γ-PGA Production Research Articles

The role of structural evolution in the complexation and flocculation of heavy metals by the microbial product poly-γ-glutamic acid

The process and mechanism of heavy metal flocculation with extracellular polymeric substances (EPS) secreted by microorganisms, are crucial to their fate in natural environment, wastewater treatment and soil bioremediation applications. However, the structural features of EPS and the relationship between these features and the flocculation process and mechanism remain unclear. In the present study, structural features of the microbial product poly-γ-glutamic acid (γ-PGA) complexed with the heavy metal ions Pb2+ and Cu2+ were characterized and the evolution of these features was identified as having a key role in the flocculation process and mechanism. The secondary structure of the γ-PGA-Pb complex changed significantly, while that of the γ-PGA-Cu complex was only slightly altered. The significant structural change in γ-PGA-Pb was found to be responsible for the combination of residual COOH and Pb2+, promoting the bridging of inter-colloids and faster growth of hydrodynamic diameter. If the conformation changed sufficiently, such as with the γ-PGA-Pb complex in the pH range 4.6–6.2, pH had no impact on the conversion ratio. The unchanged structure of γ-PGA-Cu prevented the flocculation process, although the coordination mode of γ-PGA-Cu resulted in a higher biosorption capacity. This in-depth molecular-level study provides insight into the γ-PGA flocculation mechanism, promoting the use of γ-PGA and γ-PGA producing microorganisms for application in various remediation strategies.

Online measurement of the viscosity in shake flasks enables monitoring of γ-PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate-starvation inducible promoter Ppst.

Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA /gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.

Structural and genetic insights into a poly-γ-glutamic acid with in vitro antioxidant activity of Bacillus velezensis VCN56.

Poly-γ‑glutamic acid (γ‑PGA) produced by Bacillus species is a natural biopolymer, which is widely used in various fields including food, pharmaceuticals, and cosmetics. In this study, the screening of 19 Bacillus isolates derived from traditionally fermented foods revealed that Bacillus velezensis VCN56 was the most potent γ‑PGA producer. The maximum concentration of crude γ‑PGA was 32.9 ± 1.5g/L in the PGA-3 medium containing glycerol, citric acid, sodium glutamate, NH4Cl, and starch. The resulting γ-PGA was purified and then characterized by HPLC, FTIR, and 1H-NMR analyses. Molecular weight of purified γ‑PGA was estimated to be 98kDa with a polydisperse index of 2.04. Notably, the pure γ‑PGA showed significant in vitro antioxidant scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (72.0 ± 1.5%), hydroxyl (81.0 ± 0.6%), and superoxide (43.9 ± 0.8%) radicals at the concentration of 4mg/mL. Using whole-genome sequencing, the genetic organization of pgs operon responsible for γ‑PGA biosynthesis in B. velezensis VCN56 differs from those in other Bacillus genomes. Further genome analysis revealed metabolic pathways for γ-PGA production and degradation. For the first time, the present study provides a better understanding of γ-PGA with a promising antioxidant activity produced by B. velezensis at the phenotypic, biochemical, and genomic levels, which hold potential applications in the foods, cosmetics, and pharmaceutical industries.

The combined application of γ-PGA-producing bacteria and biochar reduced the content of heavy metals and improved the quality of tomato (Solanum lycopersicum L.).

Plant growth-promoting bacteria and biochar have been widely used as immobilizers to remediate heavy metal contaminated soil. However, few studies have unraveled the effect and synergistic mechanism of combined application of plant growth-promoting bacteria and biochar on in situ heavy metal contaminated soil remediation and plant yield and quality improvement under heavy metal pollution stress. In this study, the effects of biochar, γ-PGA-producing bacteria (Bacillus amyloliquefaciens strain W25) and their combined application on Cd and Pb immobilization, γ-PGA production in soil filtrate, the bacterial community in rhizosphere soil, physicochemical properties of soil, heavy metal uptake, and quality and yield of tomato in heavy metal-contaminated soil were investigated. The application of W25, biochar, and their combinations significantly reduced Cd content in mature tomato fruits by 22-60%, increased the single fruit weight and lycopene content by 7-21% and 23-48%, respectively, and the combination of biochar and W25 had the best effect. All the treatments significantly reduced DTPA-Cd and DTPA-Pb contents in rhizosphere soil (42-53% and 6.5-35%), increased the pH value and the activities of urease-alkaline phosphatase of soil, but did not affect the expression of heavy metal transporter gene LeNRAMP1 in tomato roots. Biochar + W25 increased the relative abundance of plant growth-promoting bacteria such as Bacillus and Streptomyces. Biochar-enhanced plant growth-promoting bacteria to settle and colonize in soil significantly improved the ability of strain W25 to produce γ-PGA, and immobilized Cd in soil filtrate. The combination of biochar and plant growth-promoting bacteria ensures safe crop production in heavy metal-contaminated soil.

Efficient molasses utilization for low-molecular-weight poly-γ-glutamic acid production using a novel Bacillus subtilis strain

BackgroundPoly-γ-glutamic acid (γ-PGA) is a biopolymer and has various applications based on its biocompatibility, non-toxicity, and edibility. Low-molecular-weight (Mw)-γ-PGA has promising applications in agriculture and pharmaceuticals. It is traditionally produced by enzymatic hydrolysis. Cost-effective bioproduction of low-Mw-γ-PGA is essential for commercial application of γ-PGA.ResultsBacillus subtilis 242 is a newly isolated low-Mw-γ-PGA-producing strain. To develop cost-effective production of γ-PGA using this newly isolated strain, cane molasses and corn steep liquor were used to produce γ-PGA. The concentration of cane molasses was optimized and 100 g/L cane molasses resulted in high γ-PGA production. The effects of yeast extract and corn steep liquor on γ-PGA yield were investigated. High concentration of γ-PGA was obtained in the medium with corn steep liquor. A concentration of 32.14 g/L γ-PGA was achieved in fed-batch fermentation, with a productivity of 0.67 g/L/h and a percentage yield (gγ-PGA/gglutamate) of 106.39%. The Mw of γ-PGA was 27.99 kDa.ConclusionThis study demonstrated the potential application of B. subtilis 242 for cost-effective production of low-Mw-γ-PGA from cane molasses.

Construction of a mutant Bacillus subtilis strain for high purity poly-γ-glutamic acid production

To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. The construction of strain GH16 was achieved by knocking out five genes encoding extracellular proteins and an operon from Bacillus subtilis G423. We then analyzed the amount of protein impurities in the γ-PGA produced by the resulting strain GH16/pHPG, which decreased from 1.48 to 1.39%. Subsequently the fla-che operon, PBSX, as well as the yrpD, ywoF and yclQ genes were knocked out successively, resulting in the mutant strains GH17, GH18 and GH19. Ultimately, the amount of protein impurities was reduced from 1.48 to 0.83%. In addition, the amount of polysaccharide impurities in the γ-PGA was also decreased from 2.21 to 1.93% after knocking out the epsA-O operon. The high purity γ-PGA producer was constructed, and the resulting strain was a promising platform for the manufacture of other highly pure extracellular products and secretory proteins.

γ-Polyglutamic Acid Production, Biocontrol, and Stress Tolerance: Multifunction of Bacillus subtilis A-5 and the Complete Genome Analysis.

Bacillus subtilis A-5 has the capabilities of high-molecular-weight γ-PGA production, antagonism to plant pathogenic fungi, and salt/alkaline tolerance. This multifunctional bacterium has great potential for enhancing soil fertility and plant security in agricultural ecosystem. The genome size of B. subtilis A-5 was 4,190,775 bp, containing 1 Chr and 2 plasmids (pA and pB) with 43.37% guanine-cytosine content and 4605 coding sequences. The γ-PGA synthase gene cluster was predicted to consist of pgsBCA and factor (pgsE). The γ-PGA-degrading enzymes were mainly pgdS, GGT, and cwlO. Nine gene clusters producing secondary metabolite substances, namely, four unknown function gene clusters and five antibiotic synthesis gene clusters (surfactin, fengycin, bacillibactin, subtilosin_A, and bacilysin), were predicted in the genome of B. subtilis A-5 using antiSMASH. In addition, B. subtilis A-5 contained genes related to carbohydrate and protein decomposition, proline synthesis, pyruvate kinase, and stress-resistant proteins. This affords significant insights into the survival and application of B. subtilis A-5 in adverse agricultural environmental conditions.

Poly-Gamma-Glutamic Acid Secretion Protects Bacillus subtilis from Zinc and Copper Intoxication.

ABSTRACTZinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms. Here, we show that the Gram-positive soil bacterium, Bacillus subtilis, produces the extracellular polymeric substance, poly-gamma-glutamate (γ-PGA) as a protective mechanism in response to zinc and copper excess. Furthermore, we provide evidence that zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two-component regulatory system and likely occurs at a posttranscriptional level through the small protein, PgsE. These data provide new insight into bacterial metal resistance mechanisms and contribute to our understanding of the regulation of bacterial γ-PGA biosynthesis.IMPORTANCE Zinc and copper are potent antimicrobial compounds. As such, bacteria have evolved a diverse range of tools to prevent metal intoxication. Here, we show that the Gram-positive model organism, Bacillus subtilis, produces poly-gamma-glutamic acid (γ-PGA) as a protective mechanism against zinc and copper intoxication and that zinc and copper dependent γ-PGA production occurs by a yet undefined mechanism independent of known γ-PGA regulation pathways.

Chemical pretreatment and saccharification of corncob for poly-γ-glutamic acid production by Bacillus subtilis SCP010-1

Lignocellulosic biomass is an important raw material which has been extensively studied for biotechnological applications, but research on its use for the production of γ-polyglutamic acid (γ-PGA) is rarely reported. The present study aimed to improve the release of xylose and glucose from corncob for γ-PGA production using the strain Bacillus subtilis SCP010–1, which could metabolize xylose. The effectiveness of the three chemical reagents NaOH, NH3 and H2O2 pretreatments was investigated by the response surface methodology (RSM). The results showed that maximum glucose and xylose yields of 11.8 ± 0.3 and 17.36 ± 0.6 g/L, respectively, as determined by phloroglucinol-acid and enzymatic methods, were obtained when using 0.986% of NaOH for 2.07 h. Then, the pretreatment solution was subject to further optimize conditions of enzymatic hydrolysis, and the content of glucose and xylose in the corncob hydrolysate reached respectively 15.3 ± 0.3 and 22.34 ± 0.17 g/L under synergism of hemicellulase and cellulase. Finally, the fermentation conditions for γ-PGA production by B. subtilis SCP010–1 were also optimized, and after adding supplemental nutrients to the corncob hydrolysate, a maximum γ-PGA production of 30.035 ± 0.49 g/L was achieved by fed batch fermentation with feeding corncob hydrolysate. Considering the cost of the materials used in this study, nearly 85% of reduction in cost could be achieved, hence indicating that this production plan offers good prospects for industrial production of γ-PGA. More importantly, this process allows the use of environmental-friendly biological resources.

Enhanced production of poly-γ-glutamic acid via optimizing the expression cassette of Vitreoscilla hemoglobin in Bacillus licheniformis

Poly-γ-glutamic acid (γ-PGA) is a natural polymer with various applications, and its high-viscosity hinders oxygen transmission and improvement of synthesis level. Vitreoscilla hemoglobin (VHB) has been introduced into various hosts as oxygen carrier, however, its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis. Here, we want to optimize the expression cassette of VHB for γ-PGA production. Firstly, our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation (Tat) signal peptides (SPYwbN, SPPhoD and SPTorA) into VHB expression cassette, and the best performance was attained by SPYwbN from Bacillus subtilis, the γ-PGA yield of which was 18.53% higher than that of control strain, and intracellular ATP content and oxygen transfer coefficient (KLa) were increased by 29.71% and 73.12%, respectively, indicating that VHB mediated by SPYwbN benefited oxygen transfer and ATP generation for γ-PGA synthesis. Furthermore, four promoters were screened, and Pvgb was proven as the more suitable promoter for VHB expression and γ-PGA synthesis, and γ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%. Finally, WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation, and 46.39 g/L γ-PGA was attained by glucose feeding, increased by 49.26% compared with the initial yield (31.01 g/L). Taken together, this study has attained an efficient VHB expression cassette for oxygen transfer and γ-PGA synthesis, which could also be applied in the production of other metabolites.

Efficient production of poly-γ-glutamic acid by Bacillus velezensis via solid-state fermentation and its application

A novel glutamate-dependent poly-γ-glutamic acid (γ-PGA)-producing strain Bacillus velezensis CAU263 isolated from Chinese traditional Douchi was evaluated. An efficient method of γ-PGA production was performed by this strain via solid-state fermentation (SSF) using guar meal. The maximal yield of γ-PGA reached 158.5 g/kg dry weight (DW) in 250 mL flasks and 155.1 g/kg DW in shallow tray, respectively. The molecular weight of γ-PGA was 3.8 × 106 Da, the ratio of L/D-glutamic acid was 26.1% and 73.9%. Additionally, γ-PGA significantly improved the operating characteristics of wheat dough, the specific volume of bread was increased by 15.8% and the bread hardness was reduced by 44.2% owing to the addition of 0.3 g/kg γ-PGA. Thus, γ-PGA could be produced by Bacillus velezensis CAU263 at high level using guar meal and might have potential application prospect in food industry.

Enhanced production of poly-γ-glutamic acid by Bacillus licheniformis ATCC 9945a using simultaneous pulse-feedings of citrate and glutamate

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, feeding times, the addition time of calcium and manganese solutions, the pH of the added citrate solution, and the concentration of feed stock solutions of pulse-feeds on γ-PGA production were investigated. Under optimal conditions: feeding two pulses at 8 and 24 hours of 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.

Rational engineering of cofactor specificity of glutamate dehydrogenase for poly-γ-glutamic acid synthesis in Bacillus licheniformis

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis.

A novel strategy of feeding nitrate for cost-effective production of poly-γ-glutamic acid from crude glycerol by Bacillus licheniformis WX-02

Poly-γ-glutamic acid (γ-PGA) is a natural and multifunctional biopolymer. Glycerol, especially crude glycerol, was used as fermentation raw material for production of a variety of chemical products. Here, glycerol was used with nitrate addition for γ-PGA production by Bacillus licheniformis WX-02 at a 5 L fermenter scale. However, nitrite accumulation was discovered in batch fermentation, which resulted in low γ-PGA yield (19.58 g/L). Furthermore, based on transcription analysis and intracellular NADH measurement, nitrite accumulation was probably due to the low content of intracellular NADH in glycerol-based medium, which might be resulted from low efficiency of NADH generation rate and quick NADH consumption by nitrate reduction. Accordingly, different nitrate feeding strategies were designed and optimized in γ-PGA fermentation with glycerol, then applied in fermentation with crude glycerol. Finally, 35.63 g/L of γ-PGA was achieved with the constant-rate nitrate feeding of 0.38 g/(L h) by using crude glycerol, while 36.83 g/L of γ-PGA was achieved with pH-feedback controlling. Therefore, this study provided a novel strategy for cost-effective production of γ-PGA from crude glycerol.

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 12.02g/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.

Poly-γ-glutamic acid production by simultaneous saccharification and fermentation using corn straw and its fertilizer synergistic effect evaluation.

Agricultural wastes rich in lignocellulosic biomass have been used in the production of poly-γ-glutamic acid (γ-PGA) through separate hydrolysis and fermentation (SHF), but this process is complicated and generates a lot of wastes. In order to find a simpler and greener way to produce γ-PGA using agricultural wastes, this study attempted to establish simultaneous saccharification and fermentation (SSF) with citric acid-pretreated corn straw. The possibility of Bacillus amyloliquefaciens JX-6 using corn straw as substrate to synthesize γ-PGA was validated, and the results showed that increasing the proportion of glucose in the substrate could improve the γ-PGA yield. Based on these preliminary results, the corn straw was pretreated using citric acid. Then, the liquid fraction (xylan-rich) was used for cultivation of seed culture, and the solid fraction (glucan-rich) was used as the substrate for SSF. In a 10-L fermenter, the maximum cumulative γ-PGA concentration in batch and fed-batch SSF were 5.08 ± 0.78g/L and 10.78 ± 0.32g/L, respectively. Moreover, the product from SSF without γ-PGA extraction was used as a fertilizer synergist, increasing the yield of pepper by 13.46% (P < 0.05). Our study greatly simplified the production steps of γ-PGA, and each step achieved zero emission as far as possible. The SSF process for γ-PGA production provided a simple and green way for lignocellulose biorefinery and sustainable cultivation in agriculture.

Study on the mechanism of production of \u03b3-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.

Study on the reuse process of hydrolysate from γ-polyglutamic acid fermentation residues

In order to study the feasibility of using γ-polyglutamic acid (γ-PGA) waste biomass hydrolysate as nitrogen source of culture medium, waste biomass was treated with nitric acid to obtain hydrolysate. Through the orthogonal optimization experiment, the optimal conditions of biomass hydrolysis were obtained as follows: 2% nitric acid, solid-liquid ratio 4:10, 110 °C, and 24 h. Under the optimal conditions, the degree of hydrolysis reached 77.08 ± 0.48%. The fermentation medium was further optimized: biomass hydrolysate 60 mL, glucose 38 g/L, L-glutamate 25 g/L. In the optimal medium, γ-PGA yield was 30.69 ± 0.42 g/L, which was 4.49% higher than that in tryptone medium (29.37 ± 0.43 g/L). The results showed that the waste biomass hydrolysate could be used as tryptone for γ-PGA production. Moreover, the addition of waste biomass hydrolysate reduced the amount of glutamate in the medium. In addition, the waste biomass could be recycled for 2 times. This study provided a new idea for the cleaner production technology of γ-PGA.

Cultivation of Chlorella protothecoides in polyglutamic acid wastewater for cost-effective biodiesel production

The combination of gamma-polyglutamic acid (γ-PGA) wastewater treatment and oil-producing microalgae culture could not only achieve the wastewater reuse for γ-PGA production, but also provided nutrients and a large amount of water for microalgae culture. Thus the effects of wastewater concentration, different nutrients on the biomass yield and lipid content of Chlorellaprotothecoides were investigated separately. The results showed that 60% γ-PGA wastewater was very suitable for the cultivation of C.protothecoides. Only 5.86 g/L glucose, 0.73 g/L NaNO3 and 0.2 g/L K2HPO4 needed to be added in the γ-PGA wastewater. In the optimized wastewater medium, the lipid yield could reach 1.77 ± 0.03 g/L, which was 195.0% higher than that in the SE medium(0.60 ± 0.03 g/L). C.protothecoides had high absorption capacity for nitrogen and phosphorus. The nitrogen, phosphorus removal rate of wastewater reached 93.87% and 88.39%, respectively, after 7 days of culture. In addition, the wastewater treated by C.protothecoides could be reused for γ-PGA fermentation for four times. These investigations laid a foundation for reducing the pollution of γ-PGA wastewater, explored a late-model for oleaginous microalgae and γ-PGA cleaner production.

Preparation of γ-polyglutamic acid from enzymatic hydrolysate of poplar sawdust

The treatment of forestry waste has always been an environmental problem. In this study, poplar sawdust was used to produce γ-polyglutamic acid (γ-PGA). Poplar sawdust was treated with acid and then hydrolyzed with cellulose. The enzymatic hydrolysate was used to produce γ-PGA. Through single factor and orthogonal experiments, the optimum enzymatic conditions were determined as follows: solid-liquid ratio 1:5, enzyme amount 22 FPU/g, pH 5.0 and 53 min. Under the enzymatic hydrolysate medium, the γ-PGA yield reached 30.87 ± 0.44 g/L, which was 5.11% higher than that in glucose medium (29.37 ± 0.43 g/L). In addition, the amount of glucose added to the medium was reduced, which realized the comprehensive utilization of various sugars in sawdust enzymatic hydrolysate. Although the fermentation time was prolonged by 4 h, this study provided a new and feasible method for the industrial utilization of forestry waste and the clean production of γ-PGA.