Use of Corn-Steep Water Efﬂuent as a Promising Substrate for Lactic Acid Production by Enterococcus faecium Strain WH51-1

: Various challenges facing the industrial production of bio-based lactic acid (LA) such as cost of raw materials and nitrogen sources, as well as contamination risk by mesophilic and neutrophilic producers, should be overcome for the commercial production. This study aimed to investigate the feasibility of corn steep water (CSW) as a raw material for LA production using a newly thermo-alkali-tolerant lactic acid bacterium. The physicochemical characteristics of CSW were investigated. The high carbohydrates, proteins, amino acids, vitamins, essential elements, minerals, and non-protein nitrogenous compounds content conﬁrmed that the CSW is a promising substrate for LA production. Out of 67 bacterial isolates, Enterococcus faecium WH51-1 was selected based on its tolerance to high temperatures and inhibitory compounds (sodium metabisulfate, sodium chloride, sodium acetate, and formic acid). Fermentation factors including sugar concentration, temperature, inoculum size, and neutralizing agents were optimized for LA production. Lactic acid concentration of about 44.6 g/L with a high yield (0.89 ± 0.02 g/g) was obtained using 60 g/L of CSW sugar, inoculum size 10% ( v/v ), 45 ◦ C, and sodium hydroxide or calcium carbonate as a neutralizing agent. These results demonstrated the potential of strain WH51-1 for LA production using CSW efﬂuent as raw material.


Introduction
Lactic acid (LA) is one of the most extensively used chemicals because it is integrated into various applications such as food, textile, pharmaceutical, and chemical industries [1]. In addition, it is used as feedstock in the production of polylactic acid (PLA) [2], as an attractive biodegradable candidate, instead of petrochemical polymers [3]. LA can be synthesized either through chemical methods or biotechnological fermentation processes. The latter approach is preferred due to its ability to produce pure LA forms with the utilization of renewable substrates, whereas a racemic mixture of DL-LA is produced by chemical methods [4]. Almost all LA produced in the world is currently obtained by microbial fermentation processes [5,6].
The global market size for LA was valued at USD 2.7 billion in the year 2020 and is expected to expand at a compound annual growth rate (CAGR) of 8.0% from 2021 to 2028 (https://www.grandviewresearch.com/ (Accessed on 31 May 2021)). Therefore, the development of cost-effective LA fermentation processes is worth being undertaken to meet the huge rising demand. The cost of raw material represents approximately 40-70% of the total production costs; therefore, using cheap raw materials, including agricultural and industrial residues instead of expensive pure sugars, is one of the most important key factors that determine the success of the fermentation process [2,7]. 20 g/L CSW sugars and adjusted pH at 9.0 before incubation at 50 • C for 48 h. At the end of the incubation period, 100 µL of each flask was streaked on mYD agar medium and incubated at 50 • C for 24 h. The appeared bacterial colonies were re-streaked on the same media for purification. The purified bacterial isolates were grown on mYD agar medium supplemented with 5 g/L CaCO 3 . Positive acid-producing isolates were tested for catalase activity using 3% H 2 O 2 for the primary selection of lactic acid bacteria. All bacterial isolates were kept in glycerol (30%) at −80 • C.
Further screening was conducted on broth media supplemented with 20 g/L of CSW sugar and incubated at 50, 55, and 60 • C for 48 h. To investigate the effect of inhibitors on sugar consumption, LA concentration, and LA production of the selected isolates, different concentrations of sodium metabisulfate (1.0-8.0 g/L), sodium chloride (2.5-10%), sodium acetate (5-20 g/L), and formic acid (2.5-10 g/L) were separately supplemented to mYD medium containing CSW as a sole carbon source.

Characterization and Identification of Isolate WH51-1
The most potent LA bacterial producer, coded WH51-1, was subjected to molecular identification through the extraction of genomic DNA using a modified method according to Miller et al. [28]. The 16S rRNA gene fragment was amplified using genomic DNA as a template and the bacterial universal primers of 27f (5-GAGTTTGATCACTGGCTCAG-3) and 1492r (5-TACGGCTACCTTGTTACGACTT-3) in a polymerase chain reaction (PCR) [29]. The PCR mixture contained 1 × PCR buffer, 0.5 mM MgCl 2 , 0.25 mM dNTP, 2.5 U Taq DNA polymerase (QIAGEN), 0.5 µM of each primer, and 1 µg of genomic DNA. The PCR was conducted in a DNA Engine Thermal Cycler (PTC-200, Bio-Rad, Hercules, CA, USA) at the following conditions: hot starting performed at 94 • C for 3 min, followed by 30 cycles of 94 • C for 0.5 min, 55 • C for 0.5 min, and 72 • C for 1 min, with an extension performed for 10 min at 72 • C. The PCR product was commercially sequenced using both primers at Sigma Company (Cairo, Egypt) by using an ABI 3730xl DNA sequencer. The sequence was then compared with those in the GenBank database through BLASTN. Multiple sequence alignment was then performed on 1290 bp of 16S rRNA gene fragments by the ClustalX 1.8 software package, and the phylogenetic tree was established with a neighbor-joining method of the Kimura 2-parameter model to calculate genetic distance as the transitional and transversional substitution rates using MEGA (version 6.1) software. The level of confidence for each branch at 1000 repeats was tested by bootstrap analysis. The chimeric formation was detected using the Uchime2_NCBI tool to find chimeras that are >3% diverged from the closest sequences. The obtained 16S rRNA gene sequence of the isolated strain was deposited in GenBank with the accession number MZ093371.

Optimization of Fermentation Conditions
All optimization experiments were conducted in 250 mL Erlenmeyer flasks containing 100 mL working volume. To determine the effect of CSW concentration on LA production by strain WH51-1, different concentrations (Viz., 20, 40, 60, 80, and 100 g/L) from the total sugar were tested in batch fermentations. The experiment was conducted at pH 9.0 and 50 • C for 48 h. The fermentation temperature was also investigated at 35, 40, 45, 50, and 55 • C using a medium containing 60 g/L CSW sugar, inoculum size 10% (v/v), at pH 9.0 for 48 h. To investigate the influence of inoculum size on LA production, different inocula sizes (2.5-12.5%, v/v) were inoculated into the fermentation medium after maintaining the previous factors. Additionally, different neutralizing agents (NaOH and CaCO 3 ) were investigated to control the pH during fermentation at pH 9.0. A 5N NaOH was added during the fermentation process while CaCO 3 was added into the medial components at 0.5 g/g-carbon source concentration.

Analytical Methods
The corn steep water samples' conductivity and total dissolved salts were determined using a digital InoLab system (InoLab PH 720, WTW GmbH, Weilheim in Oberbayern, Ger-Fermentation 2021, 7, 111 4 of 18 many), while pH was estimated by InoLab pH 720, (WTW GmbH, Weilheim in Oberbayern, Germany). Dissolved oxygen was determined using a digital WTW GmbH analyzer (model oxi197i). Chemical oxygen demand (COD) was measured using a COD Reactor (Model HACH, Loveland, CO, USA), whereas biological oxygen demand (BOD) was measured by adding standard microbial and air saturation for a 5-day incubation at 25 • C using a BOD Incubator (Velp R 10300141, Usmate Velate, Italy), followed by the determination of remaining oxygen [30].
Seventeen essential elements were determined by an Inductive Coupled Plasma Mass Spectrometer (ICP-MS) (model, Triple Quad 8800 G3663A, Stevens Creek Blvd. Santa Clara, CA, USA). The sample extract was clarified by centrifugation at 13,000 rpm for 15 min, and the supernatant was filtered with a 0.22 µm filter then acidified by using Nitric acid 69% with ratio (1%). Stock standard solutions were prepared by a single element or multielemental traceable standard solutions in acid matrices prepared specifically for plasma mass spectrometric analysis.
Protein content was estimated by the Lowry method [31], while the total sugars are estimated using the phenol sulfuric acid method [32]. Lactic acid analysis was performed using the method as described by Barker and Summerson [33] and measured at 570 nm.
Amino acids were quantified by a Sykam Amino Acid Analyzer (Sykam GmbH, Eresing, Germany) equipped with a Solvent Delivery System S 2100 (Quaternary pump with flow range 0.01 to 10.00 mL/min and maximum pressure up to 400 bar), Autosampler S 5200, Amino Acid Reaction Module S4300 (with built-in dual filter photometer between 440 and 570 nm with constant signal output and signal summary option), and Refrigerated Reagent Organizer S 4130 (Laird Thermal Systems, Durham, NC, USA). For standard preparation, a stock solution containing aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, ammonia, and arginine was used. All amino acid concentrations were 2.5 µMol/mL (except cystine which was 1.25 µMol/mL), diluted with 60 µL in a 1.5 mL vial with a sample dilution buffer, then filtered using a 0.22 µm syringe filter, then 100 µL was injected. For the sample preparation, 1.0 g of each sample was mixed with 5.0 mL hexane. The mixture was allowed to macerate for 24 h. Then, the mixture was filtered on Whatman no. 1 filter paper and the residue was transferred into a test tube where it was incubated in an oven with 10 mL 6N HCl for 24 h at 110 • C. After the incubation, the sample was filtered on Whatman no. 1 filter paper, evaporated on a rotary evaporator, and dissolved completely in a 3.0 mL dilution buffer. From this solution, the first dilution was prepared by diluting 100 µL to 1.0 mL dilution buffer, from which 100 µL was further diluted to a 1.0 mL dilution buffer, and filtered using a 0.22 µm syringe filter, then 100 µL was injected.
On the other hand, total vitamins were estimated by an HPLC system (Waters 2690 Separation Module) equipped with a Waters 996 photodiode array detector (Waters, Milford, MA, USA). For a standard preparation of water-soluble vitamins, 10 mg of 7 water-soluble vitamin reference standards (ascorbic acid, thiamine HCl, riboflavin, nicotinic acid, nicotinamide, pyridoxine HCl, and folic acid) was dissolved in 10 mL 0.05 M NaOH, diluted to a concentration of 100 µg/mL and filtered with a 0.22 µm syringe filter, then 10 µL was injected. A preparation of a stock solution of fat-soluble vitamins (vitamin E, D, and A) in 5.0 mL methanol was then diluted to obtain concentrations of 806.25 IU/mL vitamin A, 114 µg/mL vitamin E, and 400 IU vitamin D3, and filtrated using a 0.22 µm syringe filter before injection of 10 µL. The sample extract was filtered using a 0.22 µm syringe filter, then 10 µL was injected.
For estimation of the non-protein nitrogenous compound in CSW, a sample extract was clarified by centrifugation at 13,000 rpm for 15 min, and the supernatant was filtered with a 0.22 µm filter, then 10 µL was injected directly into a HPLC analyzer (Waters, Milford, MA, USA) equipped with a C18 column (3.9 × 150 mm) and a fluorescent detector, with 250 and 395 nm wavelengths of excitation and emission.
For fermentation experiments, bacterial growth was measured using the total viable count method. One mL of the fermentation medium was serially diluted and inoculated on dishes containing plate count agar media. Consumed sugar was analyzed by a phenolsulphuric acid method using glucose as the standard [32]. Intermittent samples were taken and centrifuged at 6000 rpm for 10 min to estimate LA concentration using the Barker and Summerson method [33]. The LA yield (g/g) based on consumed sugars was calculated as the ratio of LA (g/L) to CSW sugar (g/L). LA productivity (P LA , g/L/h) is the ratio of LA produced to the time of the fermentation process. Maximum LA productivity (g/L/h) was determined by the difference between LA concentrations of two respective samples divided by the time difference.  Table 1. The effluent is viscous, with a deep brown color. The pH of CSW was 4.5 ± 0.2. The TDS, COD, and BOD were 6452 mg/L, 78 mg/L, and 23 g/L, respectively. Besides this, CSW contains a high sugar content (250 g/L), high amino acid content (10.7 g/L), various water-soluble vitamins (14.5 g/L), and mineral ions that make it a promising substrate for LA fermentation. The CSW not only contains low molecular weight substances which are leached out of the corn grain, but it also contains a large number of substances derived from the degradation and fermentative conversion of proteins, carbohydrates, and nucleic acids during steeping. Steep water is primarily handled by evaporation to a concentrated thick liquor that is a complex mixture of carbohydrates, amino acids, peptides, organic compounds, heavy metals, inorganic ions, and myo-inositol phosphates [34]. Watson [35] reported that the bacterial activities and any unconverted starch in the steep water increase the color formation in the evaporators due to non-enzymatic browning.

Results and Discussion
Hull et al. [34] reported that the steeping under acidic conditions is due in part to the initial presence of SO 2 and, later, lactic acid, a major fermentation product. Typical corn wet milling effluent contains various nutrients, including 250-300 mg/L of total soluble solids (TSS) [36]. Hence, if TSS is recovered from steep water, it can be used in animal feed after drying and mixing with other fibrous milling byproducts [36]. Unfortunately, since the steep water effluent is rather high in TSS, its COD and 5-day biological oxygen demand (BOD 5 ) is high [36]. Data represented in Table 1 show that the total carbohydrate of CSW is 250.44 ± 1.3 g/L. These high levels of sugars with other nutrients in CSW can stimulate the growth of microorganisms and, hence, lead to high levels of lactic acid production [37]. However, the addition of SO 2 and elevated temperatures can inhibit microbial growth during the steeping process [11]. Thus, the existence of high sugars with other nutrients would make these waste materials promising substrates for LA fermentation by suitable, potent microorganisms.

Inorganic Ions Content of CSW
Data represented in Table 1 reveal that phosphorus was the most prevalent ion present in CSW, with a value of 4515.5 mg/kg. Data also show that magnesium, calcium, and zinc existed in CSW at a concentration of 392.65, 132.65, and 29.2 mg/kg, respectively. Boron, manganese, aluminum, and iron were found at a slight concentration that ranges from (3.8-2.8 mg/kg). However, cadmium, cobalt, and vanadium were not detectable in CSW under study.
The CSW is not only rich in organic nutrients but also contains trace nutrients such as vitamins and metal ions [38,39]. Hull et al. [34] reported that the presence of inorganic phosphate in CSW is usually due to the de-phosphorylation of inositol phosphate isomers (InsPs) by phosphatase enzymes. Liggett et al. [38] reported that the presence of calcium, iron, magnesium, phosphorus, and potassium are at high concentrations in CSW compared to other elements. Hofer et al. [14] reported that the trace elements that exist in CSW are mostly bound to proteins or other molecules.

Amino Acid Analysis of CSW
Several earlier published studies reported the effectiveness of LAB to metabolize all amino acids, but this efficacy differs greatly between species [40][41][42][43]. Therefore, the analysis of amino acids in CSW would help to study their role in the growth of LAB. Data shown in Table 2 reveal that the CSW was rich in different amino acids (aspartic acid, threonine, serine, glutamic acid, proline, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine) and ammonium as had previously been reported [44,45]. Proline and glutamic acid were the most prevalent amino acids in CSW that reach 1.071 and 1.656 g/L respectively.
The results in this study confirmed that CSW is a naturally complex medium that is suitable for the growth of most LAB isolates. Wright [46] reported that the majority of the amino acid content that exists in CSW is originated from corn rather than from fermentation products. Because of the nutritional fastidiousness of LAB, they are unable to grow unless certain amino acids are supplied in the medium [5]. The high proline content is beneficial for microbial fermentation processes. Proline can stimulate the growth of some species of LAB and may be involved in osmoregulation, as previously proven [47]. Xu et al. [48] reported that proline, at a concentration of 1.0 g/L in culture media, can be absorbed and accumulated in the cell, and hence the cell growth and pyruvate concentration improve by 59.0 and 22.1%, respectively. Tian et al. [49] reported that proline addition could protect Lacticaseibacillus paracasei under hyperosmotic stress, because it contributes to membrane stabilization, co-metabolizes as a carbon or nitrogen source for cell growth, or imports and accumulates in the cells as a compatible solute to counteract hyperosmotic stress. Tian et al. [49] reported that Lacticaseibacillus paracasei could import proline from the extracellular environment for improved sugar consumption and L-LA production. Glutathione amino acids, such as glutamic, glycine, and cystine, protect LAB against oxidative stress, osmotic stress, and acid stress [50]. Glutamate, glutamine, and arginine improve the acid resistance of LAB [45]. Additionally, arginine is an essential amino acid and a nitrogenous precursor of nitric oxide (NO) that is generated by NO synthase [51]. Lysine is an essential component of the peptidoglycan cell wall of most Gram-positive bacteria [52]. Qiao et al. [44] reported that aspartate was involved in the synthesis of lysine, indicating that lysine was found at an insufficient level, especially from the end-exponential growth phase to the stationary phase. Moreover, alanine metabolism plays an important role in the synthesis and defense of the cell wall [53]. Hofer et al. [14] found that the main amino acids in CSW are arginine, alanine, and glutamic acid with values of 44.3, 35.7 and 42.0 mg/g respectively. Liggett et al. [38] reported that over 95% of the total nitrogen in steep liquor, after hydrolysis, is accounted for by ammonia and amino acids.

Analysis of Fat-Soluble and Water-Soluble Vitamins in CSW
Vitamins are important growth factors that need to be added to the medium. They play an important role in the growth of microbial strains [54], so it was important to analyze the fat-soluble and water-soluble vitamins that exist in the CSW used in the current study. Data show the absence of fat-soluble vitamins (vitamins E, D, A) in CSW, whereas watersoluble vitamins including riboflavin, nicotinic acid, nicotinamide, and pyridoxine HCL at concentrations of 0.143, 106.31, 4.59, and 34.47 mg/100 mL, respectively, were detected in CSW effluent (Figure 1), while others including ascorbic acid, folic acid, and thiamine HCl were not detected.
The presence of nicotinic acid and riboflavin in CSW are important as reducing agents in the form of NADH/NAD and FADH/FAD, respectively, which are essential for energy generation in microbial cells [55]. Pyridoxine is a precursor of pyridoxal-5 -phosphate (PLP), which is involved in many reactions of amino acid metabolism [56]. The absence of folic acid may be due to the bad solubility of some B-vitamins in water; in addition, they compose of just a small fraction of CSW [56]. Moreover, the absence of biotin was favorable for LA fermentation, as its acts as an inhibitor of some LAB [57]. The absence of thiamine in CSW used in the current study was compatible with Wright [46], who found that CSW contains considerable amounts of B-complex vitamins, except thiamine, which is usually low or absent. This phenomenon can be attributed to the hydrolysis of thiamine by SO 2 during the steeping process. Hofer et al. [14] also found that CSW contains water-soluble vitamins at different concentrations. Tanner et al. [58] found that CSW contains riboflavin, niacin, pantothenic acid, pyridoxine, and biotin at a concentration of 5, 819, 23.8, 19.1, and 0.125 µg/g, respectively. The differences between the values among published studies maybe due to actual differences in compositions and viscosity/dilution of effluent [59]. Vitamins are important growth factors that need to be added to the medium. They play an important role in the growth of microbial strains [54], so it was important to analyze the fat-soluble and water-soluble vitamins that exist in the CSW used in the current study. Data show the absence of fat-soluble vitamins (vitamins E, D, A) in CSW, whereas water-soluble vitamins including riboflavin, nicotinic acid, nicotinamide, and pyridoxine HCL at concentrations of 0.143, 106.31, 4.59, and 34.47 mg/100mL, respectively, were detected in CSW effluent (Figure 1), while others including ascorbic acid, folic acid, and thiamine HCl were not detected. The presence of nicotinic acid and riboflavin in CSW are important as reducing agents in the form of NADH/NAD and FADH/FAD, respectively, which are essential for energy generation in microbial cells [55]. Pyridoxine is a precursor of pyridoxal-5-phosphate (PLP), which is involved in many reactions of amino acid metabolism [56]. The absence of folic acid may be due to the bad solubility of some B-vitamins in water; in addition, they compose of just a small fraction of CSW [56]. Moreover, the absence of biotin was favorable for LA fermentation, as its acts as an inhibitor of some LAB [57]. The absence of thiamine in CSW used in the current study was compatible with Wright [46], who found that CSW contains considerable amounts of B-complex vitamins, except thiamine, which is usually low or absent. This phenomenon can be attributed to the hydrolysis of thiamine by SO2 during the steeping process. Hofer et al. [14] also found that CSW contains water-soluble vitamins at different concentrations. Tanner et al. [58] found that CSW contains riboflavin, niacin, pantothenic acid, pyridoxine, and biotin at a concentration of 5, 819, 23.8, 19.1, and 0.125 μg/g, respectively. The differences between the values among published studies maybe due to actual differences in compositions and viscosity/dilution of effluent [59].

Analysis of Non-Protein Nitrogenous Components of CSW
The quantities of non-protein nitrogenous substances in CSW and their identification are critical steps because these substances can be added to fermentation media as high nutritional supplements. To achieve this goal, HPLC analysis of CSW confirmed the presence of several non-protein nitrogenous components as ethanolamine, ornithine, citrulline, taurine, γ-aminobutyric acid with a concentration of 5.12, 6.33, 7.41, 13.63, and 2.36 μg/mL, respectively (Table 3). From our data, taurine was the most prevalent non-protein nitrogenous component in CSW. Daniels [60] reported that taurine acts as a bio-stimulant in CSW. Moreover, taurine is known as a sole source of sulfur for the growth of aerobic

Analysis of Non-Protein Nitrogenous Components of CSW
The quantities of non-protein nitrogenous substances in CSW and their identification are critical steps because these substances can be added to fermentation media as high nutritional supplements. To achieve this goal, HPLC analysis of CSW confirmed the presence of several non-protein nitrogenous components as ethanolamine, ornithine, citrulline, taurine, γ-aminobutyric acid with a concentration of 5.12, 6.33, 7.41, 13.63, and 2.36 µg/mL, respectively (Table 3). From our data, taurine was the most prevalent non-protein nitrogenous component in CSW. Daniels [60] reported that taurine acts as a bio-stimulant in CSW. Moreover, taurine is known as a sole source of sulfur for the growth of aerobic microorganisms [61], as well as also being considered the main source of energy for many aerobic bacteria [62]. Denger et al. [63] reported that taurine nitrogen has been confirmed as the sole source of nitrogen for the growth of two Rhodococcus spp. in the presence of excess carbon sources. Clifford et al. [64] showed that taurine degradation can also be a significant source of other metabolically important S and N species (sulfate, sulfide, bisulfide, thiosulfate, ammonium, alanine, and other organo-sulfonates). Hull et al. [34] also found non-protein nitrogenous compounds such as γ-aminobutyric acid in CSW. Various non-protein nitrogenous components, such as ammonia, purine and pyrimidine derivatives, choline, and trigonelline were also detected in CSW [59]. Metabolic transformation of low molecular weight compounds occurs during steeping. For example, γ-aminobutyric acid is present in corn grain in low levels, whereas it is found free in steep water in high amounts. In contrast, the concentration of free glutamic acid is negligible in steep water, although this amino acid is in high concentrations in endosperm proteins and steep water proteins. Free glutamic acid is enzymatically de-carboxylated to γ-aminobutyric acid during steeping [65]. Citrulline is a non-protein amino acid and is represented as the main precursor for arginine [66]. Hwang and Lee [67] found that LAB has an arginine deaminase pathway that is involved in producing citrulline and ornithine from arginine, so we thought that presence of citrulline and ornithine in the CSW under study may have been due to the arginine deaminase pathway, and that this enhanced the bacterial growth [67]. Citrulline also plays an important metabolic intermediate in the urea cycle [68]. Citrulline could protect Streptococcus pyogenes from acid stress via the arginine deiminase pathway [69]. It also can act as a compatible solute against osmotic stress [70]. According to the previous analysis, it can be concluded that CSW is an ideal low-cost raw material for LA production by a suitable microbial producer.

Isolation and Screening of the Most Potent Lactic Acid Producers
To overcome the associated problems with LA fermentation, including substrate cost and contamination risk [25], isolation of LAB from 21 dairy products was achieved at thermo-alkaline conditions (pH 9 and 50 • C) using CSW-containing media. Out of 67 bacterial isolates, only 15 isolates had the efficacy to produce LA with a high yield (greater than 0.88 g/g). A second screening test was performed to determine the ability of these bacterial isolates to produce LA under higher temperatures at 55 • C and 60 • C. Out of these 15 bacterial isolates, only six isolates were able to tolerate 60 • C and could produce more than 10 g/L of lactic acid. Out of the previous six bacterial isolates, two isolates (coded as SSD16-1 and WH51-1) were selected as the highest producers and used to investigate the inhibitory compounds that existed in the effluent substrates on them. These isolates were catalase-negative and therefore preliminarily identified as lactic bacteria.

Effect of Inhibitors on LA Production by the Most Potent Isolates
To investigate the effect of inhibitors on sugar consumption and LA fermentation with the most potent selected bacterial isolates, different concentrations of sodium metabisulfate (1-8 g/L), sodium chloride (2.5-10, % w/v), sodium acetate (5-20 g/L), and formic acid (2.5-10 g/L) were separately supplemented to fermentation medium that contained CSW as the sole carbon source (Table 4).
Both isolates showed high LA yields ranging 0.77-0.87 g/g and 0.78-0.90 g/g in the presence of 1.0-4.0 g/L of sodium metabisulfate for SSD16-1 and WH51-1, respectively, while the productivity of LA was decreased to 0.25 and 0.31 g/g at 8.0 g/L of sodium metabisulfate for isolate SSD16-1 and WH51-1, respectively.
Similarly, a high LA yield (ranged 0.73-0.86 g/g) was achieved in the presence of 2.5-7.5% of sodium chloride for both isolates, while a lower yield (0.27 and 0.31 g/g) was achieved with 10% of NaCl for both isolates (SSD16-1 and WH51-1, respectively).
On the other hand, isolates SSD16-1 and WH51-1 showed a LA yield with values ranging 0.60-0.82 g/g and 0.57-0.87 g/g in the presence of 5.0-15.0 g/L of sodium acetate, respectively, while the productivity decreased to 0.26 and 0.36 g/g at 20.0 g/L of sodium acetate, respectively.
Lactic acid yield at 0.53-0.78 g/g and 0.62-0.72 g/g were achieved in the presence of 2.5-7.5 g/L of formic acid for isolates SSD16-1 and WH51-1, respectively, while lower LA yields at 0.25 g/g were achieved with 10 g/L formic acids for both isolates.  According to the obtained results, isolate WH51-1 was considered the most potent isolate exhibiting greater stability under high-stress/inhibitory compounds that might exist in the waste materials under study, and therefore was selected for characterization and further studies. This isolate was identified as Enterococcus faecium WH51-1 using a 16Sr RNA sequence (Figure 2). Lactic acid yield at 0.53-0.78 g/g and 0.62-0.72 g/g were achieved in the presence of 2.5-7.5 g/L of formic acid for isolates SSD16-1 and WH51-1, respectively, while lower LA yields at 0.25 g/g were achieved with 10 g/L formic acids for both isolates.
According to the obtained results, isolate WH51-1 was considered the most potent isolate exhibiting greater stability under high-stress/inhibitory compounds that might exist in the waste materials under study, and therefore was selected for characterization and further studies. This isolate was identified as Enterococcus faecium WH51-1 using a 16Sr RNA sequence (Figure 2). In the present study, the selected strain WH51-1 exhibited greater stability under high NaCl concentration. Generally, salt stress adversely affects the growth performance, survival rate, and activities of central carbon metabolism, which affect the efficiency of LA production [68]. Previous research demonstrated that salt stress led to changes in cell membrane fatty acid composition in many microorganisms including Rhodococcus erythropolis, Lacticaseibacillus paracasei, and Desulfovibrio vulgaris [71][72][73].
Moreover, corn is steeped in water containing 0.2% sulfur dioxide generated from sodium metabisulfate that attacks disulfide bonds of the protein matrix encapsulating starch granules. Once the endosperm protein matrix is dispersed, the starch granules become free, and the overall starch recovery is increased [74]. The steeping tanks on large scale contain SO2 and elevated steeping temperatures to inhibit the growth of microorganisms during the steeping process [11], so we speculate that the selected strain, WH51-1, will be more applicable for LA fermentation because it exhibited high stability under different concentrations of sodium metabisulfate. In the present study, the selected strain WH51-1 exhibited greater stability under high NaCl concentration. Generally, salt stress adversely affects the growth performance, survival rate, and activities of central carbon metabolism, which affect the efficiency of LA production [68]. Previous research demonstrated that salt stress led to changes in cell membrane fatty acid composition in many microorganisms including Rhodococcus erythropolis, Lacticaseibacillus paracasei, and Desulfovibrio vulgaris [71][72][73].
Moreover, corn is steeped in water containing 0.2% sulfur dioxide generated from sodium metabisulfate that attacks disulfide bonds of the protein matrix encapsulating starch granules. Once the endosperm protein matrix is dispersed, the starch granules become free, and the overall starch recovery is increased [74]. The steeping tanks on large scale contain SO 2 and elevated steeping temperatures to inhibit the growth of microorganisms during the steeping process [11], so we speculate that the selected strain, WH51-1, will be more applicable for LA fermentation because it exhibited high stability under different concentrations of sodium metabisulfate. To investigate the influence of initial sugar concentrations on LA production by E. faecium WH51-1, fermentation processes were conducted using different concentrations of the initial sugars (20-100 g/L) in mYD media at 50 • C for 48 h. Data obtained in Table 5 and represented graphically in Figure 3 summarize the fermentation parameters and profiles for LA production at different sugar concentrations. The maximum total viable cell was increased from 68.3 ± 2.08 × 10 10 CFU/mL at 20 g/L, reaching the maximum value of 116.6 ± 2.08 × 10 10 CFU/mL at 60 g/L of the total sugars. The cell growth was decreased to 31.3 ± 1.52 × 10 10 CFU/mL at 80 g/L, giving the lowest value of 25.0 ± 3.0 × 10 10 CFU/mL at total sugars of 100 g/L. Table 5. Effect of different concentrations of CSW on the growth, sugar consumption, LA concentration, LA yield, LA productivity, and maximum LA productivity by E. faecium WH51-1.  To investigate the influence of initial sugar concentrations on LA production by E. faecium WH51-1, fermentation processes were conducted using different concentrations of the initial sugars (20-100 g/L) in mYD media at 50 °C for 48 h. Data obtained in Table 5 and represented graphically in Figure 3 summarize the fermentation parameters and profiles for LA production at different sugar concentrations. The maximum total viable cell was increased from 68.3 ± 2.08 × 10 10 CFU/mL at 20 g/L, reaching the maximum value of 116.6 ± 2.08 × 10 10 CFU/mL at 60 g/L of the total sugars. The cell growth was decreased to 31.3 ± 1.52 × 10 10 CFU/mL at 80 g/L, giving the lowest value of 25.0 ± 3.0 × 10 10 CFU/mL at total sugars of 100 g/L.   In contrast, sugar consumption by the WH 51-1 strain was increased from its minimal value of 18.3 ± 0.37 g/L at 20 g/L to give the highest value at 60 g/L of 36.9 ± 5.22 g/L, while it decreased after that to reach 13.2 g/L at 100 g/L of the total sugars. A similar pattern was given for LA production. The final LA concentration was increased from 1.16 ± 0.55 g/L at 20 g/L, achieving the highest value of 29.1 ± 0.87 g/L at 60 g/L of the total sugars and decreased after that, reaching its minimal value of 11.0 ± 0.1 g/L at 100 g/L of the total sugars. On the other hand, LA yield showed a comparable range of 0.86-0.88 g/g of sugars consumed at 20-80 g/L of the total sugars while giving 0.82 ± 0.01 g/g of sugars consumed at a high concentration of sugars (100 g/L). LA productivity was also increased from 0.33 ± 0.01 g/L/h at 20 g/L to 0.60 ± 0.01 g/L/h at 60 g/l of the total sugars while it decreased after that, obtaining the minimal value of 0.22 g/L/h at 100 g/L. The maximum LA productivity ranged from (0.33 ± 0.02) to (0.96 ± 0.04) g/L/h giving the maximum value at 60 g/L of the total sugars.
From the previous results, a comparable LA yield and productivity were obtained at 40-60 g/L of the total sugars, while the maximum LA concentration and the maximum productivity (29.13 ± 0.87 g/L and 0.96 ± 0.04 g/L/h, respectively) were obtained when 60 g/L of the total sugars was used as the initial carbon source concentration.
Several authors also reported an increased LA concentration with the increase of initial sugar concentrations up to certain limits [2,75,76]. Interestingly, the very high concentrations of sugar resulted in increasing osmotic pressure and, hence, decreasing LA productivity [77]. Only a few LAB, such as Enterococcus faecalis CBRD01 [78], Enterococcus mundtii QU 25 [5], Lacticaseibacillus paracasei subsp. paracasei CHB2121 [79], and Lb. mutant G-03 [80], have been reported to be able to tolerate sugar concentrations greater than 60 g/L.

Effect of Inoculum Size on LA Production by Enterococcus faecium WH51-1
Inoculum size plays an important role in efficient fermentation and reduction of the lag phase duration [6]. To determine the effect of different inocula sizes on the production of LA by strain WH51-1, fermentation processes were achieved by using different values of inocula sizes ranging 2.5-12.5% in mYD media at 45 • C for 48 h. Data obtained in Table 6 show that the maximum total viable cell was increased from 78.0 ± 3.60 × 10 10 CFU/mL at an inoculum size of 2.5% (v/v) reaching the maximum value of 121.33 ± 2.88 × 10 10 CFU/mL at 10% (v/v). Cell growth was decreased to 86 ± 6.24 × 10 10 CFU/mL at 12.5%. In contrast, the sugar consumption by WH 51-1 strain was increased from 25.43 ± 1.19 at 2.5% to give the highest value of 37.43 ± 1.01 g/L at 10% (v/v) while it decreased after that to reach 26.4 ± 0.62 g/L at 12.5% (v/v). The final LA concentration was increased from 23.03 ± 0.15 g/L at an inoculum size of 2.5% (v/v) achieving the highest value of 32.86 ± 1.01 g/L at 10% (v/v) and decreased after that, reaching 23.4 ± 1.34 g/L at 12.5% (v/v). Lactic acid productivities were also increased from 0.47 ± 0.01 g/L/h at 2.5% (v/v) to 0.68 ± 0.022 g/L/h at 10% (v/v), while decreased to 0.48 ± 0.02 g/L/h at 12.5% (v/v). The maximum LA productivity ranged from (0.83 ± 0.16) to (1.11 ± 0.07) g/L/h, giving the maximum value of 10%. As a result, 10% (v/v) was selected as the optimal inoculum size for LA production by E. faecium WH51-1. Vishnu et al. [81] reported that the optimal inoculum size for LA production by Lactobacillus amylophilus GV6 was 10% (v/v). Panesar et al. [82] found that the maximum LA production was 33.72 g/L by Lacticaseibacillus casei NBIMCC 1013, which could be observed with an inoculum size of 2-4% (v/v).

Effect of Neutralizing Agents on LA Production by Enterococcus faecium WH51-1
Neutralizing agents are used during fermentation for overcoming product inhibition. Neutralizing the fermentation media prevents the progressive acidification of the medium and improves organic acid production [83]. For evaluating the effect of pH control on LA fermentation parameters by E. faecium WH51-1, CaCO 3 and NaOH solutions were used as neutralizing agents (Figure 4). The results showed that there were almost comparable total viable cells and sugar consumption in the case of neutralizing the fermentation media by NaOH (179.33 ± 3.51 × 10 10 CFU/mL and 50.06 ± 1.81 g/L, respectively) and CaCO 3 at 178.66 ± 2.51 × 10 10 CFU/mL and 49.7 ± 1.25 g/L, respectively. Moreover, there was a comparable final concentration of LA (~44.5 g/L) using NaOH or CaCO 3 as a neutralizing agent that was highly increased as compared to the fermentation without neutralizing agents at 32.8 g/L. A high LA yield at 0.89 g/g of sugar consumed was obtained. However, a slight increase in LA productivity was obtained when NaOH was used for neutralizing the fermentation media achieving 0.46 ± 0.02 g/L/h, compared to neutralization by CaCO 3 , which obtained 0.41 ± 0.01 g/L/h. Furthermore, the highest value of maximum LA productivity (1.58 ± 0.07 g/L/h) was obtained using the NaOH solution.
(0.83 ± 0.16) to (1.11 ± 0.07) g/L/h, giving the maximum value of 10%. As a result, 10% (v/v) was selected as the optimal inoculum size for LA production by E. faecium WH51-1. Vishnu et al. [81] reported that the optimal inoculum size for LA production by Lactobacillus amylophilus GV6 was 10% (v/v). Panesar et al. [82] found that the maximum LA production was 33.72 g/L by Lacticaseibacillus casei NBIMCC 1013, which could be observed with an inoculum size of 2-4% (v/v).

Effect of Neutralizing Agents on LA Production by Enterococcus faecium WH51-1
Neutralizing agents are used during fermentation for overcoming product inhibition. Neutralizing the fermentation media prevents the progressive acidification of the medium and improves organic acid production [83]. For evaluating the effect of pH control on LA fermentation parameters by E. faecium WH51-1, CaCO3 and NaOH solutions were used as neutralizing agents (Figure 4). The results showed that there were almost comparable total viable cells and sugar consumption in the case of neutralizing the fermentation media by NaOH (179.33 ± 3.51 × 10 10 CFU/mL and 50.06 ± 1.81 g/L, respectively) and CaCO3 at 178.66 ± 2.51 × 10 10 CFU/mL and 49.7 ± 1.25 g/L, respectively. Moreover, there was a comparable final concentration of LA (~44.5 g/L) using NaOH or CaCO3 as a neutralizing agent that was highly increased as compared to the fermentation without neutralizing agents at 32.8 g/L. A high LA yield at 0.89 g/g of sugar consumed was obtained. However, a slight increase in LA productivity was obtained when NaOH was used for neutralizing the fermentation media achieving 0.46 ± 0.02 g/L/h, compared to neutralization by CaCO3, which obtained 0.41 ± 0.01 g/L/h. Furthermore, the highest value of maximum LA productivity (1.58 ± 0.07 g/L/h) was obtained using the NaOH solution. As a result, NaOH seemed to be better than CaCO3 as a neutralizing agent during LA production from CSW, not only because of higher fermentation parameters but also due As a result, NaOH seemed to be better than CaCO 3 as a neutralizing agent during LA production from CSW, not only because of higher fermentation parameters but also due to the avoidance of formation of gypsum waste materials as a result of CaCO 3 supplementation [26]. Several reports have been recorded on LA fermentation processes using an NaOH solution for controlling the pH of the fermentation medium [75,84,85].

Conclusions
In this work, a cost-effective system for increased LA production from CSW was achieved under thermo-alkaline conditions for decreasing the contamination risk. A com-plete analysis of CSW indicated its suitability to be used as a raw material for LA production due to its high content of sugars, nitrogen, amino acids, trace elements, and vitamins. Enterococcus faecium WH51-1 has been proven to have a good stability toward various inhibitory compounds, and is a promising candidate for LA production from CSW effluent with minimal nutrient supplementation (5 g/L yeast extract). Optimizing the fermentation conditions increased LA production, achieving a maximum concentration of 44.6 g/L at a yield of 0.89 g/g-consumed sugar and productivity of 1.58 g/L/h using 60 g/L CSW sugar, inoculum size (10%, v/v), at 45 • C and pH 9.0 using NaOH as a neutralizing agent. Further optimization studies, including response surface statistical designs, will be investigated for improved sugar consumption and LA production from CSW effluent.