Alcoholic Fermentation of Tomato Canning Waste (TCW) from the Adrar Region in the Southwest of Algeria

1. Introduction

Organic waste recovery as a viable sustainability practice for waste management and renewable energy production. Organic waste, comprising food scraps, yard waste, and agricultural byproducts, holds potential as a resource for energy generation and valuable product creation through methods such as composting, anaerobic digestion (AD), and pyrolysis. The recovery of organic waste is sustainable due to its ability to reduce greenhouse gas emissions (GHGs), particularly methane (CH₄) from landfill decomposition, and to serve as a renewable energy source that diminishes dependence on fossil fuels. Additionally, organic waste recovery promotes resource recovery within a circular economy by converting waste into compost and biofertilizers, enhancing soil health and reducing chemical fertilizer usage. It also reduces the burden on landfills, mitigating associated pollution and conserving valuable space. Economic advantages, including new revenue streams and job creation, further support its implementation [1]. In particular, organic agricultural waste presents a significant environmental challenge in the agricultural sector, highlighting the importance of valorizing this waste biomass for bioenergy production [2].

1.1. Tomato Waste

In 2021, the global tomato production reached 189 million metric tons exhibiting a growing trend (+4% compared to 2017), cultivated on a total area of 4.84 million hectares dedicated to this crop [3]. In the Adrar region (Southwest of Algeria), the cultivation of tomatoes (Solanum lycopersicum L.) has been introduced since 1970. Its production has experienced significant growth due to the expansion of cultivated areas, which increased from 50 hectares in 1970 to 1140 hectares in 2019 [4]. The composition of the tomato is influenced by the climate (humid or dry), temperature, soil type, agricultural practices, and the timing of planting and harvesting. Tomatoes in the Adrar province are of an early nature: they are planted outdoors in late August without the need for a greenhouse, and they are harvested in December. According to statistics from the Agricultural Services Directorate of Adrar for the year 2019, c.a. 693 tons of tomatoes produced in the Adrar region were destined for processing [5]. According to Shrestha et al., [6], for every kilogram of tomatoes processed, 40 g of by-products are generated, with 20 g being waste and 20 g being tomato pomace. Furthermore, del Valle et al., [7] reported that up to 4% of the total weight of processed tomatoes could be rejected after the canning process. The solid residue that remains after processing tomatoes to create tomato juice, paste, sauce, puree, or ketchup is commonly known as tomato canning waste (TCW) [8]. The dry pomace contains 44% seeds and 56% pulp and skin, while the wet pomace has 33% seeds, 27% skin, and 40% pulp [9]. TCW's chemical components differ according to the sample that is being examined. However, it frequently falls within these ranges: 12–23% protein, 5–20% fat, 4–6% ash, 27–32% cellulose, 5–18% hemicellulose, 11–26% simple sugars, 7.6% pectin, and 31% lignin [10,11,12]. Additionally, tomato waste contains some mineral salts, including smaller concentrations of iron, manganese, and copper (15–30 mg/kg) with high levels of calcium, potassium (about 7–11 g/kg), magnesium, sodium, and phosphorus (around 2-3 g/kg) [13].Tomato waste has previously been exploited as animal feed [14]. However, a research carried out by Heuzé et al. [15] in 2015 has demonstrated that the use of organic tomato waste as livestock feed is not beneficial due to its limited nutritional value. Locally, despite the large tomato production, around 90% of TCW in the Adrar region is thrown away untreated, with harmful consequences for the environment: infiltration of liquids into the soil, risk of groundwater pollution, foul odors and GHGs. In practice, there are a number of ways to treat organic waste, including AD, alcoholic fermentation (AF), composting, and incineration [16,17]. For the TCW, classic incineration is seen as an unprofitable and polluting method. Utilizing these waste materials as compost for fertilization is also considered unprofitable because TCW is characterized as acidic, which may require additional pre-treatments and post-treatments (such as grinding, mixing with other substances, sorting, etc.) [18,19].

A number of researches are currently focusing on the energy recovery from agricultural and agro-industrial wastes to produce biofuels [20,21]. AF to produce bioethanol is widely employed and can serve as a treatment solution for this particular agro-industrial waste, TCW. This process is utilized in the agri-food industry for fermented beverages such as wine, beer, and so on. It is also used in industrial ethanol production, particularly as fuel or a basic raw material in the chemical industry [22].

1.2. State of the Art Alcoholic Fermentation

Recent studies on AF of tomato waste focuses on improving fermentation conditions to increase ethanol production while efficiently using tomato by-products. Recent research has proven a variety of procedures and settings that enhance ethanol generation from tomato pulp and other waste sources. According to studies, the best fermenting conditions for tomato pulp include a sugar content of 230 g/L and an inoculum of 5 g/L, which yields roughly 101.5 g/L of ethanol with 99.5% efficiency [23,24]. Another study found that the fermentation process had sluggish kinetics, with maximal substrate consumption rates of 4.545 g/(L·h) and ethanol productivity of 1.39 g/(L·h). Further research has shown that even tomato waste could be converted to bioethanol, with maximum ethanol concentrations of 0.17% under the right conditions [25].

The AF of tomato waste is affected by many critical parameters, including inoculum, temperature, initial sugar concentration and pH levels. These factors significantly impact final product quality, including overall alcohol content, flavor profile and other sensorial characteristics. The ideal ratio of inoculum for fermentation is generally around 5% w/w [26]. Increasing inoculum concentrations may promote faster fermentation, but not necessarily improving final product quality [27]. For fermentation temperatures, the best results are achieved between 22 °C and 34.5 °C, since specific studies indicate that 31°C is an effective temperature for maximizing viable cell numbers [28]. As far as sugar is a key factor in alcohol yield, higher concentrations can increase ethanol production. Research confirms that sugar levels between 8.8% and 14.4% provide an optimum alcohol content of around 11%. In fact, tomato waste juice must be kept at a pH of around 3.7 to optimize fermentation conditions. However, optimizing these factors can improve bioalcohol quality from tomato waste, but variations in feedstock quality, such as tomato ripeness, can also lead to significant differences in the final product, suggesting that not all factors can be controlled or predicted [29].

1.3. Alcoholic Fermentation Principles

In this metabolism, organic molecules play the role of the final electron acceptor. Nicotinamide adenine dinucleotide, or NAD+, is utilized initially by yeast as an intermediate electron acceptor before being reduced to NADH (reduced nicotinamide adenine dinucleotide). The final reduction of acetaldehyde to CO₂ and ethanol contributes to the preservation of the redox equilibrium. The NADH generated during glycolysis process, the principal mechanism for metabolising sugars to pyruvate, plays a key role in the energy balance of this conversion, as described by Equation (1):

C₆H₁₂O₆ + 2 Pi + 2 ADP → 2 C₂H₆O + 2 CO₂ + 2 ATP

(1)

The nature of the acid is of great importance, as each yeast species requires an optimal pH for its growth. The acidic pH of TCW is detrimental to bacterial development but favorable for yeast and mold proliferation [30,31]. The optimal pH for yeast growth is typically between 4.3 and 4.7 [32], with Saccharomyces cerevisiae yeast having the advantage of multiplying in an acidic environment, with the optimum pH range between 4 and 4.5 [33].

Maintaining cytoplasmic pH is essential for yeast survival. The literature generally reports pH values between 2.8 and 8, with 4.5 and 6.5 being the ideal pH range [33,34].

1.4. Scope and Outline

Given this background, this research article aims to valorize, for the first time, tomato waste (skins, seeds, and peels) generated by the "Fouggara" tomato cannery in Reggane, Adrar, in South-Western Algeria, under Saharan climatic conditions, as a substrate for bioethanol production. The experimental study involves the coupled thermal and chemical pre-treatment of this substrate to facilitate alcoholic fermentation at laboratory scale. This paper provides a detailed characterization and experimental data. Since tomato waste is an abundant biowaste in many areas, the information from this paper enables the evaluation of the feasibility of similar processes by many stakeholders, as well as the validation of integrated process models.

The paper is organized into 3 other sections; Materials and Methods (section 2) providing detailed descriptions of the substrate preparation and the experimental equipment, Results and Discussion (Section 3) presenting the experimental findings obtained on the specific feedstock and a comparison with commercial references, and, finally, Conclusions (section 4).

2. Materials and Methods

2.1. The Substrate

The substrate under investigation consists of waste collected from the "Fouggara" tomato cannery (briefly referred to as TCW), located in Reggane, Southwest of the city of Adrar, in the Southwestern region of Algeria (Figure 1a). This substrate is composed of peels, seeds, and skins (Figure 1b). To prevent aerobic degradation of the substrate and microbial contamination, a drying process is carried out immediately after collection using an indirect solar dryer (Figure 1c). The measured moisture content after drying is < 7%. Subsequently, the dried TCW is crushed using a Janke and Kunkel micro-mill to obtain particles smaller than 500 μm.

The microorganism employed for the AF is the dry industrial baker's yeast, Saccharomyces cerevisiae (Figure 2) [35].

2.2. Experimental Methodology for Alcoholic Fermentation

2.2.1. Samples Preparation

For the production of bio-alcohol through AF, 1-liter glass flask fermenters with 700 mL effective volume are employed and sealed airtight. These fermenters have 2 outlets: one for sampling and the other connected to a flask for the recovery of the produced gas, enabling the measurement of the carbon dioxide (CO₂) released. The movement of CO₂ bubbles released contributes to stirring the mixture, thus promoting AF. The reactors are incubated in a temperature-controlled water bath at 30 ± 1 °C [36].

TCW is mixed with water, in 4 different concentrations, namely 50, 75, 100, and 150 g/L. These mixes are tested in glass bioreactors to determine the optimal TCW concentration resulting in the best bioethanol production, see Figure 3.

2.2.2. Substrate Pretreatment

For the pretreatment of TCW, 2 methods are used:

• Steam explosion: to initiate the experiment, the 4 bioreactors are filled with 35, 52.5, 70, and 105 g of substrate and topped up with tap water to reach a total volume of 700 mL. The mixture undergoes thermal pretreatment in an autoclave at 140 °C and 2.5 bar for 40 minutes with 3 depressurization cycles to ensure the effective cracking of lignocellulosic bonds.
• Acid hydrolysis: following steam explosion, concentrated sulfuric acid (H₂SO₄) is added to the bioreactors in an amount equivalent to 5% of the working volume (3.5 mL in 700 mL). The treated bioreactors are then reintroduced into the autoclave for 50 minutes at 90 °C to break polysaccharide bonds and produce monomers [37]. Subsequently, to eliminate phenolic compounds that inhibit microbial activity during fermentation, calcium hydroxide (Ca(OH)₂) is added.

2.2.3. pH Adjustment

To ensure yeast proliferation and inhibit bacterial growth, the pH of the TCW should be maintained within the range of 4.3–4.7. To adjust the pH, some concentrated sulfuric acid was added.

2.3. Alcoholic Fermentation and Distillation

Sugar is necessary to reactivate the yeast, therefore 8.4 g of sugar are mixed with 0.7 grams of Saccharomyces cerevisiae yeast. Distilled water is then added to make up the volume to 100 mL. This solution is incubated in a preheated oven for 30 minutes at 30 °C.

The reactivated yeast is added to the previously adjusted bioreactors. They are incubated under anaerobic conditions in a water bath at 30 °C for 72 hours. All digesters are processed in triplicate and the results have been shown as means.

Using a 1-liter distillation column system, the fermented TCW is distilled to extract bioethanol. This system is heated using a round-bottom flask heater to the boiling temperature of ethanol at 78 °C [38,39]. The temperature is controlled at the top of the column. Vapors are taken to reflux to increase the ethanol concentration, and then they are subsequently condensed using a condenser, and the produced ethanol is collected in a glass flask.

2.4. Measurements: Targets and Equipment

AF is used to upgrade TCW for the bioethanol production process. Parameters such as ash content, protein, pH, reducing sugars, density, refractive index, Brix degree, and alcohol content during the 72 hours of AF must be monitored:

• Ash: ash content represents the total mass of mineral salts present in a fruit is calculated according to the American Society for Testing and Materials' (ASTM) standard methodology ASTM-E-1755-01 (2020) (Oxidation at 575± 25 °C standard test method for ash in biomass) [40].
• Proteins: to assess the protein content, the total nitrogen content is determined. Nitrogen is essential for protein synthesis, which plays a role in transporting sugars into the cell during AF, where they are converted into bioethanol. The protein content is determined with the Kjeldahl method, which measures total nitrogen, essential for assessing protein content during fermentation processes [41].
• pH: it is determined with a pH meter (Mettler-Toledo AG, Analytical CH-8603 Schwerzenbach, Switzerland).
• Reducing sugars: the reducing sugars are measured as prescribed in the method of Dubois and Cool (1956) [42]
• Density Density measurements are performed at 20 °C by laboratory density meter based on oscillation (OIV-MA-AS2-01A:R2012)
• CO₂: the volume of CO₂ generated is measured using the liquid displacement method
• Refractive index: it is a dimensionless quantity characteristic of a medium, describing the behavior of light in that medium. An Abbé refractometer (Standard NF ISO 2173: 2003) is used to measure refractive index.
• Brix degree/soluble solids: the total soluble solids, expressed as °Brix %, are measured from homogeneous filtered fruit juice using an Abbé refractometer. (Standard NF ISO 2173: 2003).
• Alcohol content: an alcoholmeter with a range of 0-100° (standard OIV-MA-AS312-01A: R2009) is used to measure alcoholic content.
• Yeast evolution: yeast proliferation under a microscope over the course of 72 hours of fermentation is measured. The evolution of the yeast is monitored by hemocytometric counting (Malassez Cell) of the microbial population under an optical microscope type (OPTIKA B-350, G: 100×).
• For sampling from the flasks, as shown in Figure 3, there are 2 holes, one for CO₂ removal and the other for sampling. The sample volume (after mixing and homogenization) does not exceed 3 mL, which has no influence on the total reactor volume.
• Finally, bioethanol is characterized using Agilent Cary 660 Fourier Transform Infrared (FT-IR) infrared spectrometer. Chromatographic analyses were carried out on an electronically pressure-controlled PerkinElmer Gas Chromatography (GC) (Clarus 500 series). The instrument is controlled by a “Total Chrom Tutorial, version 6.3” computer system.

3. Results and Discussion

The results of alcoholic fermentaion are presented here, from the fermentation process to bioethanol extraction.

3.1. Fermentation Parameters

Figure 4 portrays the trends of measured parameters during the 72-h test on TCW. All parameters are discussed in the following sub-paragraphs.

3.1.1. Ash Content

The value found for the TCW is 3.57% of Total Solids (TS), which is close to that found by Sarada et al., [43], with 4.55%. This high value explains the richness of tomato waste in mineral elements. According to Figure 4a, the ash values of TCW during AF decreased from 3.57% to 2.41%. This reduction in ash content during AF is the result of the mineral salts consumed by the yeast during the sugar degradation process.

Figure 4. Evolution of the substrate over 72-h fermentation: (a) ash, (b) proteins, (c) reducing sugars, (d) cells count, (e) PH, (f) refractive index, (g) density and CO₂, (h) Brix index.

Figure 4. Evolution of the substrate over 72-h fermentation: (a) ash, (b) proteins, (c) reducing sugars, (d) cells count, (e) PH, (f) refractive index, (g) density and CO₂, (h) Brix index.

3.1.2. Proteins

The total nitrogen content in TCW is 5.5%, and the protein content is estimated at 34.4%. These values are higher than those found by Persia et al. [44] and Seddiki [45]. In the study by Persia et al. (2003), the nutritional profile of dried tomato seeds is evaluated, highlighting their potential as a valuable feed ingredient. The research focuses on the chemical composition, including protein, fat, and fiber content, and assesses the seeds' overall nutritional value for poultry diets. Similarly, the study carried out by Seddiki focuses on the valorization of tomato by-products, emphasizing their potential in enhancing food security and sustainability. The research highlights innovative methods for utilizing these by-products, which are often discarded, to create value-added products (protein 24.5%).

This protein content will be used as a source of nitrogenous materials in organic or inorganic form, which is essential during yeast multiplication to meet the nitrogen requirements for the formation of amino acids, nucleotides, and certain vitamins [46]. Yeasts are capable of utilizing inorganic nitrogen in the form of ammonium ions (chloride, nitrate, phosphate, and notably sulfate, which is the ideal compound as it provides the sulfur necessary for the synthesis of certain amino acids) [47].

According to Figure 4b, it can be observed that the protein content decreases during AF. This decrease is likely a result of yeast assimilating the proteins to complete the AF.

3.1.3. Reducing Sugar Content

The levels of reducing sugars are shown in Figure 4c. The results obtained show a significant decrease during the AF process, where the sugar content drops from 5.24 to 2.02 mg/L at the end of the AF. This result agrees with the reported findings by [48]. This decrease is likely due to biomass production and sugar assimilation. Furthermore, yeast uses sugars to generate energy and support cell growth. They are capable of converting glucose into ethanol and CO₂, with the coproduction of glycerol, esters, and certain acids [48,49,50].

3.1.4. Cell Count

Yeast proliferation under a microscope over the course of 72 hours of fermentation was monitored, and the results are shown in Figure 4d. The curve indicates that the stages of Saccharomyces cerevisiae proliferation over time are typical of those of all microorganisms. This shows a conventional growth curve with a latent phase, an increasing phase, an exponential phase, a stationary phase, and a declining phase, during which the high ethanol content inhibits yeast proliferation [51]. To confirm these results, a comparison is made with this curve to the MONOD model [52], a similarity in the following notable 5 distinct phases was observed:

• Phase 1 (0-12h): Latency phase during which cells synthesize the enzymes needed to metabolize the substrate present. Cell reproduction is nearly non-existent during this phase.
• Phase 2 (12-24h): Acceleration phase where actual growth initiation occurs.
• Phase 3 (24-36h): Exponential growth phase with cell reproduction reaching its maximum at a constant rate. This is due to the richness of TCW in nutrients (mineral compounds, vitamins, carbon, and nitrogen compounds), providing an energy source [53]. Additionally, the nitrogen source is significant because it contributes to the structure of nucleic acids and proteins, important constituents of Saccharomyces cerevisiae cells [54].
• Phase 4 (36-60h): Stationary phase with cell concentration remaining relatively constant, indicating cessation of cell reproduction. This is a sign of exhaustion of nutritional sources, namely glucose, and accumulation of inhibitory products resulting from microbial metabolism, where ethanol concentration increases in the medium (Figure 12). Growth rate, metabolic activity, cell viability, and yeast production capacity are all declining. Canetta et al., [55] confirmed these phenomena in their work, especially in Saccharomyces cerevisiae.
• Phase 5 (60-72h): Decline phase with a decrease in cell mass due to autolysis and a negative growth rate. This is attributed to ethanol causing stress in yeast and becoming toxic at high concentrations. These findings have been validated by several researchers, particularly in Saccharomyces cerevisiae [54,55,56,57].

3.1.5. pH Evolution

Figure 4e shows a decrease in the pH of TCW. This pH decrease is likely due to sugar consumption and bioethanol production, as well as the acids and alcohols metabolized by microorganisms (yeasts) present in the reaction mixture, such as fatty acids, especially octanoic and decanoic acids. Furthermore, this decrease in intracellular pH can be due to either proton influx or the accumulation of reaction intermediates such as acetic acid and glycerol [58]. Additionally, some of the CO₂ produced dissolves in the mixture, contributing to the lowering of pH. This result shows that yeast is able to tolerate numerous organic acids up to pH 4.5. Nevertheless, lactic, citric, and acetic acids significantly inhibit yeast growth, as well as sorbic and propionic acids to a greater extent [53]. There are other factors that can affect pH, as described by Jones et al., [59], in which ethanol stress leads to a decrease in cytoplasmic pH, resulting in yeast death.

3.1.6. Refractive Index

The refractive index values decrease from 1.3412 to 1.3354 during fermentation (Figure 4f). This decrease can be explained by the increase in the speed of light during fermentation, which is directly related to the decrease in density [36].

3.1.7. Density

According to the results presented in Figure 4g, a significant decrease in density was observed. Density values are recorded after every 24 hours, showing a mildly decreasing trend from 1.14 to 1.03 kg/L. During the fermentation process, the loss of mass in the form of CO₂ and the conversion of glucose into alcohol account for this fall in density [60].

3.1.8. Soluble Solids Content

According to the presented results in Figure 4h, a decrease in soluble solids content is observed, primarily due to the reduction in sucrose concentration in the fermented solution, expressed in degrees Brix (°Brix). This indicator decreases from 5.5 °Brix to 1.8 °Brix.

3.1.9. Alcohol Degree

At the beginning of fermentation, the alcohol content is zero. However, after 24 hours, a value of 2° is recorded. Alcohol production becomes significant during the last 48 hours, increasing from 6° to 12° at the end of fermentation (after 72 hours). Similar observations were reported by El Ogaïdi [61], who estimated a fermentation time between 36 and 72 hours. Table 1 illustrates the changes in the appearance of the fermented mixture over time, together with the increase of the alcoholic degree. The results obtained have shown that TCW produces ethanol through AF, with a final alcohol content of 12°.

3.2. Characterization of the "Bioethanol" Fermentation Product

The results of the physicochemical properties of the bioethanol produced from TCW are presented in Table 2, in comparison of bioethanol produced from another feedstock (potato waste [62]) and the commercial reference.

The characteristics of the laboratory-produced bioethanol are similar to those of commercial ethanol. Comparatively, the density of the ethanol solution produced from TCW is 0.886 kg/L, whereas that of commercial ethanol is 0.789 kg/L. Laboratory-produced bioethanol (Figure 5) exhibits the following characteristics: volatility, flammability, clarity, and a pungent odor.

3.2.1. Characterization of Bioethanol Obtained by IR Spectrophotometry

Figure 6 represents the vibrations of the bands obtained by infrared spectrophotometry of the bioethanol produced by AF of TCW. The infrared spectrum of the final product obtained through fermentation after distillation is characterized by the vibrations of the following bands: 2977 cm^-1 and 3341 cm^-1, corresponding respectively to the C-H groups of an alkane and the specific O-H group of an alcohol. A comparison between the infrared spectra of ethanol produced from TCW and that of commercial ethanol shows agreement, with vibration bands of different groups appearing at the same frequency. Boulal et al., [23] obtained identical findings.

A comparison between the GC spectra of pure ethanol and the bioethanol produced from tomato waste (Figure 7) shows that the peaks of the ethanol molecule in the GC spectrum are visible at a retention time of 0.9. The second peak appearing is likely due to impurities in the bioethanol produced. This observation aligns with the findings of Boulal et al., [23].

4. Conclusions

In this study, the solid residue that remains after processing tomatoes to create tomato juice, paste, sauce, puree, or ketchup commonly known as tomato canning waste was used for the first time as a feedstock for bioethanol production by alcoholic fermentation using thermochemical pretreatment. TCW was obtained from the "Fouggara" tomato canning factory in Reggane/Adrar, South-West Algeria.

The obtained results are very promising and show that the bioethanol produced is characterized by an alcohol content of 12° over a process period of 72 hours with a density of 0.886 kg/L, close to that of commercial ethanol at 0.789 kg/L. In addition, its characteristics are very close to those of commercial ethanol. Several parameters were involved to obtain these results, namely the acid pH of the TCW, which favors the growth of the Saccharomyces cerevisiae yeast used in the fermentation process. The evolution of this yeast over time follows a curve typical of the MONOD model, with phases of latency, acceleration, exponential growth, a stationary phase, and a decline phase. During the stationary and decline phases, the high ethanol content inhibits yeast proliferation. The density also decreases from 1.14 to 1.03 kg/L due to the significant release of carbon dioxide (CO₂) during AF, resulting from the transformation of glucose into alcohol and the loss of mass in the form of CO₂. Reducing sugars dropped from 5.24 to 2.02 mg/L at the end of fermentation due to the biomass production and sugar assimilation. Since the results are very interesting, further research to this scope could improve the quantity and quality of the produced bioethanol to reach sustainable development goals.