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Article

Co-Fermentation of Food Waste and Municipal Sludge from the Saudi Arabian Environment to Improve Lactic Acid Production by Lactobacillus rhamnosus AW3 Isolated from Date Processing Waste

by
Naif Abdullah Al-Dhabi
*,
Galal Ali Esmail
and
Mariadhas Valan Arasu
Addiriyah Chair for Environmental Studies, Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
*
Author to whom correspondence should be addressed.
Sustainability 2020, 12(17), 6899; https://doi.org/10.3390/su12176899
Submission received: 28 July 2020 / Revised: 19 August 2020 / Accepted: 21 August 2020 / Published: 25 August 2020
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Abstract

:
Food waste and municipal sludge were used as the substrates for the biosynthesis of lactic acid in a batch fermentor. The probiotic bacterial strain Lactobacillus rhamnosus AW3 isolated from date processing waste was used to produce lactic acid in a batch fermentor. Co-fermentation enhanced the biosynthesis of lactic acid and decreased substrate inhibition more than mono-substrate fermentation. A maximum yield of 28.4 ± 0.87 g/L of lactic acid was obtained through co-fermentation of food waste and municipal sludge at an optimized ratio of 2:0.5. Lactic acid production was improved by the supplementation of fructose, peptone, and sodium dihydrogen phosphate at pH 5.5 after 48 h fermentation. This production was approximately three-fold higher than that during mono-fermentation of food waste. The tested bacterial strains were obtained from the Microbial Type Culture Collection (MTCC). Lactic acid showed potent antimicrobial activity against pathogenic organisms, such as Bacillus subtilis MTCC 5981 (14 mm), Staphylococcus aureus MTCC 737 (20 mm), Pseudomonas aeruginosa MTCC 424 (24 mm), Enterobacter aerogenes MTCC111 (19 mm), Escherichia coli MTCC 443 (18 mm), Penicillium chrysogenum MTCC 5108 (19 mm), and Aspergillus niger MTCC 282 (19 mm). The antimicrobial properties of lactic acid have significant potential to inhibit the growth of pathogenic bacteria and fungi and improve probiotic properties. The lactic acid extracted from L. rhamnosus AW3 decreased the pH value of soil (p < 0.01) and increased the availability of soil phosphorus (p < 0.01). These findings demonstrate the bioconversion of food waste and municipal sludge into lactic acid, and the recycling of food wastes in urban areas to enhance soil nutrients.

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 H2SO4 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 Mn2+, Mg2+, Cu2+, and Fe3+ have been reported to influence lactic acid production [34,35]. Cu2+ 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 Mn2+, Mg2+, Cu2+, Fe3+, Ca2+, and Co2+ 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 H2SO4 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.

3. Results and Discussion

3.1. Characterization of Lactobacillus Strains and Production of Lactic Acid

A total of seven Lactobacillus colonies from date processing waste were isolated and recovered from Rogosa agar plates and MRS agar plates as Lactobacillus species based on the catalase test and Gram staining. Each of the seven bacterial isolates was Gram positive and rod shaped and showed a negative reaction towards citrate and catalase tests. The Lactobacillus strains survived in 3% bile salts and utilized most of the tested carbohydrates. The morphological and biochemical characteristics of Lactobacillus are presented in Table 1. Microscopic observation showed that 100% of LAB was rod-shaped and to achieve taxonomic classification, 16S rDNA analysis was undertaken. The isolate AW3, identified as L. rhamnosus AW3, has potent lactic acid producing capacities. Bacteria from the genera Lactobacillus, Enterococcus, and Lactococcus produce types of lactic acids and have been used to enhance bioavailability and reduce mycotoxins [40]. Recent findings showed that probiotic Lactobacillus strains can be widely used to remove toxins such as fumonisins, trichothecenes, aflatoxins, and mycotoxins during harvest and storage [41]. These LAB species isolated from various food products can be effectively utilized in the formulation of functional foods to manage the growth of pathogenic bacteria and indirectly control various diseases that may affect consumers [42]. The lactic acid-producing ability of Lactobacillus rhamnosus has been described previously and used in various substrates. Rivas et al. [43] used corn steep liquor and spent yeast cells for the production of lactic acid. L-lactic acid production of Lactobacillus rhamnosus using cassava powder was reported by Wang et al. [44]. Recently, Chen et al. [45] used a mixed culture of Lactobacillus rhamnosus and Bacillus coagulans to optimize the medium for the production of lactic acid.
The physico-chemical factors and nutrient contents of FW and MS are described in Table 2. The culture medium was found to be rich in soluble proteins, soluble carbohydrate, suspended solids, and volatile suspended solids. The increased availability of simple carbohydrate from the food waste favored lactic acid production. FW contains various simple sugars that are useful for the production of lactic acid by various bacteria [6]. The batch fermentation system is useful for the production of lactic acid because its technical simplicity. However, fed-batch production of lactic acid has been reported previously to improve yield [46]. The fed-batch fermentation system offers various advantages such as reducing substrate inhibition and improving productivity [47]. Batch fermentation was carried out for 72 h, and lactic acid yield, sugar consumption, and biomass are described in Table 3. The maximum production of lactic acid was 28.3 ± 0.22 g/L and the biomass yield was 24.2 ± 0.13 g/L after 72 h. Lactic acid production attained 26.5 ± 0.15 g/L within 48 h fermentation and increased to 28.2 ± 0.23 g/L. In previous research, Lactobacillus rhamnosus B103 has been used for the production of lactic acid [48].
Batch fermentation has been recommended for industrial production of lactic acid, however substrate inhibition of lactic acid synthesis is the major disadvantage [49]. Hence, a feeding technique has been recommended to eliminate the inhibition of the substrate in the microbial environment. This strategy allows control of the growth rate and maximization of the lactic acid yield in batch culture. In the present analyses, all lactic acid producing organisms were classified from the genus Lactobacillus and cultured in a batch fermentor. In previous research, the probiotic L. lactis was isolated from various flours such as sorghum, wheat, and quinoa [50,51,52]. The strain Lactobacillus rhamnosus has been isolated from kidney beans, sorghum, amaranth flour, wheat, and sourdough [53,54,55]. Lactic acid production has been carried out in test tubes (15 mL) previously in aerobic conditions and lactic acid yield variation was reported. In Lactobacillus strains such as Lactobacillus delbrueckii ssp. bulgaricus CECT 5038 and CECT 4005, the lactic acid yield was lower, however the case of L. delbrueckii ssp. bulgaricus CECT 5036 showed maximum yield in micro-aerobic conditions [56]. To improve the silage process, Lactobacillus strains such as L. plantarum, L. brevis, and L. paracasei were used to reduce the concentrations of various pathogenic organisms [57].

3.2. Batch Fermentation of Lactic Acid in Relation with Other Factors

The Lactobacillus strain was cultured for 72 h and lactic acid production was assayed. Total sugar content of the fermentor declined after 72 h of incubation. Biomass production reached a maximum up to 72 h (4.2 ± 0.13 g/L wet weight) and a marginal decrease was observed after 96 h (3.73 ± 0.11 g/L) (Table 4). The pH of the culture medium was initially adjusted to 5.5. Generally, lactic acid bacteria synthesize lactic acid in a wide pH range (pH 4.0–6.0). Lactobacilli produced maximum lactic acid at a low pH value (pH 4.0) [58]. Carbohydrate content of the medium continuously declined from 63 ± 4.2 to 10 ± 1.1 after 96 h. The culture medium pH was 5.5 and carbohydrate content declined more rapidly than in previous studies. In previous research, a decreased carbohydrate level was reported at a higher pH (6.0) due to high bacterial activity and acid feedback inhibition [28,58]. In the current study, protease and α-glucosidase activity during the fermentation process was tabulated (Table 5). Both enzyme activities were low at lower pH values and at pH 6.5 enzyme activities were maximum (97 ± 3.1 and 11.2 ± 1.03 U/mL after 48 h). These findings indicate increased microbial activity at higher pHs. α-glucosidase activity was used to indirectly measure the processes of methanogenesis, acidogenesis, and hydrolysis. However, when the pH value increased in the fermentor above 7.0, protease (40 ± 0.18 U/mL) and α-glucosidase (7.5 ± 0.8 U/mL) activity decreased after 72 h. Protease production was detected from pH 4.0 to pH 7.0 and maximum enzyme production was detected at pH 8.0 (97 ± 3.2 U/mL). α-glucosidase reached a maximum in an alkaline pH range (pH 8.0 to pH 10.0) and this enzyme activity was very low in an acidic medium.

3.3. Effect of FW and MS Ratios on Lactic Acid Production

Table 3 shows the production of lactic acid by co-fermentation of FW and MS at 0:1, 1:0, 0.5:1, 1:0.5, 1:1, 1:1.5, 1.5:1, and 2.0:5 concentrations. Fermentation was performed for 48 h and lactic acid was stable at this fermentation period. The mono-fermentation of MS yielded less lactic acid (21.3 g/L), followed by the FW:MS ratio of 0.5:1 (22.5 g/L) after 24 h fermentation. The highest lactic acid concentration of 28.4 g/L was obtained when the FW and MSS ratio was 2:0.5. Co-fermentation of FW and MS enhanced lactic acid yield. These findings indicate that fermentation of FW with MS at a ratio of 2:0.5 was optimal for lactic acid productivity (Table 6). During the fermentation process, fatty acids were generated from the FW due to hydrolysis and these fatty acids decreased the pH value during the fermentation process [59]. Mixing of FW with an optimal level of MS enhanced lactic acid production. In recent years, co-fermentation of FW with other wastes has been extensively studied as a better option for energy recycling and the waste-handling process [60]. Wu et al. [61] reported that FW co-fermented with a de-oiled grease trap enhanced the yield of biogas. Co-digestion of FW with primary sludge enhanced the lactic acid yield by about 97% [62]. A 2:0.5 ratio of FW with MS enhanced lactic acid conversion by three-fold compared to mono-fermentation. Waste-activated sludge (WAS) and FS have been used to increase lactic acid production. Valorization of these wastes has attracted much attention in recent years [63]. The synergistic property of WAS and food waste was previously studied by Wu et al. [61] and Edwards et al. [64]. An optimized level of FW and MS was found to enhance lactic acid production. In addition, an optimal ratio of WAS and FW has also been reported to enhance lactic acid production. The ratio of these wastes varied based on the nutrient content and physical nature of the components [65].

3.4. Effect of pH on Lactic Acid Production

In this study, pH adjustment was not undertaken for blank experiments, and the analyzed pH range was between 4.5 and 7.0. However, these ranges may vary widely based on the type of substrate and organism used for the composting process. Tang et al. [29] used FW for lactic acid production using indigenous microbiota and the pH between 5.0 and 6.0 was tested for lactic acid production. The blank experiment yielded 22.9 ± 0.31 g/L. As shown in Figure 1, the culture medium in weak alkaline conditions enhanced lactic acid yield more than in a strong alkaline environment. Maximum lactic acid production was observed after 48 h at pH 5.5. However, lactic acid yield decreased at higher pH ranges (6.0–7.0). It was observed that increasing the pH value undermined the production of lactic acid after 24 h. Akao et al. [66] used garbage for the production of lactic acid in unsterile conditions and reported enhanced product yield at pH 5.5 in thermophilic conditions. In the case of lactic acid fermentation, temperature and pH of the medium overlapped in the bio-process. During the process of fermentation, volatile fatty acids from the FW reduced the pH of the culture medium. At higher temperatures, a low pH value was found to be optimum to enhance the product yield [67]. Volatile fatty acids were generated from food wastes and these inhibited anaerobic digestion [59]. Mixing of an appropriate concentration of FW with MS can result in a favorable pH to maximize lactic acid production. Sophora flavescens residues and food wastes at a ratio of 1:1.5 was found to be optimal to maintain pH during the fermentation process [65].

3.5. Nutrient Sources on Lactic Acid Production

Carbon sources were found to influence lactic acid production and the results are shown in Figure 2a. Lactobacillus sp. showed various fermentation characteristics with respect to carbon sources. When the bioreactor was treated with glucose, fructose, and maltose, the selected bacterial strain metabolized these carbon sources and converted then into 27.4 ± 0.4 to 29.8 ± 0.56 g/L lactic acid. This might be mainly due to acidification of the fermentation medium and the various Lactobacillus carbohydrates using the homofermentation pathway. In previous research, a co-culture of engineered Lactococcus lactis and Lactobacillus delbrueckii revealed the conversion of sugar into lactic acid [68]. In L. coryniformis, high levels of lactic acid production have been achieved with glucose supplementation [69]. Enhanced lactic acid production in a packed bed reactor has been previously achieved in a medium containing sucrose as the carbon source in Lactobacillus delbrueckii [70]. In the current study, FW and sludge supplemented with nitrogen sources enhanced lactic acid production. Figure 2b reveals the influence of nitrogen sources on lactic acid production. Inorganic nitrogen and complex nitrogen sources, namely peptone, yeast extract, ammonium sulphate, and beef, extract significantly influenced lactic acid production. Among the nitrogen sources, peptone was utilized by the strain for maximum lactic acid production. Types of organic biomass such as molasses enhanced bacterial cell growth because of high nitrogen content and calcium. The available calcium content in the molasses buffers the enzymatic action [71]. Nitrogen implied either in catabolic processes or anerobic processes is mainly available in the form of inorganic compounds, peptides, and amino acids. These sources can be generally supplemented in the form of ammonium sulphate, yeast extract, urea, and peptone [72]. The tested phosphates regulated lactic acid production. Lactic acid yield was high in the medium containing potassium dihydrogen phosphate (27.5 ± 1.1 g/L) and sodium di-hydrogen phosphate (28.3 ± 0.8 g/L). Dibasic ions, such as di-potassium hydrogen phosphate (28.7 ± 0.82 g/L), and monobasic ions, such as sodium di-hydrogen phosphate (28.91 ± 0.57 g/L), enhanced lactic acid production. These metal ions served as the co-factor for enzyme production. Mineral elements and ions provided in the form of various salts and vitamins available in yeast extract and other natural nitrogen sources act as cofactors in various enzymatic reactions [73].

3.6. Antimicrobial Properties of Crude Extract from L. rfhamnosus AW3

The cell-free sample obtained from the batch fermentation experiment was evaluated for antibacterial and antifungal properties; results are presented in Table 7. The crude sample containing approximately 34.2 g/L lactic acid with various final pH values clearly revealed antibacterial and antifungal properties against all of the tested bacterial and fungal pathogens. Antimicrobial activity was higher in the Lactobacillus strain cultured at pH 5.5 than that cultured at pH 6.0. The zone of inhibition was maximized against P. aeruginosa MTCC 424 (24 mm), S. aureus MTCC 737 (20 mm), and E. aerogenes MTCC111 (19 mm), whereas a smaller clear zone was observed with pH 6.0 (15, 14, and 15 mm zones, respectively). Antibacterial activity was also detected against E. coli MTCC 443 (18 mm) and B. subtilis MTCC 5981 (13 mm). Fungi such as A. niger MTCC 282 (19 mm) and P. chrysogenum MTCC5108 (19 mm) showed almost the same level of activity against the L. rhamnosus AW3 stain maintained at pH 5.5. At pH 6.0, antimicrobial potency of the sample decreased by about 28% compared to the pH value 5.5. Probiotic Lactobacillus strains produce proteolytic enzymes and antimicrobial compounds and vitamins [74]. The preservative role of these Lactobacillus strains is mainly due to the production of various bioactive secondary metabolites including antifungal compounds, bacteriocins, lactic acids, hydrogen peroxide, acetoin, and hydrogen peroxide [75]. The inhibition activity of lactic acid varies among Lactobacillus species. Pelaez et al. [76] reported the inhibitory activity of acetic and lactic acid produced by Lactobacillus species. In recent years, the antimicrobial potential of Lactobacillus has been reported by Manhanzva et al. [77] and Meleh et al. [78]. In addition, Zhao et al. [79] studied the correlation of the colonization of the bacterial flora and biological properties of bacteria from the genus Lactobacillus.

3.7. Lactic Acid Induced Changes in Soil Chemical and Biological Properties

Lactic acid was applied to the soil at various concentrations and resulted in a significant decrease in pH values (p < 0.01). Lactic acid in the soil medium enhanced the pH value of the soil and the soil pH reached the pH value of control. The T1 soil reached a maximum value within 14 days of incubation, whereas T4 reached a maximum value after 28 days (Table 8). The decrease in the soil pH for several weeks was mainly due to the acidity of the soil sample [80]. The applied lactic acid was degraded continuously in the soil. After seven days of treatment lactic acid concentration decreased by about 75% in T1, whereas this decrease was more than 60% in the case of T2–T4 (Table 9). The continuous degradation of lactic acid was mainly due to the activity of microorganisms in the soil and this trend was described previously [81]. Treatment of the soil sample with lactic acid enhanced P solubilization, which was caused by the sudden decrease in the soil lactic acid. The applied lactic acid improved P availability, hence allowing it to be used when required by plants. Activity of soil enzymes such as dehydrogenase and acid phosphomonoesterase was improved during the first two weeks. These two enzymes from the soil sample indirectly measure microbial activity of the soil sample. Dehydrogenase enzyme activity was found to be maximized after 14 days of treatment. However, no significant change was found in β-glucosidase activity and lactic acid did not induce this enzymatic activity.

4. Conclusions

Lactic acid was produced from food waste (FW) and municipal sludge (MS) in a batch fermentor. Lactobacillus rhamnosus AW3 isolated from date processing effluent showed an efficient conversion of wastes to lactic acid. Co-fermentation of FW and MS for lactic acid production was carried out with a three-fold increase in the overall yield (28.4 ± 0.87 g/L). Using a batch fermentor with an optimal ratio of FW and MS (2:0.5 ratio) stabilized lactic acid production. Lactic acid yield was improved by nutrient supplements and an optimal pH (5.5) after two days of fermentation. The lactic acid extracted from L. rhamnosus AW3 decreased the pH value of the soil and increased the availability of soil phosphorus, thus improving soil fertility. These findings are useful in the bioconversion of FW and MS into lactic acid and food waste recycling in urban areas.

Supplementary Materials

The supplementary is available online at https://www.mdpi.com/2071-1050/12/17/6899/s1.

Author Contributions

N.A.A.-D. and M.V.A. designed and supervised the experiment. N.A.A.-D., G.A.E., and M.V.A., performed the laboratory work. N.A.A.-D. and M.V.A. analyzed the results and drafted the manuscript. N.A.A.-D. obtained the fund for execution of the laboratory work. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through the Vice Deanship of Scientific Research Chairs.

Acknowledgments

The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through the Vice Deanship of Scientific Research Chairs.

Conflicts of Interest

The authors declare no conflict of interest.

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Sustainability 12 06899 g001 550
Figure 1. Lactic acid production by various pHs by L. rhamnosus AW3.
Figure 1. Lactic acid production by various pHs by L. rhamnosus AW3.
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Figure 2. (a) Effect of carbon sources on lactic acid production at pH 5.5 at 37 °C incubation (FR—Fructose; Gl—Glucose; LA—Lactose; M—Maltose; SU—Sucrose; ST—Starch; and C—Control). (b) Effect of nitrogen sources on lactic acid production at pH 5.5 at 37 °C incubation (AC—Ammonium chloride; AS—Ammonium sulphate; PE—Peptone; YE—Yeast extract; BE—Beef extract; CA—Casein; and CL—control).
Figure 2. (a) Effect of carbon sources on lactic acid production at pH 5.5 at 37 °C incubation (FR—Fructose; Gl—Glucose; LA—Lactose; M—Maltose; SU—Sucrose; ST—Starch; and C—Control). (b) Effect of nitrogen sources on lactic acid production at pH 5.5 at 37 °C incubation (AC—Ammonium chloride; AS—Ammonium sulphate; PE—Peptone; YE—Yeast extract; BE—Beef extract; CA—Casein; and CL—control).
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Table
Table 1. Biochemical characteristics of lactic acid bacteria isolated from date processing waste.
Table 1. Biochemical characteristics of lactic acid bacteria isolated from date processing waste.
Biochemical TestBacteria
AW1AW2AW3AW4AW5AW6AW7
Gram staining+++++++
ShapeRodRodRodRodRodRodRod
Citrate utilization test–––––––
Catalase test–––––––
Bile salt hydrolysis+++++++
Growth at temperatures+++++++
10 °C
20 °C+++++++
40 °C––+––––
40 °C––+ –
Glucose hydrolysis+++++++
Sucrose hydrolysis+++++++
Lactose hydrolysis+++++++
Dextrose hydrolysis+++++++
Xylose hydrolysis+++++++
D-arabinose–––––––
Mannose+++++++
A-cellulose–––––––
D-raffinose+++++++
Sorbital+++++++
+++++++
+; Positive results, –; Negative results.
Table
Table 2. Physico-chemical factors and nutrient content of food waste and municipal sludge.
Table 2. Physico-chemical factors and nutrient content of food waste and municipal sludge.
ParameterFood WasteMunicipal SludgeMixture
pH6.3 ± 0.126.5 ± 0.046.7 ± 0.13
Total suspended solid (mg/L)190489 ± 42716489 ± 57945972 ± 1487
Volatile suspended solid (mg/L)187681 ± 69811952 ± 39742087 ± 720
Total COD (mg/L)190275 ± 38715486 ± 29644864 ± 376
Soluble COD (mg/L)70142 ± 3026359 ± 46.99736 ± 104
Soluble protein (mg/L)5984 ± 31.54085 ± 56.54622 ± 3.6
Soluble carbohydrate (mg/L)53830 ± 2.436280 ± 109.245392 ± 284.4
Table
Table 3. Lactobacillus strains isolated from palm effluent and lactic acid yield in a batch reactor.
Table 3. Lactobacillus strains isolated from palm effluent and lactic acid yield in a batch reactor.
Lactobacillus StrainsLactic Acid Yield (g/L)
24 h48 h72 h
AW123.1 ± 20.0923.6 ± 20.1723.9 ± 20.29
AW220.98 ± 20.1721.9 ± 20.2820.52 ± 20.11
AW322.9 ± 20.1226.4 ± 20.2926.9 ± 0.1
AW4ND23.8 ± 0.1424.3 ± 0.11
AW522.3 ± 20.2225.6 ± 21.126.2 ± 0.1
AW623.3 ± 20.1424.8 ± 21.224.7 ± 20.42
AW7ND23.1 ± 20.1224.2 ± 0.13
Table
Table 4. Analysis of nutrients and lactic acid production at various fermentation periods.
Table 4. Analysis of nutrients and lactic acid production at various fermentation periods.
Fermentation Period (h) Total Sugar (g/L)Biomass (g/L)Lactic Acid (g/L)
1263 ± 4.21.3 ± 0.221.3 ± 1.11
2454 ± 1.92.15 ± 0.921.7 ± 0.13
3643 ± 2.23.4 ± 0.222.8 ± 1.2
4821 ± 1.83.6 ± 0.126.5 ± 1.15
6020 ± 3.34.1 ± 0.228.2 ± 0.23
7214 ± 1.24.2 ± 0.1328.3 ± 1.22
8413 ± 1.33.91 ± 0.1222.5 ± 1.2
9610 ± 1.13.73 ± 0.1120.3 ± 1.1
Table
Table 5. Protease and α-glucosidase activity.
Table 5. Protease and α-glucosidase activity.
pHProtease Activity (U/mL)α-Glucosidase Activity (U/mL)
48 h72 h48 h72 h
4.532 ± 2.234 ± 1.31.5 ± 0.311.9 ± 0.2
5.045 ± 1.340 ± 0.94.9 ± 0.185.3 ± 0.13
5.551 ± 0.559 ± 1.15.7 ± 1.15.6 ± 0.32
6.092 ± 4.173 ± 1.86.2 ± 0.736.8 ± 0.49
6.597 ± 3.180 ± 1.511.2 ± 1.0313.6 ± 0.23
7.041 ± 1.345 ± 1.510.3 ± 0.568.1 ± 0.12
7.540 ± 0.1837 ± 0.9 7.5 ± 0.8 6.3 ± 0.15
Table
Table 6. Co-fermentation of food waste and municipal sludge at various ratios.
Table 6. Co-fermentation of food waste and municipal sludge at various ratios.
FW:MS RatiosDry Weight of FWDry Weight of MSLactic Acid Yield (g/L)
24 h48 h
0:0101521.323.2
1:0015024.525.9
0.5:151022.523.3
01:00.510524.925.4
1:017.57.524.325.2
01:01.56922.723.5
1.5:19625.426.3
02:00.512327.328.4
Table
Table 7. Antimicrobial activity of lactic acids from L. rhamnosus AW3 extracted from a batch fermentor.
Table 7. Antimicrobial activity of lactic acids from L. rhamnosus AW3 extracted from a batch fermentor.
MicroorganismZone of Inhibition (mm) at Various pH Values
4.555.566.5
B. subtilis MTCC 59811413131011
S. aureus MTCC 7371617201415
P. aeruginosa MTCC 4242019241515
E. aerogenes MTCC 1111818191514
E. coli MTCC 4431415181718
P. chrysogenum MTCC 51081014191817
A. niger MTCC 2821312191918
Table
Table 8. Influence of lactic acid on the pH profile of the soil. Lactic acid was added at various concentrations and the pH was tested for 35 days.
Table 8. Influence of lactic acid on the pH profile of the soil. Lactic acid was added at various concentrations and the pH was tested for 35 days.
Treatment (Days)pH
T1T2T3T4
07.3 ± 0.36.7 ± 0.146.3 ± 0.215.9 ± 1.1
77.5 ± 0.217.1 ± 0.346.6 ± 0.246.1 ± 2.2
147.7 ± 0.127.3 ± 0.116.9 ± 0.126.3 ± 1
217.72 ± 0.17.6 ± 0.167 ± 0.156.4 ± 0.59
287.73 ± 0.177.73 ± 0.17.1 ± 0.316.9 ± 0.32
357.73 ± 0.157.73 ± 0.157 ± 0.227 ± 0.9
Table
Table 9. Lactic acid content of soil samples at various incubation times. Lactic acid was added at various concentrations and its quantity was determined by the HPLC method for 35 days.
Table 9. Lactic acid content of soil samples at various incubation times. Lactic acid was added at various concentrations and its quantity was determined by the HPLC method for 35 days.
Treatment (Days)Lactic Acid (%)
T1T2T3T4
0100 ± 0100 ± 0100 ± 0100 ± 0
746 ± 2.450 ± 1.564 ± 3.171 ± 3.1
1432 ± 1.928 ± 3.156 ± 1.145 ± 1.1
2118 ± 2.122 ± 1.433 ± 2.337 ± 3.1
288 ± 1.213 ± 0.925 ± 1.139 ± 2.2
350 ± 00 ± 00 ± 00 ± 0

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Al-Dhabi, N.A.; Esmail, G.A.; Valan Arasu, M. Co-Fermentation of Food Waste and Municipal Sludge from the Saudi Arabian Environment to Improve Lactic Acid Production by Lactobacillus rhamnosus AW3 Isolated from Date Processing Waste. Sustainability 2020, 12, 6899. https://doi.org/10.3390/su12176899

AMA Style

Al-Dhabi NA, Esmail GA, Valan Arasu M. Co-Fermentation of Food Waste and Municipal Sludge from the Saudi Arabian Environment to Improve Lactic Acid Production by Lactobacillus rhamnosus AW3 Isolated from Date Processing Waste. Sustainability. 2020; 12(17):6899. https://doi.org/10.3390/su12176899

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Al-Dhabi, Naif Abdullah, Galal Ali Esmail, and Mariadhas Valan Arasu. 2020. "Co-Fermentation of Food Waste and Municipal Sludge from the Saudi Arabian Environment to Improve Lactic Acid Production by Lactobacillus rhamnosus AW3 Isolated from Date Processing Waste" Sustainability 12, no. 17: 6899. https://doi.org/10.3390/su12176899

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