10-L Fermenter Research Articles

Hybrid substrate-based pH autobuffering GABA fermentation by Levilactobacillus brevis CD0817.

The probiotic fermentation of the bioactive substance gamma-aminobutyric acid (GABA) is an attractive research topic. There is still room for further improvement in reported GABA fermentation methods based on a single substrate (L-glutamic acid or L-monosodium glutamate). Here, we devised a pH auto-buffering strategy to facilitate the fermentation of GABA by Levilactobacillus brevis CD0817. This strategy features a mixture of neutral monosodium L-glutamate plus acidic L-glutamic acid as the substrate. This mixture provides a mild initial pH; moreover, the newly dissolved L-glutamic acid automatically offsets the pH increase caused by substrate decarboxylation, maintaining the acidity essential for GABA fermentation. In this study, a flask trial was first performed to optimize the GABA fermentation parameters of Levilactobacillus brevis CD0817. The optimized parameters were further validated in a 10L fermenter. The flask trial results revealed that the appropriate fermentation medium was composed of powdery L-glutamic acid (750g/L), monosodium L-glutamate (34g/L [0.2mol/L]), glucose (5g/L), yeast extract (35g/L), MnSO4·H2O (50mg/L [0.3mmol/L]), and Tween 80 (1.0g/L). The appropriate fermentation temperature was 30°C. The fermenter trial results revealed that GABA was slowly synthesized from 0-4h, rapidly synthesized until 32h, and finally reached 353.1 ± 8.3g/L at 48h, with the pH increasing from the initial value of 4.56 to the ultimate value of 6.10. The proposed pH auto-buffering strategy may be popular for other GABA fermentations.

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817

Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.

Valorization of the pelagic Sargassum horneri for co-production of erythritol and alginate oligosaccharides

Pelagic Sargassum is invasive macroalgae with huge biomass. To produce bulk chemicals with profit from the biomass, innovative strategies need to be developed. In this study, maximum saccharification yield of Sargassum horneri biomass was obtained with the combined use of 3% alginate lyase and 3% cellulase, releasing 20.83 g/L glucose and 1.73 g/L mannitol at a 1:6 feed ratio. Subsequently, the crude S. horneri hydrolysate (pH 3.0) was proved most suitable for erythritol production of Yarrowia lipolytica strain. After 60 h fermentation in a 10-L fermenter, the erythritol concentration reached 18.42 g/L with a yield of 0.82 g/g; while the concentration of alginate oligosaccharides (AOS) was 37.56 g/L. Finally, AOS with a purity of 93.4% were obtained by ethanol precipitation, and erythritol was harvested via crystallization. This proposed strategy demonstrates the feasibility of transforming invasive Sargassum into two high-value chemicals for the first time.

The multiple effects of REG1 deletion and SNF1 overexpression improved the production of S-adenosyl-l-methionine in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is often used as a cell factory for the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications. However, SAM production by S. cerevisiae is negatively influenced by glucose repression, which is regulated by a serine/threonine kinase SNF1 complex. Here, a strategy of alleviating glucose repression by deleting REG1 (encodes the regulatory subunit of protein phosphatase 1) and overexpressing SNF1 (encodes the catalytic subunit of the SNF1 complex) was applied to improve SAM production in S. cerevisiae. SAM production, growth conditions, glucose consumption, ethanol accumulation, lifespan, glycolysis and amino acid metabolism were analyzed in the mutant strains.ResultsThe results showed that the multiple effects of REG1 deletion and/or SNF1 overexpression exhibited a great potential for improving the SAM production in yeast. Enhanced the expression levels of genes involved in glucose transport and glycolysis, which improved the glucose utilization and then elevated the levels of glycolytic intermediates. The expression levels of ACS1 (encoding acetyl-CoA synthase I) and ALD6 (encoding aldehyde dehydrogenase), and the activity of alcohol dehydrogenase II (ADH2) were enhanced especially in the presence of excessive glucose levels, which probably promoted the conversion of ethanol in fermentation broth into acetyl-CoA. The gene expressions involved in sulfur-containing amino acids were also enhanced for the precursor amino acid biosynthesis. In addition, the lifespan of yeast was extended by REG1 deletion and/or SNF1 overexpression. As expected, the final SAM yield of the mutant YREG1ΔPSNF1 reached 8.28 g/L in a 10-L fermenter, which was 51.6% higher than the yield of the parent strain S. cerevisiae CGMCC 2842.ConclusionThis study showed that the multiple effects of REG1 deletion and SNF1 overexpression improved SAM production in S. cerevisiae, providing new insight into the application of the SNF1 complex to abolish glucose repression and redirect carbon flux to nonethanol products in S. cerevisiae.

Efficient production of mannosylerythritol lipids by a marine yeast Moesziomyces aphidis XM01 and their application as self-assembly nanomicelles

Mannosylerythritol lipids (MELs) are one of the most promising biosurfactants because of their excellent physicochemical properties, high environmental compatibility, and various biological functions. In this study, a mangrove yeast strain Moesziomyces aphidis XM01 was identified and used for efficient extracellular MEL production. The MEL titer reached 64.5 ± 0.7 g/L at flask level within 7 days with the optimized nitrogen and carbon source of 2.0 g/L NaNO3 and 70 g/L soybean oil. Furthermore, during a 10-L two-stage fed-batch fermentation, the final MEL titer reached 113.6 ± 3.1 g/L within 8 days, with prominent productivity and yield of 14.2 g·L−1·day−1 and 94.6 g/g(glucose and soybean oil). Structural analysis indicated that the produced MELs were mainly MEL-A and its fatty acid profile was composed of only medium-chain fatty acids (C8–C12), especially C10 acids (77.81%). Further applications of this compound were evaluated as one-step self-assembly nanomicelles. The obtained MEL nanomicelles showed good physicochemical stability and antibacterial activity. In addition, using clarithromycin as a model hydrophobic drug, the MEL nanomicelles exhibited high loading capacity and could be used for the controlled and sustained drug release in low-pH environments. Therefore, M. aphidis XM01 is an excellent candidate for efficient MEL production, and the prepared MEL nanomicelles have broad application prospects in the pharmaceutical and cosmetic fields.

Liamocin overproduction by the mutants of Aureobasidium melanogenum 9-1 for effectively killing spores of the pathogenic fungi from diseased human skin by Massoia lactone.

Liamocins and Massoia lactone have many applications. In this study, the glucose-derepressed mutant Δcrea5 in which the CREA gene was removed could produce 36.5g/L of liamocins. Furthermore, overexpression of the MSN2 gene in the mutant Δcrea5 made the transformant M60 produce 41.4g/L of liamocins and further overexpression of the GAL1 gene in the transformant M60 rendered the transformant G40 to produce 49.5 ± 0.4g/L of liamocins during the 10-L fermentation while their wild type strain 9-1 made only 26.3g/L of liamocins. The expressed transcription activators Msn2 and Gal1 were localized in the nuclei, promoting expression of the genes responsible for liamocins biosynthesis and sugar transport. Massoia lactone prepared from the produced liamocins could actively kill the spores of the pathogenic fungi from the diseased human skin by inhibiting spore germination and causing cellular necrosis of the fungal spores.

A novel two-stage heterotrophic cultivation for starch-to-protein switch to efficiently enhance protein content of Chlorella sp. MBFJNU-17

This work aimed to firstly establish an efficient and novel two-stage cultivation process to produce microalgal biomass rich in protein using a heterotrophic Chlorella sp. MBFJNU-17 strain. In the first-stage cultivation, to reduce the glucose and urea utilization, microalga achieved a high biomass at 40 g/L glucose and 1 g/L urea; meantime, the expression from starch biosynthesis genes of microalga was up-regulated under nitrogen-starvation conditions for starch accumulation (55.01%). In the second-stage cultivation, based on the over-compensation effect, Chlorella cells after the first-stage cultivation were further treated at 5 g/L glucose and 3 g/L urea to up-regulate starch degradation, central carbon metabolism and urea absorption genes expression to drive intracellular starch-to-protein switch for biosynthetic protein (59.75%). Moreover, microalga performed similar characteristics in a 10-L fermenter by the established process. Taken together, Chlorella sp. MBFJNU-17 was the promising candidate to produce high-value biomass enriched in protein by the established two-stage cultivation.

A novel and efficient strategy mediated with calcium carbonate-rich sources to remove ammonium sulfate from rare earth wastewater by heterotrophic Chlorella species

This work was the first time to establish the desired approach with two heterotrophic Chlorella species for ammonium sulfate (AS)-rich rare earth elements (REEs) wastewater treatment by heterotrophic cultivation. The results showed that these two Chlorella species treated by 6 g/L CaCO3 performed the best ability to remove NH4+-N and SO42- of REEs wastewater. Moreover, the established process performed similar features in REEs wastewater treatment by replacing CaCO3 with eggshell powder (ESP) and oyster shell powder (OSP) enriched in CaCO3. Furthermore, microalgae treated by ESP/OSP in a 10-L fermenter showed 837.39 mg/(L·d) NH4+-N and 1,820 mg/(L·d) SO42- removal rates. The developed kinetic models could be well fitted to the experimental data obtained by the 10-L fermenter. Taken together, the established process mediated with two Chlorella species and ESP/OSP by heterotrophic cultivation was the great potential for AS-rich REEs wastewater treatment in a cost-effective manner.

Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors

The regulation of single gene transcription level in the metabolic pathway is often failed to significantly improve the titer of the target product, and even leads to the imbalance of carbon/nitrogen metabolic network and cofactor network. Global transcription machinery engineering (gTME) can activate or inhibit the synergistic expression of multiple genes in specific metabolic pathways, so transcription factors with specific functions can be expressed according to different metabolic regulation requirements, thus effectively increasing the synthesis of target metabolites. In addition, maintaining intracellular redox balance through cofactor engineering can realize the self-balance of cofactors and promote the efficient synthesis of target products. In this study, we rebalanced the central carbon/nitrogen metabolism and redox metabolism of Corynebacterium glutamicum S9114 by gTME and redox cofactors engineering to promote the production of the nutraceutical N-acetylglucosamine (GlcNAc). Firstly, it was found that the overexpression of the transcription factor RamA can promote GlcNAc synthesis, and the titer was further improved to 16 g/L in shake flask by using a mutant RamA (RamAM). Secondly, a CRISPR interference (CRISPRi) system based on dCpf1 was developed and used to inhibit the expression of global negative transcriptional regulators of GlcNAc synthesis, which promoted the GlcNAc titer to 27.5 g/L. Thirdly, the cofactor specificity of the key enzymes in GlcNAc synthesis pathway was changed by rational protein engineering, and the titer of GlcNAc in shake flask was increased to 36.9 g/L. Finally, the production of GlcNAc was scaled up in a 50-L fermentor, and the titer reached 117.1 ± 1.9 g/L, which was 6.62 times that of the control group (17.7 ± 0.4 g/L), and the yield was increased from 0.19 g/g to 0.31 g/g glucose. The results obtained here highlight the importance of engineering the global regulation of central carbon/nitrogen metabolism and redox metabolism to improve the production performance of microbial cell factories.

Continuous H2/CO2 fermentation for acetic acid production under transient and continuous sulfide inhibition

Waste gas fermentation powered by renewable H2 is reaching kiloton scale. The presence of sulfide, inherent to many waste gases, can cause inhibition, requiring additional gas treatment. In this work, acetogenesis and methanogenesis inhibition by sulfide were studied in a 10-L mixed-culture fermenter, supplied with CO2 and connected with a water electrolysis unit for electricity-powered H2 supply. Three cycles of inhibition (1.3 mM total dissolved sulfide (TDS)) and recovery were applied, then the fermenter was operated at 0.5 mM TDS for 35 days. During operation at 0.5 mM TDS the acetate production rate reached 7.1 ± 1.5 mmol C L−1 d−1. Furthermore, 43.7 ± 15.6% of the electrons, provided as H2, were distributed to acetate and 7.7 ± 4.1% to butyrate, the second most abundant fermentation product. Selectivity of sulfide as inhibitor was demonstrated by a 7 days lag-phase of methanogenesis recovery, compared to 48 h for acetogenesis and by the less than 1% electrons distribution to CH4, under 0.5 mM TDS. The microbial community was dominated by Eubacterium, Proteiniphilum and an unclassified member of the Eggerthellaceae family. The taxonomic diversity of the community decreased and conversely the phenotypic diversity increased, during operation. This work illustrated the scale-up potential of waste gas fermentations, by elucidating the effect of sulfide as a common gas impurity, and by demonstrating continuous, potentially renewable supply of electrons.

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.

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-l-methionine production in Saccharomyces cerevisiae

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L−1 in a 250-mL flask and 8.86 g L−1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

Improvement of Polymer Grade L-Lactic Acid Production Using Lactobacillus rhamnosus SCJ9 from Low-Grade Cassava Chips by Simultaneous Saccharification and Fermentation

The present study aims to examine the process for L-lactic acid production from low-grade cassava chips (LGC) using a two-step fermentation approach (TSF) and simultaneous saccharification and fermentation (SSF) by proficient, newly isolated Lactobacillus rhamnosus strain SCJ9. The optimized medium composition revealed by response surface methodology for TSF was 166 g/L LGC hydrolysate and 20 g/L yeast extract (YE), while other medium components were fixed (g/L) as follows: tween80 (2.0), (NH4)2HPO4 (2.0), CH3COONa∙3H2O (6.0), (NH4)2HC6H5O7 (2.0), MgSO4∙7H2O (0.5), and MnSO4∙H2O (0.3). Based on the optimization conditions, the maximum experimental L-lactic acid of 134.6 g/L was achieved at 60 h fermentation time with a production efficiency of 89.73%, 0.95 g/g yield and 2.24 g/L/h productivity. In contrast, L-lactic acid production by SSF under optimized concentrations of thermostable-α-amylase (AA) and glucoamylase (GA) gave maximum L-lactic acid of 125.79 g/L at only 36 h fermentation time which calculated to the production efficiency, yield and productivity of 83.86%, 0.93 g/g and 3.49 g/L/h, respectively. The L-lactic acid production obtained from SSF was significantly improved when compared to TSF based on lower enzyme loading usage, shorter hydrolysis time and increase in production efficiency and productivity. Furthermore, there were no significant differences in the production by SSF between experiments conducted in laboratory bottle and 10-L fermenter. The results indicated the success of up-scaling for L-lactic acid production by SSF which could be developed for a further pilot-scale production of L-lactic acid.

Utilizing Gelatinized Starchy Waste from Rice Noodle Factory as Substrate for L(+)-Lactic Acid Production by Amylolytic Lactic Acid Bacterium Enterococcus faecium K-1.

To valorize starchy waste from rice noodle factory, bioconversion of gelatinized starchy waste (GSW) to value-added product as L(+)-lactic acid, the monomer for polylactate synthesis, was investigated using amylolytic lactic acid bacterium, Enterococcus faecium K-1. Screening for appropriate nitrogen source to replace expensive organic nitrogen sources revealed that corn steep liquor (CSL) was the most suitable regarding high efficacy for L(+)-LA achievement and low-cost property. The successful applying statistic experimental design, Plackett-Burman design incorporated with central composite design (CCD), predicted the maximum L(+)-LA of 93.07g/L from the optimized medium (OM) containing 125.7g/L GSW and 207.3g/L CSL supplemented with CH3COONa, MgSO4, MnSO4, K2HPO4, CaCl2, (NH4)2HC6H5O7, and Tween80. Minimizing the medium cost by removal of all inorganic salts and Tween80 from OM was not an effect on L(+)-LA yield. Fermentation using the optimized medium without minerals (OM-Mi) containing only GSW (125.7g/L) and CSL (207.3g/L) in a 10-L fermenter was also successful. Thinning GSW with α-amylase from Lactobacillus plantarum S21 increased L(+)-LA productivity in the early stage of 24-h fermentation. Not only showing the feasible bioconversion process for GSW utilizing as a substrate for L(+)-LA production, this research also demonstrated the efficient model for industrial starchy waste valorization.

High-efficiency expression of a superior β-mannanase engineered by cooperative substitution method in Pichia pastoris and its application in preparation of prebiotic mannooligosaccharides

β-mannanase with high specific activity is a prerequisite for the industrial preparation of prebiotic mannooligosaccharides. Three mutants, namely MEI, MER, and MEIR, were constructed by cooperative substitution based on three predominant single-point site mutations (K291E, L211I, and Q112R, respectively). Heterologous expression was facilitated in Pichia pastoris and the recombinase was characterized completely. The specific activities of MER (7481.9 U mg−1) and MEIR (9003.1 U mg−1) increased by 1.07- and 1.29-fold from the initial activity of ME (6970.2U mg−1), respectively. MEIR was used for high-cell-density fermentation to further improve enzyme activity, and the expression levels achieved in the 10-L fermenter were significantly high (105,836 U mL−1). The prebiotic mannooligosaccharides (<2000 Da) were prepared by hydrolyzing konjac gum and locust bean gum with MEIR, with 100% and 76.40% hydrolysis rates, respectively. These characteristics make MEIR highly attractive for prebiotic development in food and related industries.

Preparation of pH-Responsive Alginate-Chitosan Microspheres for L-Valine Loading and Their Effects on the A40926 Production.

The glycopeptide A40926 biosynthesized by Nonomuraea gerenzanensis is a precursor of the second generation glycopeptide antibiotic dalbavancin. The skeleton of this glycopeptide consists of seven amino acids and is biosynthesized by the NRPS gene module. L-valine, a branched amino acid, is also a significant precursor for A40926 production. This study details the use of pH-responsive alginate-chitosan microspheres loaded with L-valine prepared by internal emulsification gelation. The effects of process and formulation variables on microsphere size, loading capacity, and encapsulation efficiency were investigated. Then, effects on A40926 production by the pH-responsive microspheres were evaluated in a 10-L fermenter. Results demonstrated that use of the pH-responsive microspheres could improve A40926 yield from 465 to 602mg L-1 in a 10-L scale fermenter.

Cellular lipid production by the fatty acid synthase-duplicated Lipomyces kononenkoae BF1S57 strain for biodiesel making

Lipomyces starkeyi has been regarded as one of the model oleaginous yeasts. However, in this study, Lipomyces kononenkoae BF1S57 strain was found to be able to yield 60.06% (w/v) lipids (11.3 g/L lipids) from 90.0 g/L glucose, its cell mass was 18.8 ± 0.3 g/L within 96 h of a 10-L fermentation. Furthermore, the genome of L. kononenkoae has two copies of the genes encoding the β-subunit and α-subunit of fatty acid synthase, respectively. This may be related to high lipid contents in its cells. Over 94% of the fatty acids of the extracted lipids produced by L. kononenkoae BF1S57 strain was C16:0, C16:1, C18:0, C18:1 and C18:2 fatty acids. The properties of the prepared biodiesels in this study totally met the requirements of US biodiesel ASTM D6751 and EU biodiesel EN 14214. All these results demonstrated that the produced cellular lipids could be used as raw materials for biofuel production.

Increasing L-threonine production in Escherichia coli by overexpressing the gene cluster phaCAB.

L-Threonine is an important branched-chain amino acid and could be applied in feed, drugs, and food. In this study, L-threonine production in an L-threonine-producing Escherichia coli strain TWF001 was significantly increased by overexpressing the gene cluster phaCAB from Ralstonia eutropha. TWF001/pFW01-phaCAB could produce 96.4-g/L L-threonine in 3-L fermenter and 133.5-g/L L-threonine in 10-L fermenter, respectively. In addition, TWF001/pFW01-phaCAB produced 216% more acetyl-CoA, 43% more malate, and much less acetate than the vector control TWF001/pFW01, and meanwhile, TWF001/pFW01-phaCAB produced poly-3-hydroxybutyrate, while TWF001/pFW01 did not. Transcription analysis showed that the key genes in the L-threonine biosynthetic pathway were up-regulated, the genes relevant to the acetate formation were down-regulated, and the gene acs encoding the enzyme which converts acetate to acetyl-CoA was up-regulated. The results suggested that overexpression of the gene cluster phaCAB in E. coli benefits the enhancement of L-threonine production.

Consistent production of kojic acid from Aspergillus sojae SSC-3 isolated from rice husk.

A consistent kojic acid producing fungal strain has been isolated from rice husk using glucose-peptone medium. The isolate was identified as Aspergillus sojae SSC-3 on 18S rDNA analysis. A. sojae was capable of producing substantially good amount of kojic acid, however the production was varying from batch to batch. In order to obtain consistent, repeated and high levels of kojic acid, monospore isolation procedures was adopted. The highest production of kojic acid obtained was 12 ± 2 g/L in 120h with sucrose (10%) and yeast extract (0.5%) as carbon and nitrogen source respectively. The process was scale up to 10L fermenter size which repeatedly resulted in the production of 18 ± 2 g/L of kojic acid in 96h. Kojic acid was recovered (> 82%) from the fermentation broth with > 99% purity. Best to our knowledge this is the first report were kojic acid production is reported from Aspergillus sojae strain.

Enhanced Production of Protopanaxatriol from Ginsenoside Re and Rg1 Using a Recombinant Bacterial β-glucosidase

20(S)-protopanaxatriol (PPTA), an aglycone of ginseng saponins, is rarely found and is present in a limited amount; it exhibits various pharmaceutical activities, including memory improvement, and anti-diabetic and anti-cancer effects. An enzymatic method was developed to improve 20(S)-protopanaxatriol aglycone [20(S)-PPTA] production using recombinant β-glucosidase (BgpA) cloned from Terrabacter ginsenosidimutans Gsoil 3082T. The optimal hydrolytic activity was obtained at pH 7.0 and 37°C. The enzyme could hydrolyze the rutinoside and glucose moieties at the C6 and C20 positions, respectively, of ginsenosides Re and Rg1 to produce 20(S)-PPTA through the formation of ginsenoside F1 as an intermediate. For gram-scale production, the enzyme reaction was performed in a 10-L fermenter for 48 h at a substrate concentration of 10 mg/mL. Finally, 5.45 g 20(S)-PPTA was produced from 36 g PPT type ginsenoside mixture with 95% purity by chromatography. This study thus reveals an industrial application of a minor component, 20(S)-PPTA.