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Application of Box-Behnken Design for Optimization of Poly-?-Glutamic Acid Production by Bacillus licheniformis SAB-26
Abstract
Optimization of poly-γ-glutamic acid (PGA) produced by the glutamate-independent Bacillus licheniformis SAB-26 has been earned out using response surface methodology. Predicted maximum PGA yield (57.9 g L -1) was calculated using non-linear optimization algorithm powered by Microsoft Excel, where the optimized medium composition was (g L-1): casein hydrolysate, 12.2; ammonium sulfate, 20; K2HPO4, 27 and KH2PO4, 27 after 72 h. A verification experiment was carried out to compare predicted and experimental results, where experimental yield reached (59.9 g L-1) in a good sign of model validity. Pulsed feeding experiment of citrate at different time intervals revealed increasing the polymer production to 88 g L-1.
... They were able to obtain 88 g/L of product concentration using two pulsed feeding of citrate at different time intervals. [10] A culture medium named C was introduced primarily for the Bacillus licheniformis ATCC 9945 a strain. Another medium named E medium was recommended later to further increase c-PGA production by this strain (both theses culture mediums are displayed in Table 1). ...
... Therefore, to further increase c-PGA production, two-pulse feeding was considered for further investigation. These results are consistent with the investigation of the effect of different numbers of feeding pulses on the reported production amount by Abdel-Fattah et al. [10] that increasing the feeding pulse numbers from 2 to 4 reduced c-PGA production. The production enhancement of the two-pulse strategy is attributed to the fact that enough nutrients are provided to create a sudden stimulus. ...
... The reference also argued that a two pulse feeding was best for maximum production (although using a different bacterial strain). [10] Table 2 (experiments rows 7,15,16) shows the effect of changing the addition time of calcium (Ca) and manganese (Mn) of the culture medium from common 24 hours to 8 and 0 hours on c-PGA production. It can be seen that by changing the addition time of manganese and calcium ions from 24 to 8 hours, there is not much change in the c-PGA production, but further reducing it to the start of the fermentation process increased production by more than 10% to 60 g/L. ...
Poly-γ-glutamic acid (γ-PGA) is a versatile biopolymer with widespread applications in the food, pharmaceutical, and medical industries. One of the main challenges in expanding γ-PGA industrial applications is the high cost of production. Developing an efficient and low-cost fermentation process such as bacterial cultivation with pulsed feeding can significantly reduce production costs. Thus, initially, a new pulsed feeding strategy of citrate and glutamate was developed for γ-PGA production enhancement in the fed-batch culture of Bacillus licheniformis ATCC 9945a. Then, the effects of pulse number, feeding amount in each pulse, feeding times in pulse-feeds, the addition time of calcium and manganese solutions to the culture medium, the pH of the added citrate solution, the concentration of feedstock solutions of pulse-feeds on γ-PGA production were investigated. Under optimal conditions: feeding two pulses at 8 and 24 hours of batch culture, 20 g citrate and glutamate per liter of culture medium per pulse (about 52 mL of each of citrate and glutamate feeding solutions prepared with a concentration of 384 g/L by adding distilled water) about 88±4 g/L of γ-PGA was obtained. It is one of the highest values ever reported for γ-PGA production with Bacillus licheniformis ATCC 9945a, of course with a much simpler process than the other fed-batch processes.
... The MFC analysis and its electrical power measurement were carried out. Current (I) Optimization of the anolyte composition in MFC using the Plackett-Burman design (PBD) For the evaluation of the relative significance of 10 variables, the Plackett-Burman experimental design was used according to a previous study (Abdel-Fattah et al., 2007) [20] with some modifications. These factors include growth medium components and other physical parameters. ...
... The MFC analysis and its electrical power measurement were carried out. Current (I) Optimization of the anolyte composition in MFC using the Plackett-Burman design (PBD) For the evaluation of the relative significance of 10 variables, the Plackett-Burman experimental design was used according to a previous study (Abdel-Fattah et al., 2007) [20] with some modifications. These factors include growth medium components and other physical parameters. ...
The production of bioelectricity via the anaerobic oxidation of organic matter by microorganisms is recently receiving much interest and is considered one of the future alternative technologies. In this study, we aimed to produce electrical current by using facultative halophilic archaeon Natrialba sp. GHMN55 as a biocatalyst at the anode of a microbial fuel cell (MFC) to generate electrons from the anaerobic breakdown of organic matter to produce electrical current. Since the MFC's performance can be affected by many factors, the Plackett-Burman experimental design was applied to optimize the interaction between these factors when tested together and to identify the most significant factors that influence bioelectricity generation. We found that the factors that significantly affected electrical current generation were casein, inoculum age, magnet-bounded electrodes, NaCl, resistor value, and inoculum size; however, the existence of a mediator and the pH showed negative effects on bioelectricity production, where the maximum value of the 200 mV voltage was achieved after 48 h. The optimum medium formulation obtained using this design led to a decrease in the time required to produce bioelectricity from 20 days (in the basal medium) to 2 days (in the optimized medium). Also, the overall behavior of the cell could be enhanced by using multiple stacked MFCs with different electrical configurations (such as series or parallel chambers) to obtain higher voltages or power densities than the single chambers where the series chambers were recorded at 27.5 mV after 48 h of incubation compared with 12.6 mV and 1.1 mV for parallel and single chambers, respectively. These results indicate that the order of preferred MFC designs regarding total power densities would be series > parallel > single.
... Furthermore, β12, β13, and β23 represent the cross-product coefficients, while β11, β22, and β33 represent the quadratic coefficients. To determine the optimal predicted response and coefficients for the variables, Microsoft Excel 2007 was used for the calculations [20,21]. ...
Extreme halophilic archaea that can live in high saline environments can offer potential applications in different biotechnological fields. This study delves into the fascinating field of halophilic archaea and their ability to produce biosurfactants. Some strains of haloarchaea were isolated from Wadi El-Natrun and were screened for biosurfactants production in a standard basal medium using emulsification index assay. Two strains were chosen as the potential strains for surface tension reduction. They were identified as Natrialba sp. BG1 and N3. The biosurfactants production was optimized and the produced emulsifiers were partially purified and identified using FTIR and NMR. Sequential statistical optimization, Plackett–Burman (PB) and Box–Behnken Designs (BBD) were carried out using 5 factors: oil, NaCl, casamino acids, pH, and inoculum size. The most significant factors were used for the next Response Surface Methodology experiment. The final optimal conditions for biosurfactants production were the inoculum size 2% pH 11 and NaCl 250 g/L, for Natrialba sp. BG1 and inoculum size 2.2%, pH 10 and NaCl 100 g/L for Natrialba sp. N3. The produced biosurfactants were tested for wound healing and the results indicated that Natrialba sp. BG1 biosurfactants is more efficient than Natrialba sp. N3 biosurfactants. Biosurfactants extracts were tested for their cytotoxic effects on normal cell line as well as on different cancer cells using MTT assay. The findings demonstrated that varying concentrations of the biosurfactants (31.25, 62.5, 125, 250, 500 and 1000 µg/mL) exhibited cytotoxic effects on the cell lines being tested. Additionally, the outcomes unveiled the presence of anti-inflammatory and antioxidant properties for both biosurfactants. Consequently, they could potentially serve as natural, safe, and efficient novel agents for combating cancer, promoting wound healing, and providing anti-inflammatory and antioxidant benefits.
... This design helps to illustrate and identify the effect of each parameter on the bioprocess and their interaction with each other (El-Hamid et al., 2018). It has also been found to be helpful in attaining increased gene expression and enzyme production (Abdel-Fattah et al., 2007;Halder et al., 2013;Meng et al., 2015). The screened variables/components significantly affecting the biodegradation process can then be further optimized using response surface methodology (RSM) (Darvishmotevalli et al., 2019). ...
... and β44 are quadratic coefficients27,54 . Variables maximal predicted response and coefficients calculations were carried out using Microsoft Excel 2007.Recovery of the biosurfactant. ...
Halophilic archaea is considered an promising natural source of many important metabolites. This study focused on one of the surface-active biomolecules named biosurfactants produced by haloarchaeon Natrialba sp. M6. The production trend was optimized and the product was partially purified and identified using GC–Mass spectrometry. Sequential optimization approaches, Plackett–Burman (PB) and Box–Behnken Designs (BBD) were applied to maximize the biosurfactants production from M6 strain by using 14 factors; pH, NaCl, agitation and glycerol; the most significant factors that influenced the biosurfactant production were used for Response Surface Methodology (RSM). The final optimal production conditions were agitation (150 rpm), glycerol (3%), NaCl (20.8%), pH (12) and cultivation temperature (37°C). GC–Mass spectrometry for the recovered extract revealed the presence of a diverse group of bipolar nature, hydrophobic hydrocarbon chain and charged function group. The majority of these compounds are fatty acids. Based on results of GC–MS, compositional analysis content and Zetasizer, it was proposed that the extracted biosurfactant produced by haloarchaeon Natrialba sp. M6 could be a cationic lipoprotein. The antiviral activity of such biosurfactant was investigated against hepatitis C (HCV) and herpes simplex (HSV1) viruses at its maximum safe doses (20 μg/mL and 8 μg/mL, respectively). Its mode of antiviral action was declared to be primarily via deactivating viral envelopes thus preventing viral entry. Moreover, this biosurfactant inhibited RNA polymerase- and DNA polymerase-mediated viral replication at IC50 of 2.28 and 4.39 μg/mL, respectively also. Molecular docking studies showed that surfactin resided well and was bound to the specified motif with low and accepted binding energies (ΔG = − 5.629, − 6.997 kcal/mol) respectively. Therefore, such biosurfactant could be presented as a natural safe and effective novel antiviral agent.
... and β44 are quadratic coefficients27,54 . Variables maximal predicted response and coefficients calculations were carried out using Microsoft Excel 2007.Recovery of the biosurfactant. ...
The biosurfactant applications produced by haloalkaliphilic archaea
... The confidence level was analyzed to determine the good fit of the equation. 17,31 ...
Background:
Gamma-polyglutamic acid (γ-PGA) is a microbially produced non-toxic peptide biopolymer which is gaining grounds in many biotechnological fields and has a wide range of applications.
Objectives:
In this study, the characteristics of γ-PGA produced by Bacillus megaterium isolated from an oil seed were determined, while the nutritional requirements of the bacterium were optimized using a predictive 15 factor-16 run Plackett-Burman experimental design.
Material and methods:
The main effect of each factor, the interaction and quadratic effects of the factors on optimized production were determined from Box-Benkhen model using Dell Statistica v. 12 and 13 software. Bacillus megaterium UP47 produced the highest γ-PGA (16.33 g/L) out of 56 spore-forming Bacillus strains isolated from soil, water and fermented food samples.
Results:
Hydrolysates of the produced γ-PGA had a retention factor which corresponded to the L-glutamic acid standard (retention factor (rf) 0.35), while high-definition fourier transform infrared (FT-IR) spectroscopic imaging showed characteristic peaks representative of the active bonds present in γ-PGA. The γ-PGA at a concentration as low as 50 mg/100 mL exerted antimicrobial inhibitions against test pathogens. A 2.00 w/v γ-PGA solution had 11 mm and 13 mm inhibition zones against Staphylococcus aureus and Shigella dysenteriae, respectively. A second order polynomial equation for prediction of γ-PGA was derived as: γPGA yield = 3316.061 - 449.708A + 9.036A2 - 139.813B + 3.095B2 - 7.699C - 0.164C2 + 13.116AB - 0.087AB2 - 0.248A2B + 3.781AC - 0.076A2C - 0.394BC. It showed an increase in γ-PGA yield with increasing L-glutamic acid and biotin, but a decrease with yeast extract.
Conclusions:
Bacillus megaterium UP47 had a maximum γ-PGA yield of 54 g/L and 62 g/L, respectively, from the Plackett-Burman and Box-Benkhen design, thereby resulting in an appreciable increase in polymer yield after the optimization process with a 95% confidence level.
Poly (γ-glutamic acid) is a versatile biopolymer that can be used on an industrial scale if efficient methods are developed to increase production. In this study, first, based on the central composite design method of the response surface module, the effect of operational variables including temperature in the range of 30-44 °C, pH 4.5-8.5, and stirring in the range of 600-1000 rpm on poly (γ-glutamic acid) production was investigated in the batch fermentation of Bacillus licheniformis ATCC 9945a for the first time. Under optimal conditions viz. T of 37.4 °C, pH of 6.6, and agitation rate of 784.2 rpm, 15.5 g/L γ-PGA was obtained. According to the statistical analyses, adjusted R2 was 0.9572, and analysis of variance explicated that T-T, pH-pH, and agitation-agitation effects indicated the lowest p-values and had the most significant influence on biopolymer synthesis. Under the optimal conditions, glutamate (a novel feed) pulse feeding (as poly (γ-glutamic acid)-based monomer) was optimized, for the first time, using the one-factorial method to achieve a maximum of 42.13 g/L of biopolymer production by the two-pulsed feeding method. The chemical confirmation and physical characterization of the product indicated a pure poly (γ-glutamic acid) sample suitable for biological, biomedical, and biopharmaceutical applications.
Poly-γ-glutamic acid (γ-PGA) is a beneficial, biocompatible, and biodegradable biopolymer. These properties have been led to the development of the use of this compound in various industries such as bio-medicine, biopharmaceutical, biotechnology, and tissue engineering. The limitation of the industrial development of γ-PGA is the high cost of its production. To reduce γ-PGA production costs, various strategies are used, such as culture medium optimization using inexpensive compounds, the development of efficient cultivation processes of batch and fed-batch. In this research, first, an efficient batch culture medium was developed to produce γ-PGA of Bacillus licheniformis ATCC 9945a. Then, the γ-PGA production increased by the pulsed feeding method and its optimization. By optimal culture medium development, the production of this product in batch culture was increased from 11 g/L to 47 g/L. Then, using the optimized pulsed feeding strategy of citrate (γ-PGA precursor), γ-PGA production was increased to 59.5 g/L, which is one of the highest production values reported with this strain. To optimize two-pulse feeding, the effect of feeding times, stock citrate solution concentration, and time of calcium and manganese solutions addition on γ-PGA production were investigated and optimized. Finally, FTIR confirmed the chemical structure of poly gamma glutamic acid, and the study of γ-PGA morphological properties with SEM showed a nanostructure ideal for biological applications.
Statistically based experimental designs were applied to optimize the production of avicelase by a recently isolated endospore forming strain, WL1, that showed 99.8% 16S rDNA identity to Geobacillus stearothermophilus. Seven culture parameters were examined for their significance as effectors of avicelase expression using the Plackett-Burman factorial design. Concentrations of avicel, yeast extract and ammonium sulfate were the most significant factors affecting the process of enzyme production. The second optimization step was to figure out the levels of these three independent variables that generate maximum avicelase activity, using the Box-Behnken design. Maximal enzyme activity (0.8 U mL-1), which is approximately two folds the activity expressed in the basal medium, has been predicted at concentrations of (g L-1): Avicel (42), yeast extract (3.6) and ammonium sulfate (0.8). A verification experiment was accomplished and revealed approximately 99% model validity.
A locally isolated thermostable Bacillus strain producing polyglutamic acid (PGA) was characterized and identified based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to Bacillus licheniformis. To evaluate the effect of different culture conditions on the production of PGA, Plackett-Burman factorial design was carried out. Fifteen variables were examined for their significance on PGA production. Among those variables, K(2)HPO(4), KH(2)PO(4), (NH(4))(2)SO(4) and casein hydrolysate were found to be the most significant variables that encourage PGA production. A correlation between cellular growth, PGA and the produced traces of polysaccharides was illustrated. An inverse relationship practice between cell dry weights and the produced PGA was demonstrated. On the other hand, a direct proportional relation was shown between polysaccharides on one side and cell dry weight and produced PGA on the other. The pre-optimized medium, based on statistical analysis, showed a production of 33.5 g/l PGA, which is more than three times the basal medium.
The biosynthesis by specific micro-organisms of polyamides consisting of glutamic acid repeating units that are linked between the à-amino and γ-carboxylic acid functionalities, (γ-PGA), have been known for some time. Reviews by Housewright1 and Troy2 on this interesting biopolymer have been published.
A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS-PAGE, and measurement of viscosity, and calculation of the D/L ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.
Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35gl–1 with a productivity of 1gl–1h–1 by pulsed-feeding of citric acid (1.44gh–1) and l-glutamic acid (2.4gh–1) when citric acid was depleted from the culture medium.
Bacillus subtilis C1, a bacterium capable of producing a glycerol and γ-PGA bioconjugate, was studied. C1 is a glutamate independent bacterium, which produces the γ-PGA derivative in the absence of l-glutamate. A large amount of the bioconjugate, 21.4 g/l, was produced when the bacteria were cultured in medium T at 37 °C, 150 rpm for 6 days. The number-average molecular weight (Mn) of the bioconjugate was over 1 × 107 as determined by gel permeation chromatography, amino acid analysis showed only glutamic acid peak and the 1H NMR spectrum showed chemical shifts of both γ-PGA and glycerol. The d-glutamate content was over 97% in every bioconjugate produced under the conditions used and the d-glutamate content was indifferent to the Mn2+ concentrations under study. During bioconjugate production, an intracellular glutamate racemase activity was detected, suggesting the enzyme is involved in the d-glutamate supply. The molecular weight of the bioconjugate varied with salt concentration in the medium; the molecular weight decreased by a factor of approximately 10 from 7.94 × 106 to 0.73 × 106 Da at 4-day cultivation time for cultures, which contained 0.05 and 5% NaCl, respectively. The viscosity of the bioconjugate produced by C1 is significantly higher than that of γ-PGA itself. This bioconjugate is the first example of γ-PGA derivative directly produced by microbes; thus, it is a unique biomaterial.
Bacillus licheniformis ATCC 9945A was grown on Medium E in batch fermentations in which the pH was maintained at 5.5., 6.5, 7.4, and 8.25. The effects of pH on cell growth, carbon source utilization, and gamma-polyglutamic acid (gamma-PGA) production, molecular weight, and polymer stereochemistry were determined. The gamma-PGA yield was highest (15 g/L, 96 h growth time) at pH 6.5. The increase in gamma-PGA formation at pH 6.5 corresponded with a relatively high specific production rate at high gamma-PGA concentration (0.09 h(-1), approximately 15 g/L gamma-PGA). In contrast, the specific gamma-PGA production rates at fermentor pH values of 5.5 and 7.4 decreased significantly for gamma-PGA fermentor yields > approximately 5 g/L. Interestingly, alteration of the medium pH had little to no significant effects on the product quality as measured by stereochemical composition and molecular weight. While glutamate and glycerol utilization were similar as a function of pH, citrate consumption increased at pH 6.5, indicating that the formation of gamma-PGA from citrate at pH 6.5 was of increased importance. The effect of aeration was evaluated by increasing the agitation speed (250 to 800 rpm) and aeration rate (0.5 to 2.0 L/min) at pH 6.5, the pH of maximal gamma-PGA production. Increased aeration resulted in doubling of the cell dry weights (2 to 4 g/L), increasing gamma-PGA yields (6.3 to 23 g/L by 48 h) and increasing in the maximum gamma-PGA-specific production rate (0.09 to 0.11 h(-1)). Other effects of increased agitation included a rapid depletion of glutamate and citrate (by 50 h) and a decrease in product molecular weight. Despite the increase in agitation and aeration, oxygen limitation of the culture was not avoided, because the partial pressure decreased to <1.0% by 29 h.