3.1 Enzymatic hydrolysis of fruit biomass
A higher concentration of reducing sugars was obtained with banana biomass using the enzyme Pectinex Ultra SP-L. The banana biomass had an initial concentration of RS of 109.39 mg/mL and after the hydrolysis the concentration of RS reached 268.08 mg/mL. Pectin is the second most important structural component of bananas that affects their mechanical, structural, and functional properties causing firmness. It is a complex carbohydrate with homogalacturonon chains (smooth regions) and rhamnogalacturonan regions (hairy regions) [31]. As banana ripening advances, more soluble pectin is generated [32]. We added whole banana (peel and pulp) in the advanced stage of ripening to the hydrolysate. In the high stage of ripening, the pectin content in the peel is lower and pectin is more soluble, making facilitating enzymatic hydrolysis. In addition, pectin is soluble in water and acid media [31] and the pH was adjusted to 4,5 to facilitate hydrolysis. Furthermore, we also tested others pectinases (Pectinex tropical, Pectinex Pulp, and Pectinex Clear) and these enzymes did not produce higher concentration of reducing sugars compared to Pectinex Ultra sp. However, the other pectinases act on 4-deoxi-6-O-metil-alfa-D-galacto-4-enurosonil (Pectinex Ultra Tropical) and (1,4)-alfa-Dgalacturonana methil ether (Pectinex Ultra Pulp), and is expected that pectin is approximately 65% in the form of homogalacturonan and only 20–35% in other forms. Thus, the Pectinex Ultra sp enzyme is more specific for the hydrolysis of banana biomass, whic has pectin as the major carbohydrate in the homogalacturonan form.
[33] reported that pectin in banana peels can be extracted by approximately 54% in dry mass (or 10% in wet basins). Thus, pectin in banana biomass is a potential substrate for generating sugars for further fermentation, and our hydrolysis results show that pectin esterase can generate high concentrations of RS.
Another highlighted result was the hydrolysis of banana biomass by amylase. After hydrolysis, the biomass had an RS concentration of 181 mg/ml. However, the substrate of the amylase (starch) is higher in unripe bananas - approximately 70–80% [19] and is reduced to less than 1% in overripe bananas [34]. However, the starch content of the banana biomass was sufficient to achieve a high concentration of reducing sugars after hydrolysis.
The pretreatmenteatment of apple biomass with Pectinex Ultra sp. produced the higher concentration of RS (150.22 mg/mL) after 180 min of hydrolysis when compared to the other enzymes utilized. However, the initial concentration of RS in apple biomass was lower than that for banana biomass, and thus, the final concentration of RS in apple was lower than that in banana biomass. Pectin is a major multifunctional component usually found in association with other structural constituents such as cellulose and lignin present in the cell walls of plants [35], including apples. The concentration of pectin in apples is in the range of 0.5–1.6% [36] and the current technology to produce apple juice already includes an enzymatic treatment of the cell walls to increase the extraction yield, reducing sugars, soluble dry matter content, and galacturonic acid [37]. In addition, the initial presence of free sugars makes apple waste a potential biomass for the production of bioproducts [24], including bioethanol. [38] applied a pretreatmenteatment of apple biomass to release sugars for the production of bioethanol. The pretreatmenteatment included heating followed by acidification with citric acid and further enzymatic treatment using CellicCTec®2 (a mixture of cellulase, β-glucosidases, and hemicellulases) and Viscozyme® L (a mixture of beta-glucanases, pectinases, hemicellulases, and xylanases). The apple hydrolysate contained 153 g/L of RS. In contrast, we used only one enzyme, and obtained a similar concentration of of 150.22 mg/L RS. This yield indicates that apple biomass can be used to produce bioethanol. Apple waste is a good source of biomass. Several studies have apple pomace to produce bioethanol. Different strategies have been proposed to improve ethanol production, such as the combination of heating and enzymatic hydrolysis of lignocellulose material [38], removal of lignin using organosolv [23] or direct fermentation of cocultures [24]. However, all strategies to release sugars and further fermentation are still conducted at the laboratory scale, as the final concentration of ethanol is not enough to compete with first generation ethanol production. Therefore, further studies are necessary to improve the production steps.
The hydrolysis of mango biomass produced three times the initial concentration of RS using Pectinex Clear and Saczyme enzymes. The initial RS concentration was approximately 20 mg/mL, and after 180 min of hydrolysis, the value was 59 mg/mL. The sugar released was only 10.7% of the initial mass of mango biomass. These enzymes use different substrates to produce free sugar. Pectinex Clear acts on the (1,4)-alfa-D-galacturonic acid chain, and Sacyzime is a glucoamylase acting on (1,4)- e (1,6)-alfa-D-glucose at the non-reducing ends of polysaccharides. We used both peel and pulp of mango, and the peel comprised 46% cellulose and 28% hemicellulose [22]. However, the enzyme Cellulacast released only 14 mg/mL of RS after 180 min of hydrolysis [22]. They performed an alkaline pretreatmenteatment to remove lignin before the enzymatic reaction with cellulase to release 58 mg/mL of glucose from mango peel, but 24 h were necessary to achieve this concentration. Thus, the use of pulp and peel wastes from mangoes can be a good source to obtain sugars for fermentation, as only 180 min was sufficient to generate a similar concentration.
Two pectin esterases (Pectinex Ultra SP and Pectinex Clear) and glucoamylase (Saczyme) increased the initial concentration of sugars from ~ 50 to ~ 90 g/L in papaya biomass. Papaya peel is rich in pectin, consists of low methyl-esterified, linear Ca2+-cross-linked homogalacturonan with high molar mass pectin, and can be a potential source of this polysaccharide [10]. In addition, the cell-wall of papaya is a good source of pectin, as it constitutes of 35% pectin, 30% cellulose, 30% hemicellulose, and 5% protein [39]. Thus, this polysaccharide could be a source of sugar after the action enzymatic hydrolysis using esterases of pectin. However, we did not observe the release of sugars following hydrolysis with a cellulase (Cellulaclast). Comparing the three enzymes, Pectinex Ultra SP required only 60 min to hydrolyze and to reach the maximum concentration of RS, and both Saczyme and Pectinex Clear required 120 min and 180 min, respectively, to reach the same concentration of RS. [40] crushed papaya waste to produce bioethanol without any pretreatment in a waste:water ratio of 3:1, and the concentration of soluble solids was 4.02%. On the other hand, our enzymatic treatment produced twice this value, as the concentration of RS was approximately 90 mg/mL. Thus, enzymatic treatment can be an alternative to increase the yield of bioethanol production from papaya wastes, as the enzymes Pectinex Ultra SPL, Pectinex Clear, and Celluclast produced high concentrations of RS.
The Pectinex Ultra-SPL enzyme showed high conversion rates in fruit biomass, except for mango. The conversion rate was higher than 5 mg/g/min in the banana biomass at the beginning of hydrolysis. The concentration of reducing sugars increased from 109 mg/L to 268 mg/L in 30 min (Fig. 1). Subsequently, the concentration of RS was stable, and thus, the productivity decreased with time. Pectinex Ultra-SPL is a polygalacturonase, and the simplified mechanism of pectin hydrolysis is shown in Fig. 3. Banana peel is a good source of pectin [41] and its hydrolysis generates sugars for fermentation. In addition, homogalacturonan is the major pectin form in the cell walls of plants [31]. Thus, it is expected that Pectinex Ultra –SPL produces more sugars than other pectin esterases that hydrolyze pectin by the de-esterification of methoxylated galacturonic acid (Pectinex Ultra Pulp and Pectinex Ultra Tropical).
Pectin is usually found in association with other compounds, such as cellulose, lignin, or polyphenols, present in the cell walls of plants [31]. The association with other cell wall compounds s can impair the access of the enzyme to its substrate, and reduce the production of sugars. Some studies [42, 43] have applied an alkaline pretreatmenteatment in mango fruit to decrease the degree of polymerization and crystallinity of the biomass, swell fibers, and disrupt the lignin structure. We preferred the direct application of enzymes as sustainability points to a lower application of chemical treatment in the biomass, but the release of sugars was not high. Mango stem bark is a source of lignocellulosic material as it comprises 46.8% cellulose (estimated as glucan), 28.1% hemicellulose, and 23.2% lignin [43]. However, the Celluclast enzyme (a cellulase) did not release enough sugars for further fermentation, as we used both pulp and peel from mango and did not perform alkaline pretreatmenteatment.
The productivity rate using apple biomass was the second-best for generating RS. The best productivities were achieved by two pectin esterases (Pectinex Ultra SPL and Pectinex Ultra Clear). In fact, pectin esterases are well known enzymes to hydrolyze pectin in the apple matrix as the clarification of apple juice involves the treatment with a mixture of polygalacturonases, pectinase and glucoamylase for 100 min at 55°C followed by ultrafiltration (UF) using tubular membranes of molecular weight cut-off (MWCO) of 100 kDa [44]. Our enzymatic treatment confirmed this finding.
The Ultraflo Max enzyme (an endo-beta-glucanase) also produced reducing sugars, but the hydrolysis rate was lower than that of pectin esterases, as the concentration of homogalacturonan is higher in the fruits than beta-glucans.
Although fruit residues are characterized as biomass composed of cellulose, hemicellulose, and lignin, which can be used for the production of second-generation biofuels, they present some particularities in their chemical composition, which is important for the enzymatic hydrolysis process. Bananas, apples, mangoes and papayas are composed of pectin, fiber, starch, protein and total reducing sugars, such as fructose, sucrose and glucose [45, 23, 46, 47] and each enzyme acts on different substrates.
The hydrolysis of bananas using the enzyme Pectinex Ultra SP-L was the best condition for obtaining RS. Furthermore, the enzyme Pectinex Ultra SP-L was also the best enzyme to hydrolyze apple and papaya biomass. Thus, we performed fermentation with biomass from all fruits. Figure 4 shows the initial, final, and released RS by enzymatic hydrolysis.
3.2 Production of bioethanol from hydrolized fruit biomass
Afterwards, banana, apple, mango, and papaya biomass was hydrolyzed by the enzyme Pectinex Ultra SP-L and subjected to fermentation with two yeast strains to evaluate the production of bioethanol: S. cerevisiae CAT-1 and S. cerevisiae Angel. Figure 5 shows the concentrations of RS and ethanol over the course of the fermentation, and Table 1 shows the ethanol yields and ethanol productivity. In general, banana waste produced higher ethanol concentrations (28–31 g/L) and higher productivity (0.442 g/L.h), and papaya produced low ethanol concentrations (9–12 g/L). However, mango waste showed a higher ethanol yield with regard to sugar consumption.
Both yeasts (S. cerevisae Angel and S. cerevisae CAT-1) used to the fermentation of the hydrolyzed biomass produced ethanol. However, there was no optimal strain for all fruit biomasses. Similar concentrations of ethanol were produced by both strains using banana and mango biomass (Figs. <link rid="fig4">5</link>-a and 5-c). The CAT-1 strain produced more ethanol from apple biomass, and the Angel strain produced more ethanol from papaya biomass. Thus, there was no standard behavior regarding ethanol yield.
The banana biomass produced higher concentrations of ethanol; The initial RS concentration of 151.9 mg/L increased after hydrolysis to 359.08 mg/L. The strain Angel used 97% of RS and produced 31.87 g/L of ethanol whereas the CAT-1 produced 28.02 g/L. The yeast CAT-1 consumed 98% of RS, and most of sugars were used in the first 12 h of fermentation. If the global production of banana is approximately 114 million tons [48, 49] it is estimated that 460 kg of banana per ton is rejected. Therefore, banana waste has the potential to produce 3.3 billion liters of ethanol. A total of 7.1 million tons of bananas were produced in Brazil in 2020 [50], and there is potential to produce 200 million liters of ethanol per year.
[50] used banana waste composed of a mixture of pulp, peel, and pseudostem in a proportion of 1:2:10 to produce ethanol. They used acid and enzymatic processes with the application of cellulose and hemicellulose, and the biomass contained 150 g/L of RS. After the fermentation, similar to our results, the broth contained37.8 g/L of ethanol, but the higher concentration of ethanol was reached after 48 h of fermentation. [48] suggested that the minimum concentration of ethanol should be 40 g/L to reduce the cost of the distillation step. As we used a ratio of 1 g/g (biomass to water) for hydrolysis, we believe that a slight adjustment in the ratio of biomass to water can be done to reach a concentration of 40 g/L, and, thus, reduce de cost.
The hydrolyzed apple biomass produced 20.11 g/L of ethanol using S. cervisae Angel and the consumption of sugars was 96%. On the other hand, the CAT-1 strain produced 15.80 g/L of ethanol, and the consumption of RS was only 76%. Thus, the performance of different strains of S. cerevisae can vary for the same substrate, and different ethanol yields can be obtained. [38] studied apple pomace as a biomass to produce bioethanol. After pretreatmenteatment by heating and enzymatic hydrolysis of lignocellulose material, the consumption of RS was 84% and the ethanol concentration was 51 g/L after 72 h of fermentation by S. cerevisae Ethanol Red® with an yield of 0.398 g of ethanol for each gram of sugar consumed. However, the xylose released during the hydrolysis of lignocellulosic biomass cannot be used by S. cerevisae; therefore, some studies have focused on the application of cocultures using S. cerevisiae and filamentous fungi such as Trichoderma and Aspergillus species to produce bioethanol [24]. In addition, approximately 10% of the fermented sugars are consumed by the microorganisms for cell mass synthesis, yeast maintenance, and side reactions that produce lactic acid, acetic acid, and glycerol [51]. Thus, the search for high productivity and high ethanol concentration must continue, as different fruit wastes can be used as substrates, and different microorganisms can be used for fermentation.
The fermentation of hydrolisated mango fruit by both strains of S. cerevisiae produced approximately 15 and 16 g/L of ethanol, and the RS consumption was 91–92%. Several studies have been conducted to produce bioethanol from mango waste. [52] produced a fermented broth by S. cerevisae with 15% (v/v) of ethanol using mango waste. [43] dried mango stem bark residues and applied alkaline pretreatment to the waste. After fermentation with S. cerevisiae using simultaneous saccharification and fermentation, the authors reported an ethanol concentration of 43 g/L and a productivity of 0.914 g/L/h. [53] supplemented mango waste with leachate from vermicomposting to add nutrients to the broth and produced 44 g/L of ethanol after fermentation. We did not add any nutrient supplements as we focused on the application of fruit waste without the addition of chemicals, but the enzyme treatment.
Hydrolyzed papaya waste produced the lowest concentration of ethanol. S. cerevisiae CAT-1 consumed 95% of RS, but the concentration of ethanol in the fermented broth was only 9.32 g/L. S cerevisiae Angel consumed 95% of RS and produced 12.40 g/L of ethanol. [40] tested the direct fermentation of several fruit wastes, such as banana, apple, papaya, and orange, to produce bioethanol. The authors identified that banana has a higher concentration of soluble solids (10.32 Brix) and produced the highest concentration of ethanol (12% v/v). However, using chromatography, the authors identified several co-generated products, including acetaldehyde, acetone, methanol, ethyl acetate, propanol, isobutanol, isoamyl acetate, and isoamyl alcohol. Acetaldehyde is a byproduct of fermentation and can be used to generate high alcohols, such as propanol, isobutanol, and isoamyl alcohol.