1. Introduction
Biomass valorization is the process of converting biomass into highly useful materials including fuels and chemicals. Many valorization methods have shown significant promise in meeting various industrial demands. In recent years, interest has increased in recycling various types of waste, including food waste, due to large-scale generation and the application of organic substances to numerous uses, such as, biogas, animal feed, and compost [
1]. The FAO estimated that about one-third of food generated for human consumption was wasted or lost globally, and the average waste generation was about 1.3 billion tons per year [
2]. The amount of generated waste varies widely among the human population based on the types of food used for consumption. Food waste generation has increased continuously and waste accounts for 32% of the total food produced globally [
3]. Food waste in the global food supply chain has been extensively analyzed by food supply chain experts. The global food supply chain varies widely depending on types of food waste, post-harvest waste, and behavioral changes of consumers [
4]. In European Union, food waste generation increased every year and it changed in each stage of food supply chain. At household level, 25% of food waste generated in the food supply chain and this percentage increased as 40% during postharvest and processing stage. In food supply chain another 40% food waste generated during the retail and consumer levels [
3]. In Spain and, more broadly, Europe, the food industry is a major industrial sector and its generated waste is aerobically digested for biogas production [
5]. In North America, including United States, Mexico, and Canada, an estimated food loss and waste was about 170 million tons. In the United States of America, the estimated food loss and waste was 30–50% based on the types of food waste generated. In India, the annual production of food and vegetable wastes reaches 5.6 million tons. Vegetable and fruit wastes contain simple carbohydrates, and high solid and moisture contents increase the availability of nutrients to microorganisms. Depending on the characteristics of food and vegetable waste and on the existing market demand, the most relevant valorization options are production of exopolysaccharides, enzymes, extraction of various bioactive compounds, synthesis of biopolymers, bioplastics, and production of biofuels [
6]. Food waste is one of the major environmental problems, with waste and losses generated at every stage of the food supply chain. There are many waste management methods for the safe disposal of food waste, however these have various problems such as environmental pollution, toxic by-products, and high costs. Recently, the pyrolysis method has been recommended for the utilization of food waste for the production of novel products. This method is used for the production of biochar, syngas, and bio-oil [
7]. Food supply chain begins with the agriculture and livestock sector, which generated various by-products. This early stage of supply chain produces food waste and food loss in the form of very low-quality products and products with no commercial value. Food waste generation continues even at the final stage (bad storage or preservation). These substrates are highly suitable to be treated by anaerobic digestion because of high moisture content and solid and simple carbohydrates [
8]. Numerous organic waste recycling methods have been reported, however, none of these methods can eliminate the whole food waste problem in modern cities. In urbanized areas, high transportation costs, scattered food waste generation sources, and low selling prices of various regenerated products are impediments to the waste recycling process [
9]. Hence, the generation of high value-added products from food wastes is highly desirable.
Food waste is produced during food processing, production, retail, wholesale, and consumption. Food waste consists of 5–10% proteins, 10–40% lipids (
w/w), and 30–60% starch [
2,
10]. The use of food waste as a feedstock in fuel, material, and chemical production has been proposed and demonstrated to eliminate nutrient-rich food waste [
11]. Food waste is used in generic fermentation, and by fungi such as
Aspergillus oryzae and
Aspergillus awamori in submerged fermentation [
12]. Fungal hydrolysis has a large number of social and environmental advantages including no unpleasant smell or air pollution and low energy intensity compared to the traditional method food waste recycling process. This process is useful for the production of various bio-based value-added products. In addition, it allows recycling of various types of food wastes using hydrolysate as a feedstock for the fermentation process. Pleissner et al. [
13] used
Halomonas boliviensis for the production of polyhydroxybutyrate. Mixed food waste hydrolysate has also been used as the nutrient medium for the cultivation of
Chlorella pyrenoidosa for algal biomass production [
9]. Generally, the low concentration of end products and prolonged fermentation was not advantageous. However, Kwan et al. [
14] reported that the production of lactic acid from food waste as a bulking agent was profitable due to the high lactic acid yield and short fermentation time. Lactic acid has various uses in the beverage and food sector, in addition to the chemical and pharmaceutical industries, and its polymerization abilities are an advantage in the formulation of polymer poly(lactic acid) [
15]. Solid-state fermentation has been performed using starter culture with the addition of enzymes [
16], a single microorganism [
17], or isolated indigenous bacterial/fungal strains [
18].
Lactic acid can be derived from various natural sugars and applied in the production of many value-added products and various chemicals [
19]. Food waste (FW) contains simple sugars, which have significant potential for use as a simple medium for lactic acid production. The production of poly(lactic acid) by lactic acid contributes about 35% of the total bioplastic market due to its favorable material performance and eco-friendly approach [
20]. Lactide is an intermediate product of poly(lactic acid) with numerous applications as surfactants, printing toners, coatings, adhesives, and polymer additives [
21]. In recent years, these two products have been derived from corn and sugar beet [
22]. Food wastes such as brewer’s spent grains, wheat bran, corn stalks, coffee mucilage, whey, and kitchen waste have been used as the feedstock for the production of lactic acid [
23]. Bioconversion processes, such as direct fermentation, open fermentation, simultaneous fermentation and hydrolysis, and fermentation and enzymatic hydrolysis have been reported using various lactic-acid-producing bacteria [
17]. Food wastes are a mixture of various residues, and the heterogeneity of the biomass leads to uncertain results when increasing the scale of production of lactic acid. Several investigations have shown that production of lactic acid can be achieved via fermentation of food waste using various microbial consortia at higher temperatures [
24]. Most previous studies have reported low yields of lactic acid. However, the acidification and hydrolysis processes have been improved by synergistic properties of various microorganisms. Recently, co-fermentation of food waste with wastewater sludge was used for the production of lactic acid and achieved lactic acid stabilization at room temperature and at alkaline pH [
25]. To reduce the production cost of lactic acid, various types of biomass, such as potato peel, fruit and vegetable wastes, and municipal solid waste, have been used to improve the yield [
26,
27,
28]. Due to its high yield and organic content, food waste has been used as a suitable substrate for lactic acid production [
29]. The anaerobic process has four steps: Hydrolysis, acidogenesis, acetogenesis, and methanogenesis. Lactic acid production is achieved by acidogenesis and hydrolysis. Process parameters such as pH, inoculums, C/N ratio, substrate, and temperature significantly influence lactic acid production [
28,
29]. An acidic pH range (4–5) effectively promotes the hydrolysis of various food wastes for the production of lactic acid [
28]. In the present investigation, the impact of environmental and nutrient factors on lactic acid fermentation in a batch bioreactor was studied. Furthermore, the influence of lactic acid on soil characteristics was analyzed.
2. Materials and Methods
2.1. Substrate
Food waste (FW) was obtained from municipal food wastes at Riyadh, Saudi Arabia. The food wastes consisted of meat, chicken bone, bread, egg, lobster shell, and rice. It was crushed with a mechanical blender into small pieces to enhance composting after the manual separation of bones from the FW using forceps. The final substrate slurry was filtered using a sieve with 1 mm thickness and stored at 4 °C in a refrigerator. Municipal sludge (MS) was collected from a wastewater treatment facility at Riyadh. The FW and MS mixture was completely sterilized for 30 min at 121 °C to eliminate indigenous microbial flora from the food and sludge. The prepared slurry was applied to the reactors for the fermentation process. The physico-chemical properties of the substrate were analyzed.
2.2. Characterization of Lactobacillus Strain
Lactobacilli were isolated from the date effluent. Wastewater was collected from a date processing facility at Riyadh, Saudi Arabia. The sample was serially diluted and plated on Man–Rogosa–Sharpe (MRS) agar (Himedia, Mumbai, India) medium. The morphology of the bacterial isolate was studied by incubating on MRS agar medium for 48 h. Gram-staining and biochemical characteristics were studied. To identify the strain in molecular level using forward and reverse primers, 16S rDNA sequence analysis was performed [
30].
2.3. Batch Fermentation
Municipal solid sludge was previously collected from the wastewater treatment facility at Riyadh, Saudi Arabia. After the sample was incubated for 24 h at room temperature, the clear supernatant was discarded. Food waste and the concentrated sludge were mixed in a ratio of 4:1 based on volatile suspended solids. Tap water was added to maintain total COD at 44,864 ± 376 mg/L. Batch fermentation was carried out in the reactors and the working volume was fixed at 1 L. The schematic presentation of a batch fermentor is provided in the
Supplementary Figure S1. The inoculum was prepared by culturing
Lactobacillus strains in MRS broth by incubating at 37 °C under shaking condition (150 rpm for 18 h). It was stirred at 120 rpm at 35 °C. The pH of the culture medium was adjusted to be 7.0 using 5 M hydrochloric acid or 5 M sodium hydroxide [
31]. The batch fermentation experiment was performed for 72 h. A 2.5 L vessel was equipped with in- and outflow of filtered air, alkali, and inoculum ports. Culture medium was pumped into the fermentor up to a volume of 1 L. To control the pH of the medium, alkali was added continuously. The medium was mixed continuously with the use of a blade. Sampling was conducted using a sample hole. The vessel was agitated with a constant speed (220 rpm). Then, lactic acid production (g/L), total sugar (g/L), consumption of substrate, and biomass (g/L) was analyzed. Lactic acid content was analyzed using HPLC (Shimadzu Corporation, Tokyo, Japan) using a Shim-pack Fast-OA column. The chromatography column was operated at pH 2.2 and 0.005 N H
2SO
4 was used as the mobile phase. The column temperature was set as 30 °C and the flow rate was adjusted to 0.8 mL/min. A refractive index was used to determine the compound. The amount of carbohydrate content was evaluated using the phenol–sulfuric acid method as described by Dubois et al. [
32]. Reduction of the sugar level of the fermented medium was performed by 3,5-dinitro salicylic acid method [
33]. Briefly, the reagent was prepared by mixing 3,5-dinitro salicylic acid (0.63%), Rochelle salts (18.2%), phenol (0.5%), sodium hydroxide (0.5%), and sodium bisulfite (0.5%). To this reagent, 0.1 mL sample was added and kept in a boiling water bath for 5 min. The sample was cooled, and the absorbance was read at 540 nm using a UV-vis spectrophotometer.
2.4. Monitoring Lactic Acid Bacteria
The viability of lactic acid bacteria (LAB) was analyzed during the fermentation process using Man–Rogosa–Sharpe (MRS) agar medium. This medium consists of (g/L) 5 g yeast extract, 10 g beef extract, 20 g peptone, 20 g glucose, 14 g agar, 2 g CaCO3, 0.25 g MnSO4·4H2O, 0.58 g MgSO4·7H2O, 5 g CH3COONa, 2 g diammonium citrate, 2 g K2HPO4, and 1 mL Tween-80. The pH of the culture medium was adjusted to 6.5 ± 0.2. The medium was sterilized for 15 min at 121 °C. Samples were withdrawn periodically from the fermentor and overlaid with the MRS agar medium. Plates were incubated for 48 h at 37 °C in a temperature-controlled incubator. Experiments were performed in triplicate and average cell counts were considered for analysis.
2.5. Optimum FW and MS Sample
FW and MS samples were mixed at various proportions to determine the optimum concentration for lactic acid production. Six different ratios of FW and MS samples (0:1, 1:0, 0.5:1; 1:0.5, 1:1, 1:1.5, 1.5:1, and 2.0:5) were selected based on the dry weight of the sample. The culture medium was not supplemented with any other nutrients, pH maintenance, or autoclaving. About 10% (v/v) inoculum was applied for batch fermentation. The saccharification process was initiated by the addition of amylase and cellulases. After the addition of these enzymes, the fermentation process was started. The sample was stirred at 150 rpm and incubated at 37 °C for two days. Each experiment was performed in duplicate and an average value was used for data processing.
2.6. Effect of pH on Lactic Acid Production
Lactic acid production was performed in a batch fermentor and the experiment was conducted using 14 fermentation reactors. Each fermentation unit was prepared with 1000 mL culture medium and stirred mechanically at 125 rpm. This fermentor (2.5 L) contained 1 L of fermentation medium. The medium pH was controlled automatically between 4.5 and 7.0 by the addition of 10 M NaOH or 2 M hydrochloric acid. The temperature of the fermentor was maintained at 30 ± 2 °C. Samples were withdrawn every 24 h and lactic acid content was analyzed by the HPLC method.
2.7. Effect of Carbon and Nitrogen Sources on Lactic Acid Production
Lactic acid production in the medium containing additional carbon sources (1%) was studied. Carbon sources such as xylose, starch, lactose, maltose, sucrose, fructose, and glucose were incorporated. Nitrogen sources such as ammonium chloride, ammonium sulphate, peptone, yeast extract, beef extract, and casein were added at the 0.5% level. The effect of various phosphate sources (0.1%, w/v) on lactic acid production was studied. Phosphate ions such as di-potassium hydrogen phosphate, potassium dihydrogen phosphate, di-sodium hydrogen phosphate, and sodium di-hydrogen phosphate were incorporated in the bioreactor.
2.8. Effect of Metal Ions on Lactic Acid Production
Metallic ions play significant roles in activating various metabolic enzymes. Ions such as Mn
2+, Mg
2+, Cu
2+, and Fe
3+ have been reported to influence lactic acid production [
34,
35]. Cu
2+ ions also positively influence lactic acid production. During the fermentation process, the presence of copper ions inhibits the consumption of D-lactic acid to pyruvate. To analyze the influences of various metals on lactic acid production, the substrate was enriched with Mn
2+, Mg
2+, Cu
2+, Fe
3+, Ca
2+, and Co
2+ added to the culture medium at 0.01% concentration. The fermentor without any metal ions was considered a blank.
2.9. Antimicrobial Activity of Lactic Acid
Antimicrobial activity was determined using the disc diffusion method for various pathogenic bacteria, including Bacillus subtilis MTCC 5981, Staphylococcus aureus MTCC 737, Pseudomonas aeruginosa MTCC 424, Enterobacter aerogenes MTCC111, E. coli MTCC 443, Penicillium chrysogenum MTCC5108, and Aspergillus niger MTCC 282. The culture obtained from the cultivation of the Lactobacillus strain in the batch fermentor for 48 h at 37 °C was harvested and the sample wascentrifuged at 10,000× g for 10 min. The cell-free culture supernatant was further neutralized with NaOH (1 M) and the final pH was maintained at 7.0 and the sample was filtered using a 0.2 µm membrane filter. Culture media such as Mueller Hinton agar and Potato Dextrose Agar were prepared according to the manufacturer’s instructions (Himedia, Mumbai, India). A sterile 6 mm (Himedia, Mumbai, India) disc was kept on the solid agar medium and then cell free extract (25 µL) was loaded on the disc. The culture plates were incubated for 24 h and 72 h, respectively, for bacteria and fungi.
2.10. Application of Lactic Acid in Soil Amendment
One kilogram of soil was preincubated for 7 days at 25 °C at 35–45% water-holding capacity of as described by Tejada [
36] with few modifications. After 7 days of incubation, lactic acid was added at four different concentrations: T1 (0.25%,
w/w), T2 (0.5%,
w/w), T3 (0.75%,
w/w), and T4 (1%,
w/w). Lactic acid was not incorporated into the control experiment. Soil was incubated for five weeks under dark conditions at 25 °C. A sample was withdrawn every 7 days and used for biochemical analysis. The pH of the soil was determined after extraction with distilled water using a pH meter. The soil sample (1 gm) was treated with extraction buffer (10 mL) containing 0.1 N H
2SO
4 for 1 h and centrifuged at 10,000×
g for 10 min. The supernatant was filtered, and lactic acid content was analyzed. Soil phosphorus content was determined as described by Olsen et al. [
37]. Soil dehydrogenase activity was measured as described by Tabatabai [
38]. p-nitrophenyl-β-D-glucopyranoside was used as the substrate for the determination of β-glucosidase activity [
39].
2.11. Statistical Analysis
Experiments were performed in triplicate and the data are expressed as mean ± standard deviation. One-way analysis of variance was used to analyze the significance and p < 0.05 was considered statistically significant.