Dynamic immobilization of bacterial cells on biofilm in a polyester nonwoven chemostat.

Cell immobilization plays an important role in biocatalysis for high-value products. It is necessary to maintain the viability of immobilized cells for bioconversion using viable cells as biocatalysts. In this study, a novel polyester nonwoven chemostat was designed for cell immobilization to investigate biofilm formation and the dynamic balance between adsorption and desorption of cells on polyester nonwoven. The polyester nonwoven was suitable for cell immobilization, and the cell numbers on the polyester nonwoven can reach 6.5 ± 0.38 log CFU/mL. After adding the polyester nonwoven to the chemostat, the fluctuation phenomenon of free bacterial cells occurred. The reason for this phenomenon was the balance between adsorption and desorption of bacterial cells on the polyester nonwoven. Bacterial cells could adhere to the surface of polyester nonwoven via secreting extracellular polymeric substances (EPS) to form biofilms. As the maturation of biofilms, some dead cells inside the biofilms can cause the detachment of biofilms. This process of continuous adsorption and desorption of cells can ensure that the polyester nonwoven chemostat has lasting biological activity.

Kinetics-based development of two-stage continuous fermentation of 1,3-propanediol from crude glycerol by Clostridium butyricum.

BACKGROUND: Glycerol, as a by-product, mainly derives from the conversion of many crops to biodiesel, ethanol, and fatty ester. Its bioconversion to 1,3-propanediol (1,3-PDO) is an environmentally friendly method. Continuous fermentation has many striking merits over fed-batch and batch fermentation, such as high product concentration with easy feeding operation, long-term high productivity without frequent seed culture, and energy-intensive sterilization. However, it is usually difficult to harvest high product concentrations. RESULTS: In this study, a three-stage continuous fermentation was firstly designed to produce 1,3-PDO from crude glycerol by Clostridium butyricum, in which the first stage fermentation was responsible for providing the excellent cells in a robust growth state, the second stage focused on promoting 1,3-PDO production, and the third stage aimed to further boost the 1,3-PDO concentration and reduce the residual glycerol concentration as much as possible. Through the three-stage continuous fermentation, 80.05 g/L 1,3-PDO as the maximum concentration was produced while maintaining residual glycerol of 5.87 g/L, achieving a yield of 0.48 g/g and a productivity of 3.67 g/(L·h). Based on the 14 sets of experimental data from the first stage, a kinetic model was developed to describe the intricate relationships among the concentrations of 1,3-PDO, substrate, biomass, and butyrate. Subsequently, this kinetic model was used to optimize and predict the highest 1,3-PDO productivity of 11.26 g/(L·h) in the first stage fermentation, while the glycerol feeding concentration and dilution rate were determined to be 92 g/L and 0.341 h-1, separately. Additionally, to achieve a target 1,3-PDO production of 80 g/L without the third stage fermentation, the predicted minimum volume ratio of the second fermenter to the first one was 11.9. The kinetics-based two-stage continuous fermentation was experimentally verified well with the predicted results. CONCLUSION: A novel three-stage continuous fermentation and a kinetic model were reported. Then a simpler two-stage continuous fermentation was developed based on the optimization of the kinetic model. This kinetics-based development of two-stage continuous fermentation could achieve high-level production of 1,3-PDO. Meanwhile, it provides a reference for other bio-chemicals production by applying kinetics to optimize multi-stage continuous fermentation.

Dynamic flux balance analysis of 1,3-propanediol production by clostridium butyricum fermentation.

To study the relationship between the yield of 1,3-propanediol (1,3-PDO) and the flux change of the Clostridium butyricum metabolic pathway, an optimized calculation method based on dynamic flux balance analysis was used by combining genome-scale flux balance analysis with a kinetic model. A more comprehensive and extensive metabolic pathway was obtained by optimization calculations. The primary extended branches include: the dihydroxyacetone node, which enters the pentose phosphate pathway; the α-oxoglutarate node, which has synthetic metabolic pathways for glutamic acid and amino acids; and the serine and homocysteine nodes, which produce cystathionine before homocysteine enters the methionine cycle pathway. According to the expanded metabolic network, the flux distribution of key nodes in the metabolic pathway and the relationship between the flux distribution ratio of nodes and the yield of 1,3-PDO were analyzed. At the dihydroxyacetone node, the flux of dihydroxyacetone converted to dihydroxyacetone phosphate was positively correlated with the yield of 1,3-PDO. As an important intermediate product, the flux change in the metabolic pathway of α-oxoglutarate reacting with amino acids to produce glutamic acid is positively correlated with the yield. When pyruvate was used as the central node to convert into lactic acid and α-oxoglutarate, the proportion of branch flux was negatively correlated with the yield of 1,3-PDO. These studies provide a theoretical basis for the optimization and further study of the metabolic pathway of C. butyricum.

Salting-out extraction of recombinant κ-carrageenase and phage T7 released from Escherichia coli cells.

Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.

One-pot synthesis of fuel precursor from acetoin fermentation broth using ionic liquid-based salting-out extraction system.

BACKGROUND: The development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C-C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work. RESULTS: A one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 â„ƒ, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction. CONCLUSIONS: With EOAB and K2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE.

The pretreatment of the sustainable biomass feedstock of Pennisetum giganteum for biorefinery using deep eutectic solvents.

In this study, Pennisetum giganteum (PG) was investigated as lignocellulosic feedstock to be pretreated by the acidic and basic deep eutectic solvents (DESs) to generate monomeric sugars. The basic DESs showed excellent efficiency of delignification and saccharification. ChCl/MEA can remove 79.8 % lignin and reserve 89.5 % cellulose. As a result, 95.6 % glucose and 88.0 % xylose yield were obtained, significantly enhanced 9.4 and 15.5 times in contrast with those of the unpretreated PG. The 3D microstructures of raw and pretreated PG were constructed for the first time to better investigate the pretreatment effect on its structure. The increasing porosity (20.5 %) and the reducing CrI (42.2 %) contributed in enhancing enzymatic digestion. Moreover, the recyclability of DES indicated that at least 90 % DES was recovered and 59.5 % lignin still can removed with 79.8 % glucose were obtained after five recycling cycles. Meanwhile, 51.6 % lignin was recovered throughout the recycling process.

Two-Step Enzymolysis of Antarctic Krill for Simultaneous Preparation of Value-Added Oil and Enzymolysate.

Antarctic krill is a crucial marine resource containing plenty of high-valued nutrients. However, krill oil as a single product has been developed by the current solvent extraction with high cost. From the perspective of comprehensive utilization of Antarctic krill, this study proposed a novel two-step enzymolysis-assisted extraction in attempt to produce value-added oil and enzymolysate simultaneously. After two-step chitinase/protease hydrolysis, the lipid yield increased from 2.09% to 4.18%, reaching 112% of Soxhlet extraction. The method greatly improved the yields of main components while reducing the impurity content without further refining. After optimization, the oil contained 246.05 mg/g of phospholipid, 80.96 mg/g of free eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and 0.82 mg/g of astaxanthin. The by-product enzymolysate was abundant in water-soluble proteins (34.35 mg/g), oligopeptides (13.92 mg/g), amino acids (34.24 mg/g), and carbohydrates (5.79 mg/g), which was a good source of functional nutrients. In addition, both oil and enzymolysate showed high antioxidant capacity. This novel method could simultaneously provide oil and enzymolysate amounting for 58.61% of dried krill.

Biosynthetic Profile in the Co-culture of Aspergillus sydowii and Bacillus subtilis to Produce Novel Benzoic Derivatives.

Microbial co-culture simulates the natural ecosystem through the combination of artificial microbes. This approach has been widely applied in the study of activating silent genes to reveal novel secondary metabolites. However, there are still challenges in determining the biosynthetic pathways. In this study, the effects of microbial co-culture on the morphology of the microbes were verified by the morphological observation. Subsequently, through the strategy combining substrate feeding, stable isotope labeling, and gene expression analysis, the biosynthetic pathways of five benzoic acid derivatives N1-N4 and N7 were demonstrated: the secondary metabolite 10-deoxygerfelin of A. sydowii acted as an inducer to induce B. subtilis to produce benzoic acid, which was further converted into 3-OH-benzoic acid by A. sydowii. Subsequently, A. sydowii used 3-OH-benzoic acid as the substrate to synthesize the new compound N2, and then N1, N3, N4, and N7 were biosynthesized upon the upregulation of hydrolase, hydroxylase, and acyltransferase during co-culture. The plate zone analysis suggested that the biosynthesis of the newly induced compounds N1-N4 was mainly attributed to A. sydowii, and both A. sydowii and B. subtilis were indispensable for the biosynthesis of N7. This study provides an important basis for a better understanding of the interactions among microorganisms, providing new ideas for studying the biosynthetic pathways of the newly induced secondary metabolites in co-culture.

Extractive adsorption of 1,3-propanediol on a novel polystyrene macroporous resin enclosing medium and long-chain alcohols as extractant.

Extractive adsorption is an integrated separation method employing a novel resin with both particle and liquid characteristics in terms of adsorption and extraction. In this study, the novel extractive adsorption polystyrene-divinylbenzene (PS-DVB) macroporous resin was synthesized by suspension polymerization, in which n-octanol (OL-PS-DVB) or mixed alcohols of n-octanol, undecyl alcohol, and tetradecyl alcohol (MA-PS-DVB) were added as porogen and enclosed in the resin skeleton after the reaction. The characterization of the two novel resins of OL-PS-DVB and MA-PS-DVB showed that they have large specific surface areas of 48.7 and 17.4 m2/g, respectively. Additionally, the two synthesized resins have much higher static adsorption capacities of 1,3-propanediol (511 and 473 mg/g) and dynamic adsorption capacities (312 and 267 mg/g) than traditional resins, because extractants enclosed in the resin can increase the adsorption capacity. Through Langmuir equation, the theoretical static maximum adsorption capacity of the mixed alcohols resin is 515 mg/g at 298 K and Gibbs free energy change of adsorption was -3781 J/mol, indicating that the adsorption process was spontaneous. In addition, the sorbent concentration effect in the resin was generated at high 1,3-propanediol (1,3-PDO) concentrations. The fitting of the Flocculation model can reveal that there is a possible relation between adsorption and flocculation. Compared to OL-PS-DVB, MA-PS-DVB showed better performance in the recovery yield of 1,3-PDO and other byproducts, the removal rates of the inorganic salt and protein, and the efficiency of recycled resin. For MA-PS-DVB, the recovery of 1,3-PDO, butyrate acid, acetic acid, and residual glycerol was 97.1%, 94.7%, 93.3%, and 90.3%, respectively. Simultaneously, the resin of MA-PS-DVB could remove 93.8% of inorganic salts and 90.9% of proteins in the concentrated fermentation broth. The two synthesized resins of OL-PS-DVB and MA-PS-DVB still had 90% or 92% of capacity for extractive adsorption of 1,3-propanediol after 10 times of recycling, which exhibited potential application in the separation of 1,3-propanediol from fermentation broth.

In situ carbon dioxide capture to co-produce 1,3-propanediol, biohydrogen and micro-nano calcium carbonate from crude glycerol by Clostridium butyricum.

BACKGROUND: Climate change caused by greenhouse gas emission has become a global hot topic. Although biotechnology is considered as an environmentally friendly method to produce chemicals, almost all biochemicals face carbon dioxide emission from inevitable respiration and energy metabolism of most microorganisms. To cater for the broad prospect of biochemicals, bioprocess optimization of diverse valuable products is becoming increasingly important for environmental sustainability and cleaner production. Based on Ca(OH)2 as a CO2 capture agent and pH regulator, a bioprocess was proposed for co-production of 1,3-propanediol (1,3-PDO), biohydrogen and micro-nano CaCO3 by Clostridium butyricum DL07. RESULTS: In fed-batch fermentation, the maximum concentration of 1,3-PDO reached up to 88.6 g/L with an overall productivity of 5.54 g/L/h. This productivity is 31.9% higher than the highest value previously reports (4.20 g/L/h). In addition, the ratio of H2 to CO2 in exhaust gas showed a remarkable 152-fold increase in the 5 M Ca(OH)2 group compared to 5 M NaOH as the CO2 capture agent. Green hydrogen in exhaust gas ranged between 17.2% and 20.2%, with the remainder being N2 with negligible CO2 emissions. During CO2 capture in situ, micro-nano calcite particles of CaCO3 with sizes in the range of 300 nm to 20 µm were formed simultaneously. Moreover, when compared with 5M NaOH group, the concentrations of soluble salts and proteins in the fermentation broth of 5 M Ca(OH)2 group were notably reduced by 53.6% and 44.1%, respectively. The remarkable reduction of soluble salts and proteins would contribute to the separation of 1,3-PDO. CONCLUSIONS: Ca(OH)2 was used as a CO2 capture agent and pH regulator in this study to promote the production of 1,3-PDO. Meanwhile, micro-nano CaCO3 and green H2 were co-produced. In addition, the soluble salts and proteins in the fermentation broth were significantly reduced.

Recycling reaction and separation for FACylation of loxenatide by trade-off between miscibility and immiscibility of reactants and product in methanol solution.

The growing demand and scale of production for fatty acid chain modified (FACylated) polypeptide has sparked the interest in novel production technologies. In this study, a recycling reaction and separation process was proposed and applied to the fatty acid chain modification (FACylation) of loxenatide (LOX), which was based on the difference in solubility between reactants and FACylated product. Especially, the mixed PBS-Methanol (MeOH) solution was designed to meet the demands for FACylation of LOX as well as separation of FACylated LOX and residual modifier. In order to ensure the efficient FACylation, a mixed 10% PBS-90% MeOH (v/v) solution was chosen to provide a good miscibility for two reactants, LOX and N-tetradecylmaleimide (C14-MAL). On the other hand, the immiscibility between reactant (C14-MAL) and FACylated product (N-tetradecyl-Loxenatide (C14-LOX)) could realize the separation of C14-LOX when the MeOH concentration was less than 30% (v/v). Based on this strategy, the recycling reaction and separation process for FACylation of LOX was established by adjusting the MeOH concentration in the mixed solution. The reaction yield and recovery of C14-LOX exceeded 97% and 94%, and the excess reactant C14-MAL could be recycled with a recovery of more than 80%. Furthermore, after purification by reversed-phase chromatography, C14-LOX showed good pharmacokinetic and pharmacodynamic properties in vivo. This study will have great application prospects in industrial production of C14-LOX.

Pretreatment of Jerusalem artichoke stalk using hydroxylammonium ionic liquids and their influences on 2,3-butanediol fermentation by Bacillus subtilis.

Pretreatment of lignocellulose is a vital step for biological production of bio-chemicals and bio-fuels. In this work, the pretreatment of Jerusalem artichoke stalk (JAS) by hydroxylammonium ionic liquids was evaluated based on pretreatment efficiency including polysaccharide recovery and enzymatic digestibility, and influence of ionic liquids on 2,3-butanediol fermentation using Bacillus subtilis. The results showed ethanolammonium acetate (EOAA) was efficient in JAS pretreatment, and maximum cell density was increased 25% when EOAA concentration was not greater than 0.3 mol/L in medium, while the total concentration of acetoin and 2,3-butanediol was 15% greater than the control at 0.1 mol/L EOAA. After the pretreatment under optimized conditions of 170 °C for 5-h and liquid-solid ratio of 18, about 87% cellulose and 75% hemicellulose were recovered, and glucose yield of 64% and xylose of 66% were obtained after 24-h hydrolysis of JAS residue by cellulase (15 FPU/g) with solid loading of 10 wt%.

Effects of carboxymethyl chitosan adsorption on bioactive components of Antarctic krill oil.

High acid value (AV) and fluorine content of Antarctic krill oil (AKO) extracted from frozen krill by ethanol limit its product development. In this study, a method was proposed to reduce the AV and fluorine content of AKO by carboxymethyl chitosan (CMCS) adsorption. The optimal adsorption condition was 12.5% (w/v) of CMCS at 30℃ for 15 min. At this condition, AV and fluorine content decreased by 78.0% and 61.4%, respectively. It is interesting that CMCS adsorption showed specificity to particular substances. Although free fatty acids content showed a significant reduction, free EPA and DHA, phospholipid and astaxanthin remained almost constant. Moreover, CMCS adsorption showed no influence on neuroprotective activity of AKO against H2O2-induced neuro-damage of PC12 cells. The reclaimed CMCS showed an undiminished antimicrobial activity against both Gram-positive and Gram-negative bacteria. The CMCS adsorption shows a potential development for refining AKO and other oils in food industry.

Co-culture of Aspergillus sydowii and Bacillus subtilis induces the production of antibacterial metabolites.

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool to activate the biosynthetic gene clusters to generate new compounds. However, without optimization of fermentation conditions, the antagonism between the microbes often interferes with the production of secondary metabolites. In this study, the fermentation conditions of co-culture of Aspergillus sydowii and Bacillus subtilis were optimized by response surface methodology to increase the production of active metabolites against Staphylococcus aureus. After optimization, the inhibitory rate of the co-culture extract was 74.62%, which was 29.20% higher than that of the initial conditions. Meanwhile, a total of 15 newly biosynthesized metabolites were detected only in optimized co-culture, occupying 13.2% of all detected metabolites. The structures of the 12 metabolites with high variable importance in projection score were elucidated by the established LC-MS/MS approach integrated with various metabonomic tools. Among them, 7 metabolites were newly induced and the content of other 5 metabolites increased by 1.1-2.4 folds in optimized co-culture. The bioassay of metabolites in co-culture against S. aureus indicated that compounds (-)- (7S)- 10-hydroxysydonic acid, serine sydonate and macrolactin U' contributed much to the increment of antibacterial activity. This study demonstrated that optimizing the fermentation conditions of co-culture was beneficial to changing the metabolite profile and effective to induce the biosynthesis of active metabolites.

Optimization and feedback control system of dilution rate for 1,3-propanediol in two-stage fermentation: A theoretical study.

In utilizing glycerol to produce 1,3-propanediol by microbial fermentation, the problems of low utilization rate and poor production performance need to be addressed. Based on the analysis of a mathematical model for 1,3-propanediol production from glycerol by Klebsiella pneumoniae, this study theoretically investigated the effects of the dilution rate and the initial glycerol concentration in a two-stage fermentation process and the feasibility of applying the feedback control methods. First, the optimal operation conditions of initial glycerol concentration and dilution rate were obtained. Through the use of feedback control theory, a control strategy for dilution rate was designed and optimized to shorten the settling time (time required for fermentation to reach stability) from 60.92 to 36.68 h for the first reactor, and from 53.66 to 22.68 h for the second reactor. In addition, the yield of 1,3-propanediol in both two reactors reached up to 0.5 g·g-1 . The simulation results indicated that the feedback control strategy for dilution rate increased the product concentration, reduced the residual glycerol in the fermentation broth, and greatly improved the performance of the fermentation. A feeding strategy of automatic control for dilution rate has been established and will be applied as an effective guiding scheme in automatic continuous fermentations for production of 1,3-propanediol.

Simultaneous solid-liquid separation and primary purification of clavulanic acid from fermentation broth of Streptomyces clavuligerus using salting-out extraction system.

Clavulanic acid (CA) is usually used together with other ß-lactam antibiotics as combination drugs to inhibit bacterial ß-lactamases, which is mainly produced from the fermentation of microorganism such as Streptomyces clavuligerus. Recently, it is still a challenge for downstream processing of low concentration and unstable CA from fermentation broth with high solid content, high viscosity, and small cell size. In this study, an integrated process was developed for simultaneous solid-liquid separation and primary purification of CA from real fermentation broth of S. clavuligerus using salting-out extraction system (SOES). First, different SOESs were investigated, and a suitable SOES composed of ethanol/phosphate was chosen and further optimized using the pretreated fermentation broth. Then, the optimal system composed of 20% ethanol/15% K2HPO4 and 10% KH2PO4 w/w was used to direct separation of CA from untreated fermentation broth. The result showed that the partition coefficient (K) and recovery yield (Y) of CA from untreated fermentation broth were 29.13 and 96.8%, respectively. Simultaneously, the removal rates of the cells and proteins were 99.8% and 63.3%, respectively. Compared with the traditional method of membrane filtration or liquid-liquid extraction system, this developed SOES showed the advantages of simple operation, shorter operation time, lower process cost and higher recovery yield of CA. These results demonstrated that the developed SOES could be used as an attractive alternative for the downstream processing of CA from real fermentation broth.

Three-liquid-phase salting-out extraction of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-rich oils from Euphausia superba.

The TLPSOES parameters were optimized by response surface methodology using Box-Behnken design, which were 16.5% w/w of ammonium citrate, 17.5% w/w of ethanol, and 46% w/w of n-hexane at 70 min of stirring time. Under optimized conditions the extraction efficiency attained was 90.91 ± 0.97% of EPA, 90.02 ± 1.04% of DHA, and 91.85 ± 1.11% of KO in the top n-hexane phase. The highest extraction efficiency of proteins and flavonoids, i.e. 88.34 ± 1.35% and 79.67 ± 1.13%, was recorded in the solid interface and ethanol phase, respectively. The KO extracted by TLPSOES system consisted of lowest fluoride level compared to the conventional method and whole wet krill biomass. The TLPSOES is a potential candidate for nutraceutical industry of KO extraction from wet krill biomass.

Metabolism, morphology and transcriptome analysis of oscillatory behavior of Clostridium butyricum during long-term continuous fermentation for 1,3-propanediol production.

BACKGROUND: Oscillation is a special cell behavior in microorganisms during continuous fermentation, which poses threats to the output stability for industrial productions of biofuels and biochemicals. In previous study, a spontaneous oscillatory behavior was observed in Clostridium butyricum-intensive microbial consortium in continuous fermentation for 1,3-propanediol (1,3-PDO) production from glycerol, which led to the discovery of oscillation in species C. butyricum. RESULTS: Spontaneous oscillations by C. butyricum tended to occur under glycerol-limited conditions at low dilution rates. At a glycerol feed concentration of 88 g/L and a dilution rate of 0.048 h-1, the oscillatory behavior of C. butyricum was observed after continuous operation for 146 h and was sustained for over 450 h with an average oscillation period of 51 h. During oscillations, microbial glycerol metabolism exhibited dramatic periodic changes, in which productions of lactate, formate and hydrogen significantly lagged behind that of other products including biomass, 1,3-PDO and butyrate. Analysis of extracellular oxidation-reduction potential and intracellular ratio of NAD+/NADH indicated that microbial cells experienced distinct redox changes during oscillations, from oxidized to reduced state with decreasing of growth rate. Meanwhile, C. butyricum S3 exhibited periodic morphological changes during oscillations, with aggregates, elongated shape, spores or cell debris at the trough of biomass production. Transcriptome analysis indicated that expression levels of multiple genes were up-regulated when microbial cells were undergoing stress, including that for pyruvate metabolism, conversion of acetyl-CoA to acetaldehyde as well as stress response. CONCLUSION: This study for the first time systematically investigated the oscillatory behavior of C. butyricum in aspect of occurrence condition, metabolism, morphology and transcriptome. Based on the experimental results, two hypotheses were put forward to explain the oscillatory behavior: disorder of pyruvate metabolism, and excessive accumulation of acetaldehyde.

Sequential fed-batch fermentation of 1,3-propanediol from glycerol by Clostridium butyricum DL07.

The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). Although Clostridium butyricum is considered to be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellent C. butyricum (DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L•h) vs. 94.2 g/L and 3.04 g/(L•h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated by C. butyricum DL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L•h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO by C. butyricum. KEY POINTS: • A novel C. butyricum strain was screened to produce 104.8 g/L 1,3-PDO from glycerol. • Corn steep liquor powder was used as a cheap nitrogen source for 1,3-PDO production. • A sequential fed-batch fermentation process was established for 1,3-PDO production. • An automatic glycerol feeding strategy was applied in the production of 1,3-PDO.

Dietary 5,6,7-Trihydroxy-flavonoid Aglycones and 1-Deoxynojirimycin Synergistically Inhibit the Recombinant Maltase-Glucoamylase Subunit of α-Glucosidase and Lower Postprandial Blood Glucose.

1-Deoxynojirimycin (1-DNJ) is the major effective component of mulberry leaves, exhibiting inhibitory activity against α-glucosidase. However, due to the low content of 1-DNJ in mulberry products, its level cannot meet the lowest dose to exhibit its activity. In this study, a combination of dietary 5,6,7-trihydroxy-flavonoid aglycones with 1-DNJ showed synergistic inhibitory activity against maltase of mice α-glucosidase and recombinant C- and N-termini of maltase-glucoamylase (MGAM) and baicalein with 1-DNJ exhibited the strongest synergistic effect. The synergistic effect of the combination was also confirmed by the maltose tolerance test in vivo. Enzyme kinetics, molecular docking, fluorescence spectrum, and circular dichroism spectrometry studies indicated that the major mechanism of the synergism is that baicalein was a positive allosteric inhibitor and bound to the noncompetitive site of MGAM, causing an increase of the binding affinity of 1-DNJ to MGAM. Our results might provide a theoretical basis for the design of dietary supplements containing mulberry products.