L-Glutamic acid monosodium

Isolation of a novel poly--glutamic acid-producing Bacillus licheniformis A14 strain and optimization of fermentation conditions for high-level production

ABSTRACT
In the present study, bacteria producing poly-c-glutamic acid were isolated from marine sands, and an efficient producer identified. c-PGA was rapidly screened by thin-layer chromatography and UV spectrophotometer assay. Media optimization was carried out, and for the cost-effective production of c-PGA, monosodium glutamate was used as the substrate for the synthesis of c-PGA instead of glutamic acid. Lastly, Plackett–Buman design (PB) and Response surface method- ology (RSM) were used to determine significant media components and their interaction effect to achieve maximum c-PGA production. With this integrated method, a bacterial strain with a high yield of c-PGA was obtained rapidly, and the production was increased up to 37.8 g/L after optimization.

Introduction
Poly-c-glutamic acid (c-PGA) is an anionic homo-poly- amide composed of D-glutamic acid and L-glutamic acid connected with amide bonds between c-carboxyl acid and a-amino groups; c-PGA is biodegradable, non-immuno- genic, edible, water-soluble and nontoxic to humans. Because of these properties, it has wide-ranging applications in the field of food, medicine, cosmetics, agriculture, and waste-water treatment.[1–4] Gram-positive bacteria, especially those belonging to the genus Bacillus, are the significant producers of c-PGA.[5] Bacillus subtilis and B. licheniformis are reported to be the efficient producers of c-PGA.[6,7] The Gram-negative bacterium Fusobacterium nucleatum, the archaebacterium Natrialba aegyptiaca, and eukaryotes such as Hydra vulgaris and Cnidariasp. are also capable of pro- ducing c-PGA.[8–11]c-PGA producing strains are divided into two groups, L-glutamic acid-dependent and -independent strains, depending on the requirement of L-glutamic acid for bio- polymer production.[12] L-glutamic acid-dependent strains, such as B. subtilis and B. licheniformis, are most widely studied because of their ability to biosynthesize c-PGA at high levels on the addition of L-glutamic acid in the medium.[7,13] Other factors that influence c-PGA production include carbon and nitrogen sources, aeration, agitation, pH, and temperature.[1,14]Low-cost substrates and efficient strains are prerequisites for commercially viable fermentative production of biopoly- mers.[15,16] Though c-PGA can also be produced by chemical synthesis and biotransformation, these routes are not cost-effective and lead to environmental pollution.[17,18] Here, we report the isolation of a novel poly-c-glutamic acid-producing bacterium and optimization of fermentation medium using a low-cost substrate for high-level production of this biopolymer.

Samples were collected from marine sands of the Mumbai region, India. Bacillus species were isolated by streaking samples on HiCrome Bacillus agar media (HIMEDIA Laboratories, Mumbai, India), which contains (per liter) peptone 10 g, meat extract 1 g, D-mannitol 10 g, sodium chloride 10 g, chromogenic mixture 3.20 g, phenol red 0.025 g and agar 15 g (pH 7.1). After incubation at 30◦ C for
24 h, mucoidal growth and color of the colony were observed, and isolated colonies were selected for further screening.Isolated colonies were inoculated in a 25 ml screening medium consisting of (per liter) glucose 60 g, citric acid 5 g, monosodium glutamate 25 g, MgSO4·7H2O 0.5 g, MnSO4·H2O 0.1 g, K2HPO4 1.0 g, CaCl2·2H2O 0.2 g and FeCl3·6H2O 0.02 g (pH 7.0) in 250 ml baffled flasks and incubated aerobically at 150 rpm and 37 ◦C for 48 h.The presence of c-PGA in the highly viscous broth was con- firmed using UV spectrophotometry analysis (see below). The isolate producing the highest amount of c-PGA was identified using 16S rRNA sequencing and taxonomical characterization using EzBiocloud[19] and based on phylo- genetic analysis named Bacillus licheniformis A14 (GenBank accession number MH763850).Bacillus licheniformis A14 was grown in 25 mL of screening medium in a 250 ml baffled Erlenmeyer flask and incubated aerobically at 37 ◦C and 150 rpm for 24 h. A 5% (v/v) inocu- lum from this seed culture was inoculated in 25 ml of the same medium in a 250 ml baffled flask cultured aerobically in a rotary shaker at 28 ◦C and 150 rpm for 96 hours experiment described above. Variables that were significant at a 95% confidence level (p < 0.05) from the regression ana- lysis were selected to estimate the significant effect of each variable.[20] Experiments were designed using Design-Expert 10.0.3 (Stat-Ease, USA). (Table 2). Response Surface Methodology (RSM) I-optimal design was carried out to find out optimum concentrations of vari- ables for c-PGA production. Twenty-two experiments were carried out, and c-PGA production was determined. The variables used for optimization were monosodium glutam- ate, citric acid, MgSO4, and MnSO4. Statistical significance was established at the 95% confidence level (p < 0.05) and the interaction effects of each variable and predicted value were obtained from the following quadratic equation:Optimization of culture conditions using one factor at a time method Y = ß0 + X ßiXi + X biiX2 + Xn—1 Xn bijXiXj Concentrations of carbon sources (glucose and glycerol) and nitrogen sources (ammonium sulfate, ammonium chloride, ammonium nitrate, and sodium nitrate) were varied, and the effect on c-PGA production was determined.The effect of pH and temperature on c-PGA production was tested between pH 6.0 and 8.0 and 25 ◦C and 40 ◦C, respectively. To study the effect of seed age on the fermenta- tion process, inocula of different ages (8 h, 10 h, 16 h, 20 h, and 24 h) were used. Inoculum volumes (v/v) tested were 1, 2, 5, and 10%. c-PGA concentration was determined at vari- ous time points, from 6 h to 96 h.Nine variables(glucose, citric acid, monosodium glutamate, (NH4)2SO4, MgSO4·7H2O, MnSO4·H2O, K2HPO4, CaCl2·2H2O, and FeCl3·6H2O) were designated as X1, X2, X3, X4, X5, X6, X7, X8, and X9, respectively. X10 and X11 are dummy variables (Table 1). All variables were tested at a low level (—) and a high level (+). Culture conditions were those that were optimized using one factor at a time Y is the predicted response; b0 is a constant; bi is the lin- ear coefficient; bii is the squared coefficient, and bij is the interaction coefficient. Regression analysis and response sur- face plots were achieved using Design-Expert version 10.0.3(Stat-Ease, USA). Ethanol precipitation was used to extract c-PGA.[21] The culture broth after fermentation was diluted using deionized water, and pH was reduced to 3 with 85% orthophosphoric acid. Cells were separated by centrifuging at 7280 RCF for 20 min at 4 ◦C. Four volumes of cold ethanol were added to the supernatant, and the mixture was kept for 2 h at 4 ◦C. c-PGA was obtained by centrifugation at 7280 RCF for 15 minutes at 4 ◦C, dissolved in deionized water, and centri- fuged to remove any insoluble matter. The aqueous c-PGA solution was desalted by dialysis against distilled water for 24 h with three water exchanges and finally lyophilized to prepare pure c-PGA.Absorbance at 216 nm (UV/Vis spectrophotometer, EquiptronicsVR EQ-826, India) was used to determine the c-PGA concentration. A standard curve was obtained by plot- ting the average blank corrected absorbance of the standard at 216 nm against its concentration (20–200 lg/ml).[22] Purified c-PGA was hydrolyzed using 6 M HCl at 100 ◦C for 18 h in an airtight tube. Residual HCl was removed by evap- oration, and the hydrolyzed product was dissolved in dis- tilled water. Thin-layer chromatography (TLC) was performed on a silica plate (Merck, Germany) with a solvent system 96% ethanol-water (67:37, w/w) and butanol-acetic acid-water (3:1:1, w/w).[23] Amino acids were detected by spraying the plate with 0.2% ninhydrin in acetone. Results and discussion Fifty-six Bacillus isolates from soil samples were screened for c-PGA production using UV spectroscopy, and an iso- late that produced the highest concentration of c-PGA was selected. Based on 16S rRNA gene sequencing, the strain was found to be 99.92% similar to Bacillus licheniformis ATCC 14580(T) and was therefore designated as Bacillus licheniformis A14.In one factor at a time optimization, only one factor which is to be optimized is changed while keeping other media components and culture conditions constant. The optimal values of all cultivation parameters were illustrated in Figure 1. Optimal c-PGA and biomass production were determined to be at pH 7, in agreement with prior studies, which employed pH between 6 and 7[21,23] while 33◦ C was determined to be the optimal temperature. An inoculum of 16 h culture at an inoculum size of 5% led to maximum c-PGA production. c-PGA production was found to com- mence after 12 h, reaching its maximum 48 h, after which it starts decreasing due to assimilation.[24] Glucose and glycerol were evaluated as carbon sources, and the former was determined to lead to maximum c-PGA and biomass production. Glucose and glycerol are most often used for c-PGA production.[1,25] Amongst the inorganic nitrogen sources analyzed, ammo- nium sulfate supported maximum c-PGA and biomass pro- duction. Most studies have employed inorganic nitrogen sources for c-PGA production since the availability of free ammonium is higher in inorganic nitrogen sources.[26,27] It was observed from the above optimization studies that, as the cell density increases, the c-PGA production also increases. Therefore, much work has to be carried out on the nutritional requirements to improve cell density for increased c-PGA production. The citric acid in the medium plays a significant role in the polymerization of glutamic acid. Both citric acid and glu- tamic acid are precursors of the polymer, while glucose or glycerol acts as co-substrate.[26,28,29] In the present study, monosodium glutamate was used as a substrate instead of L-glutamate as it is relatively cheaper.Plackett–Burman design was used to determine which media component influences c-PGA production. The citric acid (X2), monosodium glutamate (X3), MgSO4 (X5), and MnSO4 (X6) had significant effects on c-PGA production (p > 0.05) Table 2. The model was highly significant since the “Pred R-Squared” of 0.8008 agrees with “Adj R-Squared” of 0.8935. The “Adeq Precision” ratio of 14.073 indicates an adequate signal. Usually, a signal to noise ratio of greater than 4 is desirable. These four media components play a substantial role in the production of c-PGA since monoso- dium glutamate acts as a substrate, while citric acid is imperative in the polymerization of glutamate. Chen et al. reported that the stereochemical and biosynthesis of c-PGA were substantially affected by Mn2+ and Mg2+ concentra- tion.[30] Also, the Mn2+ plays a crucial role in improving cell growth and viability and supports the utilization of dif- ferent carbon sources.[17] Therefore, the MgSO4 and MnSO4 in the medium act as a cofactor for c-PGA synthetase. The selected variables were further optimized using Response Surface Methodology (RSM).

Response Surface Methodology (RSM) I-optimal design was used to determine the optimum concentration of different variables for c-PGA production. I-optimal design is random, exact, and helps in minimizing the integral of the prediction variance across the factor space. The variables used for opti- mization were monosodium glutamate (A), citric acid (B), MgSO4 (C), and MnSO4 (D), which were selected based on the optimization carried out using Plackett-Burman design. A quadratic design matrix with actual values and responses is shown in Table 3. The p-values were used to confirm the significance of each one of the coefficients and to determine the interaction of tested variables.[31] Values of “Prob> F” less than 0.05 indicates that the model terms are significant.Therefore, from the results, it was observed that A, B, BC, A2, B2, and C2 are significant model terms. The absolute equation in conditions of coded factors is:c — PGA = +38.01 + 10.80 * A + 1.43 * B + 0.65 * C +0.54 * D + 0.21 * AB + 0.47 * AC +0.31 * AD — 0.95 * BC — 6.545E — 003 * BD —0.47 * CD — 7.38 * A2 — 2.80 * B2 —1.84 * C2 + 0.98 * D2

Figure 1. Optimization of culture conditions on c-PGA production. (A) Effect of pH; (B) Effect of temperature; (C) Effect of seed age; (D) Effect of inoculums size; (E). Effect of the incubation period; (F) Effect of carbon source; (G) Effect of inorganic nitrogen source.The ANOVA for the model is summarized in Table 4. The Model F-value of 126.79 implies that the quadratic model is significant. The “Adeq Precision” ratio of 30.204 indicates an adequate signal. Usually, a signal to noise ratio of greater than 4 is desirable. The contour plot was plotted for the pair-wise combination of selected variables, which was highly significant (Figure 2). The contour plot helps in the depiction of the response plotted alongside a combin- ation of numeric factors. From the central point of the con- tour plot, the best possible process was identified. The “point prediction” was also studied to find the optimum value of the combination of the four media components for the maximum production of c-PGA from the RSM tool. All predicted values were experimentally confirmed (Table 5). It was determined that the fermentation medium containing (per liter) monosodium glutamate 60 g, citric acid 19.9 g, MgSO4 0.10 g, and MnSO4 0.051 g yielded maximum bio- mass and c-PGA production of 37.8 g/L. This combination has high desirability of 0.99. The production profile of poly- c-glutamic acid under optimized condition was elucidated in (Figure 3). The titer of c-PGA from the newly isolated strain is com- paratively higher than that reported in the literature under L-Glutamic acid monosodium laboratory-scale cultivation using newly isolated strains.