Enhanced Poly-γ-Glutamic Acid Production by a Newly Isolated Bacillus halotolerans F29.

Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications. In this study, we isolated a novel γ-PGA-producing strain, Bacillus halotolerans F29. The one-factor-at-a-time method was used to investigate the influence of carbon sources, nitrogen sources, and culture parameters on γ-PGA production. The optimal carbon and nitrogen sources were sucrose and (NH4)2SO4, respectively. The optimal culture conditions for γ-PGA production were determined to be 37 °C and a pH of 5.5. Response surface methodology was used to determine the optimum medium components: 77.6 g/L sucrose, 43.0 g/L monosodium glutamate, and 2.2 g/L K2HPO4. The γ-PGA titer increased significantly from 8.5 ± 0.3 g/L to 20.7 ± 0.7 g/L when strain F29 was cultivated in the optimized medium. Furthermore, the γ-PGA titer reached 50.9 ± 1.5 g/L with a productivity of 1.33 g/L/h and a yield of 2.23 g of γ-PGA/g of L-glutamic acid with the optimized medium in fed-batch fermentation. The maximum γ-PGA titer reached 45.3 ± 1.1 g/L, with a productivity of 1.06 g/L/h when molasses was used as a carbon source. It should be noted that the γ-PGA yield in this study was the highest of all reported studies, indicating great potential for the industrial production of γ-PGA.

Production and molecular weight variation of poly-γ-glutamic acid using a recombinant Bacillus subtilis with various Pgs-component ratios.

Poly-γ-glutamic acid (PGA) has been of interest as a sustainable biopolymer in industrial applications. PGA biosynthesis in Bacillus subtilis is catalyzed by a transmembrane protein complex comprising PgsB, PgsC, and PgsA. To determine the Pgs component responsible for PGA overproduction, we constructed recombinants in which the promoter of the host-derived pgs gene was replaced with another host-derived gene promoter. These recombinants were then transformed using high-copy-number plasmids with various pgs-gene combinations to enhance Pgs component in different ratios. Subsequently, PGA production was investigated in batch cultures with l-glutamate supplemented medium. The recombinant strain enhanced with pgsB alone significantly overproduced PGA (maximum production 35.8 g/L) than either the pgsC- or pgsA-enhanced strain. The molecular weight of the PGA produced with the pgsB-enhanced strain was also greater than that for the pgsC- or pgsA-enhanced strain (approximately 10-fold). Hence, PgsB enhancement alone contributes to PGA overproduction with increased molecular weight.

Engineering Bacillus licheniformis as industrial chassis for efficient bioproduction from starch.

Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization.

Heterologous synthesis of poly-γ-glutamic acid enhanced drought resistance in maize (Zea mays L.).

Drought stress is the main factor restricting maize yield. Poly-γ-glutamic acid (γ-PGA), as a water-retaining agent and fertilizer synergist, could significantly improve the drought resistance and yield of many crops. However, its high production costs and unclear long-term impact on soil ecology limit its large-scale application. In this study, an environmentally friendly green material γ-PGA was heterologous synthesized in maize for the first time using the synthetic biology method. The genes (PgsA, PgsB, PgsC) participated in γ-PGA synthesis were cloned from Bacillus licheniformis and transformed into maize to produce γ-PGA for the first time. Under drought stress, transgenic maize significantly increased the ear length, ear weight and grain weight by 50 % compared to the control, whereas the yield characteristic of ear weight, grain number per ear, grain weight per ear and 100-grain weight increased by 1.67 %-2.33 %, 3.78 %-13.06 %, 8.41 %-22.06 %, 6.03 %-19.28 %, and 11.85 %-18.36 %, respectively under normal growth conditions. γ-PGA was mainly expressed in the mesophyll cells of maize leaf rosette structure and improved drought resistance and yield by protecting and increasing the expression of genes for the photosynthetic and carbon fixation. This study is an important exploration for maize drought stress molecular breeding and building resource-saving agriculture.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

Systematic engineering of Bacillus amyloliquefaciens for efficient production of poly-γ-glutamic acid from crude glycerol.

Microbial production of poly-γ-glutamic acid (γ-PGA) from non-food raw materials is a promising alternative to food feedstocks-based biosynthesis. A superior cell factory of Bacillus amyloliquefaciens for the efficient synthesis of γ-PGA from crude glycerol was constructed through systematic metabolic engineering. Firstly, some phase-dependent promoters were screened from B. amyloliquefaciens, which can be used for fine regulation of subsequent metabolic pathways. Secondly, the glycerol utilization pathway and the γ-PGA synthesis pathway were co-optimized utilizing the above-screened promoters, which increased the titer of γ-PGA by 1.75-fold. Then, the titer of γ-PGA increased to 15.6 g/L by engineering transcription factors degU and blocking competitive pathways. Finally, combining these strategies with an optimized fermentation process, 26.4 g/L γ-PGA was obtained from crude glycerol as a single carbon source (a 3.72-fold improvement over the initial strain). Overall, these strategies will have great potential for synthesizing other products from crude glycerol in B. amyloliquefaciens.

SwrA as global modulator of the two-component system DegSU in Bacillus subtilis.

The two-component system DegSU of Bacillus subtilis controls more than one hundred genes involved in several different cellular behaviours. Over the last four decades, the degU32Hy allele, supposedly encoding a constitutively active mutant of the response regulator DegU, was exploited to define the impact of this system on cell physiology. Those studies concluded that phosphorylated DegU (DegU∼P) induced degradative enzyme expression while repressing flagellar motility and competence. Recent experiments, however, demonstrated that flagella expression is enhanced by DegU∼P if SwrA, a protein only encoded by wild strains, is present. Yet, to promote motility, SwrA must interact with DegU∼P produced by a wild-type degU allele, as it cannot correctly cooperate with the mutant DegU32Hy protein. In this work, the impact of DegSU was reanalysed in the presence or absence of SwrA employing a DegS kinase mutant, degS200Hy, to force the activation of the TCS. Our results demonstrate that the role of SwrA in B. subtilis physiology is wider than expected and affects several other DegSU targets. SwrA reduces subtilisin, cellulases and xylanases production while, besides motility, it also positively modulates competence for DNA uptake, remarkably relieving the inhibition caused by DegU∼P alone and restoring transformability in degS200Hy strains.

Metabolic Engineering of Central Carbon Metabolism of Bacillus licheniformis for Enhanced Production of Poly-γ-glutamic Acid.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02 g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain.

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

Effects of cell physiological structure on the fermentation broth viscosity during poly-γ-glutamic acid production by Bacillus subtilis GXA-28.

The high viscosity of fermentation broth limited the further improvement of PGA titer. Our previous studies indicated that adding KCl to the medium could decrease the fermentation broth viscosity and improve the PGA titer. In order to clarify the reason, effects of cell physiological structure on the fermentation broth viscosity were investigated. Results from cell morphology observation showed that the reduction of cell aggregation caused by the weakened cross-linking between PGA and cells might be an important reason for the decrease in the fermentation broth viscosity. Besides, when 201.2 mM KCl was added to the medium, the zeta potential of cell surface decreased from - 70.48 ± 3.35 mV to - 81 ± 2.46 mV. The cell membrane integrity was reduced and permeability was enhanced. Furthermore, the percentage of lauric acid C12:0 in cell membrane increased by 12.36%, but palmitic acid C16:0 and stearic acid C18:0 decreased by 6.83% and 5.64%, respectively, which improved the fluidity of cell membrane. The above changes in cell membrane further affect the cross-linking between PGA and cells, thereby playing an important role in reducing the fermentation broth viscosity. This study provided some novel information for understanding the decrease of PGA fermentation broth viscosity by KCl.

CRISPRi-Based Dynamic Regulation of Hydrolase for the Synthesis of Poly-γ-Glutamic Acid with Variable Molecular Weights.

Poly-γ-glutamic acid (γ-PGA) is a decomposable polymer and has been useful in various industries. The biological functions of γ-PGA are closely linked with its molecular weight (MW). In this study, we established an efficient method to produce variable MWs of γ-PGA from renewable biomass (Jerusalem artichoke) by Bacillus amyloliquefaciens. First, a systematic engineering strategy was proposed in B. amyloliquefaciens to construct an optimal platform for γ-PGA overproduction, in which 24.95 g/L γ-PGA generation was attained. Second, 27.12 g/L γ-PGA with an MW of 20-30 kDa was obtained by introducing a γ-PGA hydrolase (pgdS) into the platform strain constructed above, which reveals a potential correlation between the expression level of pgdS and MW of γ-PGA. Then, a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was further designed to regulate pgdS expression levels, resulting in γ-PGA with variable MWs. Finally, a combinatorial approach based on three sgRNAs with different repression efficiencies was developed to achieve the dynamic regulation of pgdS and obtain tailor-made γ-PGA production in the MW range of 50-1400 kDa in one strain. This study illustrates a promising approach for the sustainable making of biopolymers with diverse molecular weights in one strain through the controllable expression of hydrolase using the CRISPRi system.

Simultaneous production of poly-γ-glutamic acid and 2,3-butanediol by a newly isolated Bacillus subtilis CS13.

Bacillus subtilis naturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-γ-glutamic acid (γ-PGA) production. 2,3-BD is a promising platform chemical in various industries, and co-production of the two chemicals has great economic benefits. Co-production of γ-PGA and 2,3-BD by a newly isolated B. subtilis CS13 was investigated here. The fermentation medium and culture parameters of the process were optimized using statistical methods. It was observed that sucrose, L-glutamic acid, ammonium citrate, and MgSO4·7H2O were favorable for γ-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 °C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/L L-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO4·7H2O was obtained by response surface methodology (RSM). The results show that the titers of γ-PGA and 2,3-BD reached 27.8 ± 0.9 g/L at 24 h and 57.1 ± 1.3 g/L at 84 h with the optimized medium, respectively. γ-PGA and 2,3-BD production by B. subtilis CS13 was significantly enhanced in fed-batch fermentations. γ-PGA (36.5 ± 1.1 g/L, productivity of 1.22 ± 0.04 g/L/h) and 2,3-BD concentrations (119.6 ± 2.8 g/L, productivity of 2.49 ± 0.66 g/L/h) were obtained in the optimized medium with feeding sucrose. The co-production of 2,3-BD and γ-PGA provides a new perspective for industrial production of γ-PGA and 2,3-BD. Key points • A strategy for co-production of γ-PGA and 2,3-BD was developed. • The culture parameters for the co-production of γ-PGA and 2,3-BD were studied. • RSM was used to optimize the medium for γ-PGA and 2,3-BD co-production. • 36.5 g/L γ-PGA and 119.6 g/L 2,3-BD were obtained from the optimum medium in fed-batch fermentation.

Poly-γ-glutamic acid production by Bacillus subtilis 168 using glucose as the sole carbon source: A metabolomic analysis.

The industrially relevant biopolymer poly-γ-glutamic acid (γ-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for γ-PGA synthesis were constructed. Efficient γ-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter Pveg or with the xylose-inducible promoter Pxyl. The carbon yield for γ-PGA increased by 129% to 0.131 C-mol C-mol-1 when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced γ-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a d-glutamic acid monomer content of 49-62%. To elucidate the consequences of γ-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in γ-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for γ-PGA synthesis from glucose.

Efficient Biosynthesis of Low-Molecular-Weight Poly-γ-glutamic Acid Based on Stereochemistry Regulation in Bacillus amyloliquefaciens.

Low-molecular-weight poly-γ-glutamic acid (LMW-γ-PGA) has attracted much attention because of its many potential applications in food, agriculture, medicine, and cosmetics. Enzymatic degradation is an efficient way for the synthesis of LMW-γ-PGA. However, the stereochemistry of γ-PGA limits the degradation of γ-PGA. This study identifies the role of γ-PGA synthase (pgsA) and glutamate racemase (racE) in the regulation of γ-PGA stereochemistry and demonstrates their combinational use for LMW-γ-PGA synthesis. First, the expression of pgsA and racE was enhanced, leading to improvements both in the molecular weight (Mw) and the d-glutamate proportion of γ-PGA. Then, an optimal combination of pgsA, racE, and γ-PGA hydrolase pgdS was constructed by exchanging the gene origins for the synthesis of LMW-γ-PGA. Finally, the Mw of γ-PGA was decreased to 6-8 kDa, which was much lower compared with the case without stereochemistry switching (20-30 kDa). This study provides a novel strategy to control the Mw of γ-PGA based on stereochemistry regulation and lays a solid foundation for synthesis of LMW-γ-PGA.

High-level production of poly-γ-glutamic acid from untreated molasses by Bacillus siamensis IR10.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer and has been applied in many fields. Bacillus siamensis SB1001 was a newly isolated poly-γ-glutamic acid producer with sucrose as its optimal carbon source. To improve the utilization of carbon source, and then molasses can be effectively used for γ-PGA production, 60cobalt gamma rays was used to mutate the genes of B. siamensis SB1001. RESULTS: Bacillus siamensis IR10 was screened for the production of γ-PGA from untreated molasses. In batch fermentation, 17.86 ± 0.97 g/L γ-PGA was obtained after 15 h, which is 52.51% higher than that of its parent strain. Fed-batch fermentation was performed to further improve the yield of γ-PGA with untreated molasses, yielding 41.40 ± 2.01 g/L of γ-PGA with a productivity of 1.73 ± 0.08 g/L/h. An average γ-PGA productivity of 1.85 g/L/h was achieved in the repeated fed-batch fermentation. This is the first report of such a high γ-PGA productivity. The analysis of the enzyme activities showed that they were affected by the carbon sources, enhanced ICDH and GDH, and decreased ODHC, which are important for γ-PGA production. CONCLUSION: These results suggest that untreated molasses can be used for economical and industrial-scale production of γ-PGA by B. siamensis IR10.

Optimized production of poly (γ-glutamic acid) (γ-PGA) using Bacillus licheniformis and its application as cryoprotectant for probiotics.

Bacteria produce poly (γ-glutamic acid) (γ-PGA), a polymer of l- or d-glutamic acid, as a defense response and have gained importance due to their applications in food and pharmaceutical industry. In the present investigation, production of γ-PGA using cost-effective carbon substrate, characterization of the produced polymer, and its application as cryoprotectant for selected freeze-dried probiotic bacteria were investigated. Central composite rotatable design of response surface methodology was used to study the main and the interactive effects of medium components: rice bran and casein peptone concentration. Rice bran at 35% (w/v) and casein peptone at 7.5% (w/v) were found to be optimal at an initial pH of 7.5, and incubation temperature of 37°C for 48 H produced 8.2 g/L γ-PGA on dry weight basis. The thermal properties such as melting temperature, heat of fusion, and thermal stability were also studied. Ten percent (w/v) of γ-PGA with 10 percent of sodium alginate (w/v) protected viability of Bifidiobacterium bifidum NCDC 235 and B. adolescentis NCDC 236 during freeze drying at -80 ËšC for 48 H. The γ-PGA synthesized by the reported bacterium with GRAS status is suitable for food and biomedical applications.

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

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

Biosynthesis of poly-γ-glutamic acid in Escherichia coli by heterologous expression of pgsBCAE operon from Bacillus.

AIMS: Poly-γ-glutamic acid (γ-PGA) is an excellent water-soluble biosynthesis material. To confirm the rate-limiting steps of γ-PGA biosynthesis pathway, we introduced a heterologous Bacillus strain pathway and employed an enzyme-modulated dismemberment strategy in Escherichia coli. METHODS AND RESULTS: In this study, we heterologously introduced the γ-PGA biosynthesis pathway of two laboratory-preserved strains-Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 into E. coli, and compared their γ-PGA production levels. Next, by changing the plasmid copy numbers and supplying sodium glutamate, we explored the effects of gene expression levels and concentrations of the substrate l-glutamic acid on γ-PGA production. We finally employed a two-plasmid induction system using an enzyme-modulated dismemberment of pgsBCAE operon to confirm the rate-limiting genes of the γ-PGA biosynthesis pathway. CONCLUSION: Through heterologously over-expressing the genes of the γ-PGA biosynthesis pathway and exploring gene expression levels, we produced 0·77 g l-1 γ-PGA in strain RSF-EBCAE(BS). We also confirmed that the rate-limiting genes of the γ-PGA biosynthesis pathway were pgsB and pgsC. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is beneficial to increase γ-PGA production and study the mechanism of γ-PGA biosynthesis enzymes.

Poly-(γ-glutamic acid) Production and Optimization from Agro-Industrial Bioresources as Renewable Substrates by Bacillus sp. FBL-2 through Response Surface Methodology.

We optimized culture conditions using Bacillus sp. FBL-2 as a poly-(γ-glutamic acid) (PGA) producing strain isolated from cheonggukjang. All experiments were performed under aerobic conditions using a laboratory scale 2.5 L fermentor. We investigated the effects of fermentation parameters (temperature, pH, agitation, and aeration) and medium components (glutamic acid, citric acid, and yeast extract) on poly-(γ-glutamic acid) production, viscosity, and dry cell mass. A non-optimized fermentation method (1.5 vvm, 350 rpm, and 37 °C) yielded PGA, viscosity, and dry cell mass at levels of 100.7 g/L, 483.2 cP, and 3.4 g/L, respectively. L-glutamic acid, citric acid, and yeast extract supplementation enhanced poly-(γ-glutamic acid) production to 175.9 g/L. Additionally, the production of poly-(γ-glutamic acid) from rice bran and wheat bran was assessed using response surface methodology (central composite rotatable design). Agricultural byproducts (rice bran and wheat bran) and H2SO4 were selected as factors, and experiments were performed by combining various component concentrations to determine optimal component concentrations. Our experimentally-derived optimal parameters included 38.6 g/L of rice bran, 0.42% of H2SO4, 28.0 g/L of wheat bran, and 0.32% of H2SO4. Under optimum conditions, rice bran medium facilitated poly-(γ-glutamic acid) production of up to 22.64 g/L, and the use of wheat bran medium yielded up to 14.6 g/L. Based on a validity test using the optimized culture conditions, poly-(γ-glutamic acid) was produced at 47.6 g/L and 36.4 g/L from these respective mediums, and both results were higher than statistically predicted. This study suggests that rice bran can be used as a potential alternative substrate for poly-(γ-glutamic acid) production.