Validation of L-Lactic Acid Production Using Companilactobacillus farciminis KUJ 25-S for Sustainable Bio-Polylactic Acid Manufacturing

Abstract

Companilactobacillus farciminis KUJ 25-S was isolated from fermented fish and identified using 16S rRNA gene sequencing with 30.0 g/L of L-LA (L-lactic acid), with 97% LA per sum of DL-LA. The characteristics of LA and its stereoisomers were confirmed using TLC, chiral-HPLC, and enzymatic techniques. Based on various conditions using liquid MRS broth (static condition, glucose 10%, NaCl 5%, 37 °C for 48 h), the highest growth and LA formation of the culture were at a low temperature (25 °C) and decreased at 37, 45, and 55 °C, respectively. The broth could grow and produce acid at an initial pH in the range 4–11, with a low initial pH of 4 promoting the highest LA formation. LA formation and growth were inversely proportional to the NaCl concentration in the 0.5–30% range. High glucose concentrations suppressed LA formation. The growth-promotion effect varied with glucose concentration (5–40%), with the optimum concentration for LA production being 20% glucose. On the other hand, if used in microoxic conditions, the absence of NaCl was more favorable to acidification than the addition of NaCl (5% NaCl). C. farciminis KUJ 25-S was proposed as a suitable method to produce L-LA based on using the appropriate line for further industrial use.

1. Introduction

Polylactic acid (PLA) is a biodegradable polymer that replaces petroleum-based products and compostable thermoplastic and can be produced from renewable sources, primarily lactic acid (LA), using fermentation methods [1,2]. Because the US Food and Drug Administration has determined that this compound is Generally Recognized as Safe, it has a large market potential in the food industry. In addition, it can be made from chemical means or by fermenting food. The global lactic acid market has been predicted to grow at a compound annual growth rate of 8.0% from 2021 to 2028 [3].

Lactic acid bacteria (LAB) have received considerable attention from researchers as industrial microorganisms because they: provide rapid and complete fermentation of inexpensive raw materials; have a low nitrogenous compound requirement; produce high yields of preferred stereospecific LA at low pH and high temperature; have low cell mass production; and produce limited quantities of other byproducts [4].

According to Benthin and Villadsen [5], only a few LAB, including Lactobacillus brevis, L. helveticus, and L. delbrueckii, are capable of producing LA that is optically pure. The complex nutritional requirements and extremely slow growth rates of LAB are the primary limitations of industrial LA fermentation methods. Therefore, it is necessary to cultivate LAB that convert carbohydrates into optically pure LA via a homo-fermentative pathway that requires only small amounts of nutrients in the medium [6].

The known homofermentative lactic acid bacterium C. farciminis has been isolated from meat and meat products [7]. Because it is part of the normal flora in the intestines of the Tilapia fish (tilapia), it is found in a variety of traditional fermented fish products [8,9]. It could be a pleomorphic strain that consistently produces R and S morphotypes with distinct phenotypes, which may explain its in vitro survival and growth abilities, as well as the modulation of exopolysaccharide synthesis and the auto-aggregation profile [10]. It may produce a large amount of L-LA, depending on how much oxygen is available. However, to our knowledge, it does not appear to have been used as the selected strain of the natural flora in fermented fish for L-LA production to produce PLA. Thus, the present study’s objective was to isolate and characterize C. farciminis KUJ 25-S from fermented fish and to determine the optimal conditions for the formation of L-LA for the production of biopolylactic acid. The conditions of aeration and micro-aeration, as well as temperature, pH, NaCl, glucose, and aeration, were the six factors used in the experimental design. Furthermore, it was hoped to propose the components for an appropriate L-LA production line to accelerate commercial production of L-LA by using effective LAB for the industrial sector.

2. Materials and Methods

2.1. Selection of Lactic Acid Bacteria from Naturally Fermented Fish

Different types of traditional fermented fish were utilized from neighborhood markets in Bangkok, Thailand. Before making 10⁻¹ to 10⁻⁶ dilutions, an initial 25 g were homogenized in 225 mL of 0.85% sterile NaCl (normal saline). Culture growth was based on the spread plate method, with 0.1 mL of the homogenized solution being spread on De Man Rogosa and Sharpe (MRS) agar (5% glucose, 10% NaCl, 2% CaCO₃, pH 5.6; [11]) from (HiMedia Laboratories Pvt. Ltd., Mumbai, Maharashtra, India). The plates were incubated under aerobic conditions at 37 °C for 48 h. An LAB colony that presented a clear zone was chosen. Each colony was isolated twice using the cross-streak plate method before being maintained on MRS agar with 5% glucose, 10% NaCl, and a slant (pH 5.6). Next, catalase-negative, Gram-positive, rod- or cocci-shaped pure LAB strains with high acid production were chosen. Using the titration method, one loop of each pure culture strain was transferred to 3 mL of MRS broth (5% glucose, 10% NaCl, pH 5.6) and incubated at 37 °C for 120 h to search for those that produced large amounts of total LA [12]. As an indicator, 2–3 drops of phenolphthalein were added to a suitable dilution of the fermentation solution supernatant. With 0.5 N sodium hydroxide, the mixed solution was titrated until a pink end point appeared. The lactic acid content was quantified according to Equation (1):

$$\mathrm{L}\mathrm{A}\left(\%\right)={\displaystyle \frac{\mathrm{N}\times \mathrm{V}\times 90.08\times 100}{1000}}$$

(1)

where N is the sodium chloride concentration and V is the volume of sodium hydroxide required for titration to reach the end point.

The top-five lactic acid producers were further screened for their L- or D-lactic acid configuration and concentration using an enzymatic method. The quantity of each LA was determined using a UV method (Megazyme 2018 D-/L-Lactic Acid (D-/L-Lactate) Rapid Assay Kit (Bray Business Park, Bray, Co., Wicklow, Ireland). Based on the L-LA ratio per total DL-LA, the highest concentration of L-LA was greater than 95%. In MRS agar and broth, the cell morphology was characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and the strain’s production of L-LA was confirmed. Thin layer chromatography (TLC), chiral-high performance liquid chromatography (HPLC), and enzymatic methods confirmed the characteristics of the LA and its stereoisomers.

C. farciminis KUJ 25-S was added to the culture collection of the Thailand Institute of Scientific and Technological Research in Bangkok. Based on De Man et al. [11], the cultures were kept in MRS broth at −20 °C with 20% sterile glycerol added.

2.2. Identification of Lactic Acid Bacteria from Naturally Fermented Fish

The LAB were analyzed according to the method of Axelsson [13] for genus identification and 16S rDNA analysis to determine the species of this genus for the purpose of microbiological identification. The Gram stain and catalase tests, as well as microscopic examination, were used to examine the morphology of the cells. The identification was confirmed by sequencing the 16S rDNA region (Macrogen Inc.; Seoul, Republic of Korea). PCR was carried out using the primers 27F 5′ (AGA GTT TGA TGA TCM TGG CTC AG) 3′ and 1492R 5′ (TAC GGY TAC CTT GTTACG ACT T) 3′. Using EF-Taq (SolGent; Daejeon, Republic of Korea), the PCR reaction was carried out with 20 ng of genomic DNA as the template in a 30 μL reaction mixture consisting of: 35 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min each were used to activate Taq polymerase, with a 10 min step at 72 °C at the end. A multiscreen filter plate (Millipore Corp.; Burlington, MA, USA) was used to purify the amplification products. A PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (maker; country) was used to carry out the sequencing reaction. Hi-Di formamide (Applied Biosystems; South San Francisco, CA, USA) was added to the DNA samples that contained the extension products. An ABI Prism 3730XL DNA analyzer (Applied Biosystems; South San Francisco, CA, USA) was used to analyze the mixture after it had been incubated for 5 min at 95 °C and subsequently 5 min on ice.

Sequences obtained from both the forward and reverse primers were assembled using the CAP3 sequence assembly program [14]. Then, the assembled contig was BLASTed to identify the species of bacteria [15]. BLAST analysis was conducted using the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) web-based BLASTn program. Best-hit sequences and sequences from other Lactobacillus species from GenBank (accessions: NR_028949, NR_042533, NR_117973, AB794060, NR_114398, LC063168, NR_044707) were taken for phylogenetic analysis. Enterococcus faecalis (accession NR_040789) was used as an outgroup. All sequences were aligned using Aliview [16] and phylogenetic analysis used maximum parsimony in MEGAX [17], with a bootstrap value of 1000 replicates.

2.3. Preparation of SEM Samples

In 0.1 M phosphate buffer (pH 7.2), bacterial cells were pre-fixed with 2.5% glutaraldehyde (ProSciTech; Townsville, Queensland, Australia) at room temperature. Then, the samples were rinsed three times for 10 min each time in 0.1 M phosphate buffer (pH 7.2). After being post-fixed for 1 h in distilled water with 1% OsO4, the samples were washed three times for 10 min each time in distilled water. Next, the samples were dehydrated for 15 min each in ethanol concentrations (20%, 40%, 60%, 80%, 100%). After being dried for 1.5 h in a critical point dryer (Quorum K850; Laughton, East Sussex, UK), the dry samples were coated with platinum using a sputter coating unit (Quorum Q150R ES; Laughton, East Sussex, UK) and imaged using a field emission scanning electron microscope (Hitachi SU8020; Schaumburg, IL, USA) for 120 s. The procedure was based on the method of [18,19]. Given the high toxicity of osmium tetroxide, all procedures involving this reagent must be performed in a certified fume hood while wearing appropriate personal protective equipment.

2.4. Preparation of TEM Samples

After collection, the bacterial pellets were rinsed three times in 0.1 M phosphate buffer (pH 7.2) after being washed twice with 0.1 M phosphate buffer (pH 7.2) and fixed in 2.5% glutaraldehyde (ProSciTech; Townsville, Queensland, Australia) for 12 h at 4 °C. After being post-fixed for 2 h in distilled water at room temperature in 1% OsO4 (ProSciTech; Townsville, Queensland, Australia), the samples were washed three times for 10 min each in distilled water. Then, the samples were dehydrated for 10 min in a graded series of 30%, 50%, 70%, 90%, and 100% acetone. For the 100% series, this was carried out three times. For the final steps, the samples were soaked in fresh Spurr’s resin three times for 3 h each, followed by polymerization at 70 °C for 8 h in a vacuum oven. After extensive dehydration, the samples were permeated with acetone at ratios of 2:1, 1:1, and 1:2 [20]. Super-slim segments of the cell pellet were cut, post-stained, and imaged using TEM (HT-7700; Hitachi Innovative America, Inc; Schaumburg, IL, USA) at 80 kV. The procedures were based on the methods of Spurr [20], Reynolds [21], and Bozzola and Russell [22]. Regarding the toxicity and handling precautions for osmium tetroxide, its use requires operation in a fume hood and appropriate personal protective equipment.

2.5. TLC

The lactic acid isomers were identified based on TLC [23] using TLC silica gel F254 aluminum sheets (20 × 20 cm), with a mobile phase of 60:2:6:10:22 (acetone:distilled water:chloroform:ethanol:ammonium-hydroxide). After spraying an indicator (a mixture of methyl red and bromothymol blue in methanol), heated air was used to dry the TLC plate for 3 min in an oven at 165 °C to allow for the development of the red color of the LA on the TLC plate. The retention factor (Rf) of the sample, which was the same as the Rf of the lactic acid standard, served as an indicator of the sample.

2.6. Enzymatic Method for D-L-Lactic Acid Determination

The amount of lactic acid was determined using a UV method (D-/L-Lactic Acid (D-/L-Lactate) (Rapid) Assay Kit; Megazyme 2018).

2.7. HPLC for D-L-Lactic Acid Determination

An HPLC system (Agilent 1100; Santa Clara, CA, USA) equipped with a UV detector at 254 nm was used to determine the concentration of the D-LA and L-LA in samples. A chiral column (SUMICHIRAL OA-5000, 5 μm, 4.6 mm i.d. × 150 mm) was used at 38 °C with 1 mmol/L copper (II) sulfate in the water as a mobile phase at a flow rate of 1 mL min⁻¹. The injection volume of the sample was 30 μL. The configuration was compared to standard D-LA and L-LA. Their quantification was performed under an external standard (0–150 μg/mL) for D-LA and L-LA.

2.8. Optimization of L-Lactic Acid Fermentation Condition

Under statically grown conditions, selected LAB were inoculated into 5 mL of MRS broth (5% NaCl, glucose 10% at 37 °C for 24 h). Scaling-up was performed by transferring the 10 mL cultures into 100 mL of MRS broth (5% NaCl, glucose 10% at 37 °C for 24 h) for 10⁻⁶–10⁻⁷ cfu/mL inoculum preparation. The cell count and viable number were determined using the trypan blue cell counting method.

In a 50 mL screw-cap tube (16 × 150 mm), a cell culture inoculum (5 mL, 10⁶–10⁷ cfu/mL) was grown in 30 mL of sterile MRS broth (10% glucose, 5% NaCl) with a final pH of 5.6. The culture was incubated at 37 °C under different static conditions, consisting of concentrations of glucose (5%, 10%, 15%, 20%, 40%; Carlo Erba Reagent; Emmendingen, Germany), pH (adjusted using 0.1 N NaOH (KemAus; Cherrybrook, NSW, Australia) or 0.1 M HCl (to 4.0, 5.6, 7.0, 11.0; KemAus; Cherrybrook, NSW, Australia), temperature (25 °C, 37 °C, 45 °C, 55 °C), and NaCl (0%, 0.5%, 5%, 10%, 30%; Carol Erba Reagent; Emmendingen, Germany) in order. Comparisons were undertaken between anaerobic conditions (37 °C for 48 h, 30 mL of MRS broth (0% and 5% NaCl, glucose 10%, and 5 mL inoculum (10⁶–10⁷ cfu/mL)) in a 50 mL screw-cap tube (16 × 150 mm)) and aerobic conditions (37 °C, 48 h, 75 mL of MRS broth (0% and 5% NaCl, glucose 10%, and 5 mL inoculum (10⁶–10⁷ cfu/mL) in 250 mL baffled flasks on a rotary shaker using 100 rpm). The optimum condition was determined based on cell dry weight (mg/mL) and the concentrations of total LA (mg/mL), L-LA (mg/mL), and D-LA (mg/mL). The condition with the highest L-LA concentration was selected.

2.9. Bacteriological Growth Analysis

Cell growth was measured using a spectrophotometer at an optical density wavelength of 600 nm (OD₆₀₀). OD₆₀₀ measurements were restricted to the reliable linear range (0–0.7), and samples exceeding this limit were diluted accordingly. The OD of the cell was converted to cell dry weigh (mg/mL) using a calibration curve of cell dry weight (Y) against OD₆₀₀ (X). Standard curves generated from triplicate biological replicates (n = 3) exhibited excellent linearity with R² ≥ 0.9990. These conditions ensured accurate quantification of cell concentration across all assays. Under static conditions, 10 mL of inoculum was grown in 100 mL of MRS broth. Then, 10 mL of cell suspension was harvested at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 14 h and transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min. The media suspension was poured off and the precipitate was washed twice with sterile water before being diluted to an OD₆₀₀ of 0.3–0.7. The precipitate was dried at 60 °C until a constant weight was recorded. The practical weight was subtracted from the known tube weight. The OD₆₀₀ (Y) and cell dried weight (X) were plotted to prepare the calibration curve.

2.10. Statistical Analysis

There were 2–3 replications of the experiments and analysis. Results were expressed as mean ± standard deviation (SD) values. The Excel^® 2019 MSO software (64-bit Version 2205; Build 16.0.15225.20278; Microsoft Corp.; Redmond, WA, USA) was used for statistical testing to compare values between treatments using ANOVA. Duncan’s test compared the conditions.

3. Results and Discussion

3.1. Selection of Lactic Acid Bacteria from Naturally Fermented Fish

LAB from various fermented fish (Pla-Chou) samples were screened and 142 were isolated. Based on the biochemical and molecular classifications (16S ribosomal RNA gene identification), the LAB samples were C. farciminis KUJ 25-S (formerly known as Lactobacillus farciminis) (Figure 1,

Table 1. BLAST results of KUJ 25-S sequences (1555 bp) showing top-three sequences as Companilactobacillus farciminis (formerly known as Lactobacillus farciminis) with >99% identity.

Scheme	E-Value	Percentage Identity	Accession Number
Companilactobacillus farciminis KCTC 3681 = DSM 20184	0	99.73%	CP017702.1
Companilactobacillus farciminis	0	99.73%	KX139182.1
Companilactobacillus farciminis KCTC 3681 = DSM 20184	0	99.73%	LC063168.1

[24]), with preliminary Lactobacillus biochemical properties (Gram-positive, catalase-negative, rod-shaped) conforming to the standard genus classification method of LAB (Tetrad formation (-); CO₂ from glucose (-); growth at 10 °C (-); growth at 45 °C (-); growth in 6.5% NaCl (+); growth in 18% NaCl (-); growth at pH 4.4 (-); growth at pH 9.6 (-); and lactic acid stereoisomer (D-LA)). L-LA production in MRS broth (static conditions at 37 °C 120 h) was 30.0 g/L of L-LA and the amount of L-LA per sum of D-LA was 97%. Strain KUJ 25-S was clearly a strain of C. farciminis, as shown by our phenotypic and phylogenetic analyses. Tilapia intestines naturally contain Lactobacillus [9] that has been reported to have been separated from culture from fermented fish [8]. According to Reuter [7], C. farciminis is a homofermentative LA bacterium. Aldolase enzymes are found in homofermentative LAB, which produce LA as the primary end product, with the process being appealing due to the low cost of commercial-scale production of LA without requiring the removal of byproducts [25]. LAB from fermented fish (Pla-Chou), were chosen because LAB can withstand some osmotic stress (NaCl and glucose) at mesophilic temperatures, which is one of the requirements for industrial use. Other requirements include: being tolerant of a variety of stresses; requiring relatively few nutrients; being genetically accessible; and being able to grow at a variety of temperatures. LAB are known to produce LA, particularly L-LA, in a cell factory [26,27,28]. The current study suggested C. farciminis KUJ 25-S was a suitable candidate.

3.2. Characterization of Lactic Acid and Its Stereoisomers from C. farciminis KUJ 25-S

The characteristics of LA and its stereoisomers were confirmed based on TLC, chiral-HPLC, and enzymatic techniques.

The LA in C. farciminis KUJ 25-S was confirmed for acidification based on TLC. The substance produced by the organism had an Rf value equal to that of the LA standard (0.14), as shown in Figure 2 and the L-LA formation (Figure 3c) was confirmed based on HPLC (Figure 3) using chiral-HPLC. The bacterium produced L-LA and D-LA with retention times of 19.972 min and 24.645 min, corresponding to the L-LA standard (Figure 3a,b) and the D-LA standard (Figure 3a), respectively, and 97% ee for L-LA. This corresponded to the enzyme method based on the specificity of the enzymes L-lactate dehydrogenase and D-lactate dehydrogenase (D-/L-Lactic Acid (D-/L-Lactate) Rapid Assay Kit; Megazyme 2018). The stereo-specificity and optical purity of LA produced from microbial cultures are largely determined by the particular microbe chosen and the specificity of its lactate dehydrogenase enzyme (LDH). L- or D-LDH stereospecific NAD-dependent enzymes change the LA structure when they transform pyruvate into the acid. D-LA, L-LA, or a mixture of the two can be produced using microbial fermentation with varying degrees of optical purity. Both enzymes have been found to be active in some LA producers, such as some species of Lactobacillus [29], which led to the formation of a racemate (DL) or LA with a low optical purity (less than 95%). The isomer’s chiral imperfections may increase the cost of purification, making its lower optical purity a serious issue for the commercial production of LA [30]. Among many factors that can affect the purity of lactic acid in fermentations are temperature, pH, aeration, feeding substrate, and the process of fermentation [29,31,32,33]. C. farciminis KUJ 25-S produced L-LA with an extremely high optical purity (>95%). This characteristic is of particular importance because it would decrease the cost of product recovery if this culture were applied to commercial production.

3.3. Cell Morphology on Solid and Liquid Media of C. farciminis KUJ 25-S

The cellular morphology of C. farciminis KUJ 25-S on solid and liquid MRS medium (glucose 10%, NaCl 5%) incubated at 37 °C for 48 h showed filamentous rods using SEM (Figure 4a) and TEM (Figure 4b), respectively. Other species, such as Lactobacillus leichmanni [34] and L. fructivorans [35], share the cellular morphology of C. farciminis KUJ 25-S as filamentous rods in solid and liquid culture. In addition, based on the aeration conditions, a distinctive cell morphology of the pleomorphic strain has been reported [10], which could be a pleomorphic strain that consistently produces R and S—two morphotypes with distinct phenotypes. This phenomenon may explain in vitro survival and growth abilities, as well as the modulation of exopolysaccharide synthesis and the auto-aggregation profile [10,36].

3.4. Effect of Temperature on Lactic Acid Formation and Growth

The effect of temperature (25 °C, 37 °C, 45 °C, 55 °C) on total acid, L-LA, and D-LA production (Figure 5a) and growth (dry weight; Figure 5b) of C. farciminis KUJ 25-S was investigated in MRS liquid medium containing 5% NaCl and 10% glucose at 37 °C for 48 h under static conditions. The optimum temperature for L-LA production was 25 °C, yielding an L-LA content of 423.96 ± 33.38 mg/mL and a maximum growth dry weight of 66.87 ± 2.43 mg/mL. Increased temperature and acidification resulted in reduced growth, with significant (p < 0.05) differences observed across temperature levels. L-LA production and growth were inversely related to temperature in C. farciminis KUJ 25-S. Our findings indicated that the fermentation temperature significantly influenced L-LA concentration, particularly between 25 °C and 37 °C, which had a notable impact on bacterial growth and L-LA production. Sriphochanart and Skolpap [37] reported similar effects using a temperature shift strategy. L-LA production and growth were suppressed at 45 °C and 55 °C; however, L-LA formation was preferred over growth at higher temperatures. Significant amounts of L-LA are produced when fermentation temperatures exceed 40 °C [18,32,38,39]. Utilizing thermophilic strains for LAB production offers several advantages, including reducing contamination risk, facilitating non-sterile fermentation to lower energy requirements for sterilizing and cooling media, and expanding the use of solid-state fermentation processes [26,40,41]. Yamini et al. [42] reported that C. farciminis KUJ 25-S was a suitable thermotolerant strain for L-LA production. C. farciminis KUJ 25-S formed L-LA within the 25–55 °C range, performing well under mesophilic fermentation, consistent with Wang et al.’s findings [43].

3.5. Effect of pH on Lactic Acid Formation and Growth

The effect of pH (4, 5.6, 7, 11) on total acid, L-LA, and D-LA production (Figure 5c) and growth (dry weight; Figure 5d) of C. farciminis KUJ 25-S was investigated in MRS liquid medium containing 5% NaCl and 10% glucose at 37 °C for 48 h under static conditions. A pH level of 4 was the optimum for L-LA production, yielding 357.0 ± 3.23 mg/mL L-LA with a dry weight of 85.99 ± 0.30 mg/mL. Increased acidification reduced growth, with significant (p < 0.05) differences observed across pH levels. LAB are known to grow across various neutral and acidic pH ranges, with C. farciminis KUJ 25-S capable of producing L-LA and growing at pH levels of 4–11. L-LA formation was enhanced at pH levels 4, 7, and 11, but growth was inversely related to L-LA production, indicating that acid production suppressed growth under pH stress. Strains of Lactobacillus are beneficial due to their high acid tolerance and ability to be modified for selective D-LA or L-LA synthesis [5,44,45]. LAB resist undesirable drops in intracellular pH by modulating cell membrane fluidity, metabolic adaptation, and stress-induced protein production [46]. An acid-resistant mutant of L. casei reportedly combatted low extracellular pH by enhancing membrane fluidity and producing more monounsaturated fatty acids [47]. Typically, neutralizing agents are used during fermentation to mitigate any negative effects on cellular metabolism caused by an LA buildup [48]. However, the large amounts of neutralizing agents (such as gypsum) used in downstream processes generate environmental problems and complicate purification in commercial operations [49]. The current study challenged C. farciminis KUJ 25-S to grow and produce L-LA at various pH levels without neutralizing agents. High L-LA formation in low pH conditions has been achieved elsewhere using acid-tolerant strains [50]. Alkaliphilic LAB, with optimum growth in the pH range 7.0–11.5, have been reported to reduce bacterial contamination by inhibiting other bacteria at high pH, facilitating easier management of fermentative output [51,52,53,54,55,56]. Alkaliphilic bacteria can create significant quantities of optically pure L-LA [15,57], and C. farciminis KUJ 25-S was similarly challenged to grow and produce L-LA in alkaline environments.

3.6. Effect of NaCl on Lactic Acid Formation and Growth

The effect was investigated of NaCl (0%, 0.5%, 5%, 10%, 30%) on the yields of total acid, L-LA, and D-LA (Figure 5e) and on the growth (dry weight; Figure 5f) of C. farciminis KUJ 25-S grown in MRS liquid medium containing 5% NaCl and 10% glucose at 37 °C for 48 h in static conditions. The optimum NaCl concentration of 0.5% produced an L-LA content of 398.16 ± 32.97 mg/mL with a dry weight of 73.05 ± 0.13 mg/mL. Growth varied with the NaCl concentration, with the maximum growth (80.061 ± 0.13 mg/mL) being at 0% NaCl. For all treatments, there were significant (p < 0.05) growth differences with the NaCl level. C. farciminis KUJ 25-S could produce L-LA and grow in the presence or absence of NaCl. Osmotic stress resistance of the culture for L-LA formation has been reported [58,59], with 30% NaCl repressing growth (<10 mg/mL) nearly 100%, as opposed to LA formation, which could still be produced. With no NaCl addition, the low cost of downstream processing to remove NaCl would be solved [60]. However, a salt-tolerant strain of an LAB is important when seaweed is used as substrate for LA production [61,62,63]. Under stress from a high salt level, growth and acidification resulted in various species of LAB, especially Lactobacilli [64] such as L. plantarum [27] and L. casei [65]. The resistance of Lactobacillus to osmotic stress is related to the saturated fatty acid-to-unsaturated fatty acid ratio in the cell membrane and the cyclopropanation of oleic acid [66]. Characterization of cellular morphology indicated that the evolved strain L. plantarum H11-ES exhibited markedly enhanced cell-membrane and cell-wall integrity, as well as increased cell length, relative to the parental strain. Whole-genome resequencing further identified mutations in poly(glycerol-phosphate) α-glucosyltransferase and ATP-binding cassette transporters, which are likely responsible for the improved salt tolerance by modulating cell-wall architecture and membrane transport processes [67].

3.7. Effect of Glucose on Lactic Acid Production

The effect was investigated of various glucose concentrations (5%, 10%, 15%, 20%, 40%) on the yields of total acid, L-LA, and D-LA (Figure 5g) and on the growth (dry weight; Figure 5h) of C. farciminis KUJ 25-S in MRS liquid medium containing 5% NaCl at 37 °C for 48 h under static conditions. Glucose levels of 10–20% in the medium enhanced L-LA formation, while levels of 5% and 40% decreased it. Growth varied significantly (p < 0.05) with glucose concentration, with higher glucose levels promoting growth and the maximum growth was observed at 40%, indicating C. farciminis KUJ 25-S’s strong adaptation to osmotic stress [28]. This ability to form L-LA in glucose aligned with the results reported in other studies on other Lactobacillus species such as L. casei [68] and L. paracasei [69,70]. For example, the production of L-LA by L. casei NRRL B-441 was inversely proportional to the initial glucose concentration, with a maximum LA concentration of 118.6 g/L using 160 g/L glucose [68]. Similarly, two studies reported L-LA production by L. paracasei with glucose of 95 g/L [69] and 195 g/L [70]. High carbon uptake rates are essential for microbial chemical production, as shown with Enterococcus faecalis [71] and L. lactis [72], and potentially with C. farciminis KUJ 25-S in the presence of glucose. LAB are suitable for industrial production due to their high sugar absorption and conversion rates, in addition to their low biomass yields [25,28]. Commonly, refined sugars, such as glucose or sucrose, are used for LA production; however, their cost poses a challenge, especially with food source carbohydrates [25,26]. Thus, it is crucial to identify alternative substrates such as lignocellulose biomass and agro-industrial wastes [25,26]. C. farciminis KUJ 25-S could be an effective candidate for L-LA production using such substrates.

For Figure 5, It should be noted that some error bars are relatively large, which likely reflects biological heterogeneity in fermentation performance among replicates under the different stress conditions tested. To enhance reliability, all experiments were conducted in three biological replicates (n = 3), and results are presented as mean ± SD. This approach ensures that the observed trends are robust while acknowledging inherent variability associated with stress-intensive conditions.

3.8. Effect of Aeration on Lactic Acid Production

The effect was investigated of NaCl concentrations (0%, 5%) on the production of total acid, L-LA, and D-LA by C. farciminis KUJ 25-S, as shown in Figure 6. The study was conducted in MRS broth with 10% glucose under both anaerobic conditions at 37 °C and aerobic conditions on a rotary shaker at 100 rpm, with 75 mL of the medium in 250 mL flasks at room temperature (30–37 °C) for 48 h. The optimum conditions for L-LA production (236 mg/mL) were 5% NaCl under aerobic conditions. However, anaerobic conditions with 0% NaCl produced a higher L-LA yield than with 5% NaCl. Typically, LAB grow well without O₂, though some can be inhibited by its presence [73]. LAB are facultatively anaerobic and produce lactic acid in the presence of glucose [74,75,76]. In the current study, C. farciminis KUJ 25-S showed a preference for producing L-LA under anaerobic conditions, particularly without NaCl, suggesting a cost-effective L-LA production method by eliminating the need for air supply or removal devices and NaCl removal during purification. The current study demonstrated the benefits of using anaerophilic LAB that can tolerate oxygen, aligning with Dedenaro et al.’s findings with C. farciminis ATCC 29644 [35]. Other studies have found aerobic conditions optimal for glucose-based L-LA production such as with L. plantarum [75] and Bacillus coagulans [77]. L. plantarum produced L-LA and D-LA during active growth in glucose broth aerobically but switched to acetate production as the glucose became depleted [75,78,79]. C. farciminis KUJ 25-S produced high L-LA levels under osmotic stress (NaCl), revealing its aerotolerance and adaptation to its ecological niche.

The observation that peak L-LA production occurred under aerobic conditions in the presence of 5% NaCl (Figure 6) may reflect a complex interplay between salt-induced metabolic adjustments and altered intracellular redox homeostasis. Salt stress is known to rewire carbohydrate metabolism and stress-response proteins in LAB, promoting energy-saving adjustments and osmoprotectant accumulation that can sustain metabolic flux toward lactic acid under otherwise inhibitory conditions [70]. Under aerobic or microaerobic conditions, certain LAB can also transition to an oxidative respiratory metabolism when electron-transport cofactors (e.g., heme or menaquinone) or the requisite genetic determinants (cyd operon) are present; such respiration can provide alternative NADH oxidation routes, reduce NADH pressure on lactate dehydrogenase (LDH), and thereby alter flux distribution and end-product profiles [80]. Moreover, experimental and genetic studies indicate that diversion of NADH from LDH toward respiratory or NADH-oxidase pathways (or modulation of LDH activity) can change the rate of lactate formation and its stereochemical outcome, offering a plausible mechanism for the strain’s high L-LA productivity under aerated and saline conditions [81]. Taken together, these lines of evidence suggest that in KUJ 25-S 5% NaCl plus aeration may promote redox balancing and membrane/transport adaptations that favor high flux through L-LDH while maintaining cellular viability—hypotheses that can be directly tested by measuring intracellular NAD⁺/NADH ratios, oxygen uptake rates, transcript/protein levels of LDH, NADH oxidases and cyd genes, and by assessing the effect of heme/menaquinone supplementation or LDH inhibition on product profiles.

4. Conclusions

In conclusion, the study revealed that C. farciminis KUJ 25-S, isolated from fermented fish, holds considerable potential for L-LA production. This strain had high tolerance to various stress conditions, including temperature, osmotic stress (glucose and NaCl), acidic and alkaline environments, and aeration. C. farciminis KUJ 25-S produced L-LA with high optical purity (>95%) across broad ranges of pH (4–11) and temperature (25–55 °C), with or without NaCl, exhibiting both aerotolerance in the presence of NaCl and glucose and anaerophilic properties in their absence. The strain’s ability to grow and produce L-LA in alkaline environments and under osmotic stress with glucose concentrations of 10–20% further supports its industrial applicability. Optimal L-LA production was observed under aerobic conditions with glucose, presenting a cost-effective method that eliminates the need for air supply or NaCl removal devices. The strain’s robust performance under acidic, saline, and osmotic stress likely arises from coordinated membrane remodeling, osmoprotectant accumulation, and redox-regulated L-lactate dehydrogenase activity, supporting high productivity and stereochemical fidelity, which underscores its suitability for industrial-scale bioprocesses [82,83,84]. The performance comparison in

Table 2. Comparison of L-LA production performance and environmental stress tolerance reported for selected LAB strains relative to C. farciminis KUJ 25-S.

Strain	Process Type/Condition/Substrate	Titer (g/L)	Yield (g/g Substrate)	Optical Purity (% L-LA)	Key Note/Stress-Relevant Trait	Reference
C. farciminis KUJ 25-S	Batch/controlled (with 5% NaCl + aeration)/glucose	265	NR	97	Candidate strain for industrial bioprocessing under harsh conditions/ Strong multi-stress tolerance (pH, NaCl, osmotic, aeration)	This study
Bacillus coagulans (arr4; highest reported example)	Exponential fed-batch (high-performance)/ glucose	206.8 (highest reported)	NR	NR	Thermotolerant, engineered/optimized process report highest titers in literature	[85]
Bacillus coagulans H-2	Long-term repeated fed-batch/glucose	203.3	NR	NR	Very high and stable titer/productivity across batches (thermotolerant).	[86]
Pediococcus acidilactici ZY271	Batch/Wheat straw (xylose)	130.8	94.9	NR	Engineering strain/NR for stress tolerance	[87]
L. plantarum	Batch/Hydrolysate from seaweed	20	NR	NR	Enhanced saline tolerance following adaptive laboratory evolution/Demonstrates improvement in stress adaptation potential	[88]

NR: Not reported. The alphanumeric suffix following the species name represents a laboratory-designated strain code.

highlights the strong industrial promise of C. farciminis KUJ 25-S. To deepen process understanding, future work will involve bioreactor-scale kinetic studies that resolve time-course trajectories of biomass accumulation, substrate utilization, and L-LA formation. These datasets will enable precise determination of µ, volumetric productivity, and Yp/s, forming a quantitative basis for mass-balance evaluation and data-driven process optimization. The resulting process insights will directly advance the downstream objective of converting biologically derived lactic acid into PLA.