capacities of the extracts indicate that these residues can be exploited beyond ethanol production, including the manufacture of bioproducts with industrial applications. To improve the viability and sustainability of the commercial processes, future studies should focus on optimizing ethanol yields and minimizing energy consumption. In short, the use of waste from Amazonian fruit farming not only contributes to the valorization of these materials but also drives regional development and environmental preservation, aligning with energy security and climate change mitigation objectives.

Author Contributions

G.V.P. and J.A.R.d.O. performed the analysis and carried out scientific writing. All authors have read and agreed to the published version of the manuscript.

Funding

We are grateful for receiving funds from CNPQ (National Council for Scientific and Technological Development) by Universal project number: 438156/2018-8, Federal University of Pará (Belém, Brazil).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are grateful for the Federal University of Pará.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 10 00496 g001
Figure 1. Flowchart of the steps developed.
Figure 1. Flowchart of the steps developed.
Fermentation 10 00496 g001
Table 1. Chemical characterization of the waste used.
Table 1. Chemical characterization of the waste used.
Component Dry Mass (%)Peach Palm PeelMango PeelAçaí Seeds
Celullose24.03 ± 1.3529.11 ± 1.4712.01 ± 1.01
Hemicelullose10.45 ± 1.5513.30 ± 2.0139.03 ± 1.97
Lignin18.48 ± 2.985.09 ± 3.0812.99 ± 2.93
Extractives (water)1.09 ± 0.866.01 ± 1.334.35 ± 1.45
Extractives (ethanol)9.47 ± 2.4611.99 ± 2.6613.01 ± 3.01
Lipids8.95 ± 0.996.35 ± 1.092.38 ± 1.03
Moisture8.31 ± 0.109.01 ± 0.088.45 ± 0.08
Protein3.11 ± 0.912.99 ± 0.554.53 ± 0.45
Ash4.01 ± 0.021.98 ± 0.052.08 ± 0.01
Table 2. Analysis of sugars, total phenolic compounds, antioxidant capacity, and fermentation inhibitors in liquors from pretreatment with dilute sulfuric acid.
Table 2. Analysis of sugars, total phenolic compounds, antioxidant capacity, and fermentation inhibitors in liquors from pretreatment with dilute sulfuric acid.
AnalysisPeach Palm PeelMango PeelAçaí Seeds
Solid Load (%)
515515515
Glucose (g/L)0.14 ± 0.01 d0.25 ± 0.02 c0.17 ± 0.02 d0.26 ± 0.02 c0.29 ± 0.01 b0.34 ± 0.01 a
Xylose (g/L)0.23 ± 0.01 f0.29 ± 0.0 e0.33 ± 0.02 d0.57 ± 0.03 c0.81 ± 0.02 b1.19 ± 0.03 a
Mannose (g/L)NdNdNdNd0.51 ± 0.02 b0.94 ± 0.05 a
TPC
(mg EAG/g DM)		17.54 ± 1.23 e43.54 ± 0.61 b48.06 ± 1.23 d69.31 ± 1.32 a14.04 ± 0.73 f42.85 ± 1.40 c
AC
(mmol TEAC/g)		1380.25 ± 263.44 b2684.44 ± 37.92 a780.74 ± 51.32 d983.70 ± 461.88 c417.78 ± 55.56 f655.31 ± 43.41 e
Furfural
(g/L)		0.002 ± 0.01 f0.005 ± 0.00 d0.003 ± 0.00 e0.009 ± 0.00 c0.011 ± 0.01 b0.034 ± 0.00 a
HMF(g/L)0.050 ± 0.00 e0.095 ± 0.00 c0.082 ± 0.009 d0.051 ± 0.010 e0.099 ± 0.004 b0.125 ± 0.00 a
Acetic acid
(g/L)		1.233 ± 0.020 f1.562 ± 0.002 e1.852 ± 0.011 d2.011 ± 0.001 c2.652 ± 0.001 b3.191 ± 0.012 a
TPC: Total phenolic compound; AC: antioxidant capacity; TPC: total phenolic compound; HMF-hydroxymethylfurfural (HMF); Nd: not detected. Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. The liquor from the pretreatment of açaí seeds had the highest amount of mannose in its composition, ranging from 0.51 to 0.94 g/L. It also demonstrated the highest glucose levels (0.29 and 0.34 g/L) and xylose (0.81 and 1.19 g/L), followed by mango peel for glucose (0.17 and 0.26 g/L) and xylose (0.33 and 0.57 g/L). Peach palm peel was the residue that demonstrated the lowest potential for releasing glucose (0.14 and 0.25 g/L) and xylose (0.23 and 0.29 g/L). It was possible to verify that solid loads of 15% allowed for a more significant release of fermentable sugars in the pretreatment liquors. Furthermore, the behavior of the acid pretreatment was compatible with the findings from the literature and promoted a more substantial release of monomers from the hemicellulosic fraction, which, in this case, was xylose.
Table 3. Quantification of carbohydrates, phenolic compounds, and antioxidant activity in liquors after fermentation.
Table 3. Quantification of carbohydrates, phenolic compounds, and antioxidant activity in liquors after fermentation.
AnalysisPeach Palm PeelMango PeelAçaí Seeds
Solid Load (%)
515515515
Glucose (g/L)0.02 ± 0.0010.04 ± 0.0090.03 ± 0.0020.07 ± 0.080.06 ± 0.010.04 ± 0.017
Xylose (g/L)0.20 ± 0.01 f0.27 ± 0.0 e0.31 ± 0.02 d0.55 ± 0.03 c0.78 ± 0.02 b1.08 ± 0.03 a
Mannose (g/L)NdNdNdNd0.31 ± 0.02 b0.54 ± 0.02 a
TPC
(mg EAG/g DM)		18.73 ± 0.5344.32 ± 2.6349.58 ± 1.9971.48 ± 1.6215.14 ± 1.0744.77 ± 0.53
AC
(mmol TEAC/g)		1581.48 ± 1282774.07 ± 15.67863.33 ± 34.51005.93 ± 22.22501.41 ± 96.20674.81 ± 16.73
Furfural (g/L)0.001 ± 0.01 f0.005 ± 0.00 d0.001 ± 0.00 e0.008 ± 0.00 c0.01 ± 0.01 b0.030 ± 0.00 a
HMF (g/L)0.01 ± 0.00 e0.075 ± 0.00 c0.034 ± 0.009 d0.039 ± 0.010 e0.059 ± 0.004 b0.101 ± 0.01 a
Acetic Acid (g/L)1.01 ± 0.02 f1.31 ± 0.002 e1.61 ± 0.011 d1.08 ± 0.001 c1.74 ± 0.001 b1.91 ± 0.012 a
Ethanol (g/L)0.061 ± 0.02 f0.114 ± 0.02 d0.07 ± 0.001 e0.119 ± 0.08 c0.183 ± 0.02 b0.276 ± 0.02 a
TPC: Total phenolic compound; AC: antioxidant capacity; Nd: not detected. Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. The glucose value in fermented liquors (Table 3) was reduced compared to Table 2 precisely due to the consumption of glucose by the yeast used to produce ethanol. On the other hand, the xylose values analyzed are practically the same as those found in pre-fermentation liquors; this results from the choice of microorganisms used to ferment the liquors since S. cerevisiae does not ferment xylose. The ethanol production from açaí liquor (0.061 and 0.276 g/L) presented exciting results since they were more significant than the theoretical percentage possible from the mass of glucose in the liquor. Since the amounts of xylose remained unchanged, fermentation of mannose fractions released during pretreatment likely occurred, with an average reduction of 40% in mannose values in the pretreatment liquor (0.51 and 0.94 g/L) observed for the fermented liquor with values of 0.31 and 0.54 g/L.
Table 4. Chemical composition of the solid fraction resulting from pretreatment with H2SO4.
Table 4. Chemical composition of the solid fraction resulting from pretreatment with H2SO4.
Component Dry Mass (%)Peach Palm PeelMango PeelAçaí Seeds
Solid Load (%)
515515515
Celullose29.22 ± 0.44 c32.01 ± 1.01 b33.33 ± 2.12 b38.01 ± 2.03 a11.75 ± 1.02 e16.43 ± 1.32 d
Hemicelullose3.62 ± 0.32 d5.18 ± 0.34 b2.98 ± 0.12 e4.32 ± 0.09 c9.75 ± 1.00 a10.63 ± 1.04 a
Lignin22.01 ± 1.03 b26.66 ± 56 a5.09 ± 0.09 f7.02 ± 0.91 e15.95 ± 0.99 d19.33 ± 0.88 c
Extractives (water)0.99 ± 0.71 e1.33 ± 0.45 d3.33 ± 0.07 c4.09 ± 0.12 b3.35 ± 1.48 c5.32 ± 0.76 a
Extractives (ethanol)5.47 ± 0.23 c4.99 ± 0.67 e5.22 ± 0.21 d6.32 ± 0.23 b15.44 ± 1.32 a14.99 ± 1.11 a
Lipids4.53 ± 0.89 d6.32 ± 0.09 a4.21 ± 0.45 d5.35 ± 0.48 c4.57 ± 0.32 e5.89 ± 0.43 b
Moisture10.01 ± 0.10 a9.54 ± 0.93 b9.41 ± 0.13 b9.88 ± 0.65 c9.77 ± 0.67 c10.03 ± 0.77 a
Protein4.23 ± 0.76 c4.56 ± 0.98 c5.33 ± 0.46 b6.21 ± 0.77 a5.85 ± 0.45 b 6.24 ± 0.55 a
Ash5.11 ± 0.54 a5.32 ± 0.78 a2.58 ± 0.09 d3.44 ± 0.54 b3.09 ± 0.05 c3.98 ± 0.23 b
Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05.
Table 5. Quantification of carbohydrates, phenolic compounds, and antioxidant capacity of enzymatic hydrolysates from the solid fraction of acid pretreatment.
Table 5. Quantification of carbohydrates, phenolic compounds, and antioxidant capacity of enzymatic hydrolysates from the solid fraction of acid pretreatment.
AnalysisPeach Palm PeelMango PeelAçaí Seeds
Solid Load (%)
515515515
Glucose (g/l)10.92 ± 0.17 d17.85 ± 0.15 b12.95 ± 0.02 c25.05 ± 0.05 a9.04 ± 0.13 e15.87 ± 0.22 b
Xylose (g/l)0.12 ± 0.01 f0.20 ± 0.02 e0.21 ± 0.08 d0.33 ± 0.06 c1.42 ± 0.11 b3.02 ± 0.21 a
Mannose (g/l)0.09 ± 0.01 d0.19 ± 0.03 cNdNd4.95 ± 0.16 b9.35 ± 0.12 a
TPC (mg EAG/g DM) 27.80 ± 3.45 f42.86 ± 1.58 e52.14 ± 1.77 d63.68 ± 0.88 c97.11 ± 0.93 b123.57 ± 1.13 a
AC (mmol TEAC/g)1392.98 ± 63.01 a1828.07 ± 56.01 a807.02 ± 72.1 c1074.29 ± 6.03 d976.61 ± 92.11 c1251.46 ± 138.01 b
TPC: Total phenolic compound; AC: antioxidant capacity. Quantification unit for phenolic compounds: milligram of gallic acid per gram of dry matter. Quantification unit for antioxidant activity: millimole of Trolox equivalent per gram of matter. Nd: not detected. Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. Based on the release of sugars in the enzymatic hydrolysate, a yield range varied from 33.39 to 76.14%, with the highest yield observed for mango peel at 15% and the lowest for peach palm peel at 5%. Mango peel was the material that allowed for the most excellent glucose release, with values ranging from 12.95 to 25.05 g/L, followed by pupunha peel. The authors of [6] analyzed the concentration of reducing sugars after enzymatic hydrolysis of pupunha peel under different conditions. They found that the concentration of solids is the parameter that most influences the release of sugars, in addition to obtaining glucose concentrations of 9.32 g/L and 15.38 g/L for solid loads of 5 and 15%, which were lower than the concentrations obtained in this work. In comparison with the other wastes used, the açaí seed released lower glucose concentrations (9.04 to 15.87 g/L) during hydrolysis with cellulase, mainly because it has lower cellulose concentrations than the other biomasses evaluated and were lower than the values found by Oliveira [10], which ranged from 20 to 40 g/L after 48 h of hydrolysis. In addition, the low glucose value of açaí in relation to the other wastes is also due to the use of cellulase for the hydrolysis step, which could be improved using mannosidases.
Table 6. Quantification of reducing sugars, phenolic compounds, and antioxidant activity of fermented hydrolysate.
Table 6. Quantification of reducing sugars, phenolic compounds, and antioxidant activity of fermented hydrolysate.
AnalysisPeach Palm PeelMango PeelAçaí Seeds
Solid Load (%)
515515515
Glucose (g/L)1.63 ± 0.050 c2.31 ± 0.003 c1.68 ± 0.005 b3.25 ± 0.010 a1.26 ± 0.006 e1.48 ± 0.005 d
Xylose (g/L)0.11 ± 0.01 f0.18 ± 0.02 e0.20 ± 0.08 d0.33 ± 0.06 c1.39 ± 0.11 b2.98 ± 0.21 a
Mannose (g/L)0.07 ± 0.01 d0.11 ± 0.03 cNdNd1.59 ± 0.16 b7.57 ± 0.12 a
TPC (mg EAG/g DM)31.11 ± 1.88 f48.21 ± 1.71 e59.32 ± 1.99 d69.01 ± 1.04 c99.45 ± 1.25 b131.87 ± 2.59 a
AC (mmol TEAC/g)1415.47 ± 63.01 b1904.12 ± 56.01 a915. 11 ± 51.21 e1099.98 ± 52.12 d1099.99 ± 85.15 d1314.54 ± 99.25 c
Ethanol (g/100 mL) 5.58 ± 0.05 d9.12 ± 0.15 b5.55 ± 0.51 e10.62 ± 0.66 a4.01 ± 0.13 f6.88 ± 0.22 c
TPC: Total phenolic compound; AC: antioxidant capacity. Quantification unit for phenolic compounds: milligram of gallic acid per gram of dry matter. Quantification unit for antioxidant activity: millimole of Trolox equivalent per gram of matter. Nd: not detected. Results are presented as the mean (n = 3) ± SD, and those with different letters are significantly different, with p < 0.05. After fermentation, the remaining glucose levels ranged from 1.48 to 3.25 g/L, which represents a glucose consumption ranging from 85.07 to 90.67% during fermentation, with the fermentation of the hydrolysates resulting in 15% of açaí seeds. In general, the percentages of xylose remained practically the same, demonstrating the already known inability of S. cerevisae to ferment pentoses [52]. Regarding mannose, it was possible to observe a slight reduction in the remaining values in the fermented product about the hydrolysates since the fermentation of mannose by S. cerevisiae follows the classic glycolytic pathway, with the initial conversion of mannose into fructose and the subsequent fermentation in ethanol and CO2. This metabolic pathway involves several vital enzymes, including hexokinase, phosphofructokinase, pyruvate decarboxylase, and alcohol dehydrogenase [53,54].
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Pantoja, G.V.; Oliveira, J.A.R.d. Chemical Analysis and Antioxidant Capacity of the Stages of Lignocellulosic Ethanol Production from Amazonian Fruit Industrial Waste. Fermentation 2024, 10, 496. https://doi.org/10.3390/fermentation10100496

AMA Style

Pantoja GV, Oliveira JARd. Chemical Analysis and Antioxidant Capacity of the Stages of Lignocellulosic Ethanol Production from Amazonian Fruit Industrial Waste. Fermentation. 2024; 10(10):496. https://doi.org/10.3390/fermentation10100496

Chicago/Turabian Style

Pantoja, Gabriela Vieira, and Johnatt Allan Rocha de Oliveira. 2024. "Chemical Analysis and Antioxidant Capacity of the Stages of Lignocellulosic Ethanol Production from Amazonian Fruit Industrial Waste" Fermentation 10, no. 10: 496. https://doi.org/10.3390/fermentation10100496

APA Style

Pantoja, G. V., & Oliveira, J. A. R. d. (2024). Chemical Analysis and Antioxidant Capacity of the Stages of Lignocellulosic Ethanol Production from Amazonian Fruit Industrial Waste. Fermentation, 10(10), 496. https://doi.org/10.3390/fermentation10100496

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