Effect of nitrogen, carbon sources and agitation speed on acetoin production of Bacillus subtilis SF4-3

Background: Currently, microbial fermentation method has become the research hotspot for acetoin production. In our previous work, an acetoin-producing strain, Bacillus subtilis SF4-3, was isolated from Japanese traditional fermented food natto. However, its conversion of glucose to acetoin was relatively low. In order to achieve a high-efficient accumulation of acetoin in B. subtilis SF4-3, main medium components and fermentation conditions were evaluated in this work. Results: The by-products analysis showed that there existed reversible transformation between acetoin and 2,3-butanediol that was strictly responsible for acetoin production in B. subtilis SF4-3. The carbon sources, nitrogen sources and agitation speed were determined to play crucial role in the acetoin production. The optimal media (glucose-H2O 150 g/L, yeast extract 10 g/L, corn steep dry 5 g/L, urea 2 g/L, K2HPO4 0.5 g/L, MgSO4 0.5 g/L) were obtained. Furthermore, the low agitation speed of 300 r/min was found to be beneficial to the reversible transformation of 2,3-butanediol for acetoin production in B. subtilis SF4-3. Eventually, 48.9 g/L of acetoin and 5.5 g/L of 2,3-butanediol were obtained in a 5-L fermenter, and the specific production of acetoin was 39.12% (g/g), which accounted for 79.90% of the theoretical conversion. Conclusions: The results indicated acetoin production of B. subtilis SF4-3 was closely related to the medium components and dissolved oxygen concentrations. It also provided a method for acetoin production via the reversible transformation of acetoin and 2,3-butanediol.

Research and industrialization of biobased materials in China / 生物工程学报

This paper reviews the research and commercialization progresses of biobased polymeric materials including polyhydroxyalkanoates (PHA), polylactides (PLA), poly (butylene succinate) (PBS) and its monomer succinate, and CO2 copolymer poly (propylene carbonate), especially these efforts made in China.

Analysis of GHB and Its Precursors in Urine and Their Forensic Application / 法医学杂志

OBJECTIVE@#To establish the method to analyze γ-hydroxybutyric acid (GHB) and its precursors 1,4-butanediol (1,4-BD) and gamma-butyrolactone (GBL) in urine through LC-MS/MS and provide evidence for related cases.@*METHODS@#GHB-d6 and MOR-d3 were used as the internal standard. The urine sample was separated by LC after protein precipitation with methanol. The electrospray ion source was for ionization. Each compound was detected through multiple-reaction monitoring (MRM) mode.@*RESULTS@#The limits of detection of GHB and its precursors 1,4-BD and GBL were 0.1, 0.1 and 2 μg/mL. The accuracy was 87.6%-98.1%. The intra-day and inter-day precisions were less than 15% and matrix effects were higher than 80%.@*CONCLUSION@#The method is high sensitive, simple, rapid, specific and with high reliability. This study has provided technical support and basic data for forensic cases involving GHB.

Effects of knockout of 2,3-butanediol synthesis key enzyme genes on 1,3-propandediol production in Klebsiella pneumoniae / 生物工程学报

2,3-butanediol (2,3-BD) is a major byproduct of 1,3-propandediol (1,3-PDO) fermentation by Klebsiella pneumoniae. To decrease the formation of 2,3-BD, the budC and budA gene, coding two key enzymes of 2,3-BD synthetic pathway in K. pneumoniae, were knocked out using Red recombination technology. The growth of the two mutants were suppressed in different level. The budC deficient strain fermentation results showed that 1,3-PDO concentration increased to 110% and 2,3-butanediol concentration dropped to 70% of the parent strain. However, the budA deficient strain did not produce 1,3-PDO and 2,3-BD, and the final titer of lactic acid, succinic acid, ethanol and acetic acid increased remarkably compared with the parent strain. Further analysis of budC deficient strain fermentation inferred that K. pneumoniae possessed the 2,3-BD cycle as a replenishment pathway. The consequence provided a new evidence for reforming low-byproduct K. pneumoniae.

Research progress in salting-out extraction of bio-based chemicals / 生物工程学报

Bio-refinery using cheap biomass focuses mainly on strain improvement and fermentation strategies whereas less effort is made on down-stream processing. Using cheap biomass more impurities are introduced into the fermentation broths than mono-sugar substrate, thus down-stream processing for bio-based chemicals becomes the key problem in industrial production. The technique called salting-out extraction (SOE) was introduced in this review, which is used to separate target products from fermentation broth on the basis of partition difference of chemicals in two phases formed by mixing salts and organic solvents (or amphipathic chemicals) with broth at suitable ratios. The effect of solvents and salts on the formation of two aqueous phases, especially short chain alcohols and inorganic salts, and the application of SOE in recovery of bio-based chemicals, such as lactic acid, 1,3-propanediol, 2,3-butanediol and acetoin were summarized. The bio-chemicals were efficiently recovered from fermentation broth, and most of the impurities (cells and proteins) were removed in the same step. This technique is promising in the separation of bio-based chemicals, especially the recovery of hydrophilic molecules with low molecular weights.

Effects of pH and oxygen supply on production of 2,3-butanediol from biodiesel-derived glycerol by Bacillus amyloliquefaciens / 生物工程学报

Bacillus amyloliquefaciens B10-127 was used to produce 2,3-butanediol (2,3-BD) from residual glycerol obtained from biodiesel synthesis. Important variables for 2,3-BD fermentation, pH and dissolved oxygen, were studied. When pH was maintained constant, the yield of 2,3-BD was inhibited. The highest 2,3-BD yields were achieved by fermentation without any pH control with an optimized initial pH 6.5. Batch fermentative production of 2,3-BD by B. amyloliquefaciens was investigated using various oxygen supply methods by changing agitation speed. Based on the analysis of three kinetic parameters including specific cell growth rate (micro), specific glucose consumption rate (q(s)) and specific 2,3-BD formation rate (q(p)), a three-stage agitation speed control strategy was proposed, aimed at achieving high concentration, high yield and high productivity of 2,3-BD. Maximum concentration of 2,3-BD reached 38.1 g/L, with the productivity of 1.06 g/(L x h), which were 14.8% and 63.1% over the best results from constant agitation speeds. In a pulse fed-batch fermentation, 2,3-BD concentration and productivity were significantly improved to 71.2 g/L and 0.99 g/(L x h), respectively. To our knowledge, these results were the highest for 2,3-BD production from biodiesel-derived glycerol.

Effect of acetic acid, furfural and 5-hydroxymethylfurfural on production of 2,3-butanediol by Klebsiella oxytoca / 生物工程学报

To get the tolerability and consumption of Klebsiella oxytoca on major inhibitors in lignocelluloses hydrolysate, we studied the effect of acetic acid, furfural and 5-hydroxymethylfurfural on production of 2,3-butanediol by Klebsiella oxytoca. The metabolites of furfural and 5-hydroxymethylfurfural were measured. The results show that when acetic acid, furfural and 5-hydroxymethylfurfural was individually added, tolerance threshold for Klebsiella oxytoca was 30 g/L, 4 g/L and 5 g/L, respectively. Acetic acid was likely used as substrate to produce 2,3-butanediol. The yield of 2,3-butanediol increased when acetic acid concentration was lower than 30 g/L. In the fermentation, more than 70% 5-hydroxymethylfurfural was converted to 2,5-furandimethanol. All furfural and the rest of 5-hydroxymethylfurfural were metabolized by Klebsiella oxytoca. It showed that in the detoxification process of 2,3-butanediol production using lignocelluloses hydrolysate, furfural should be given priority to remove and a certain concentration of acetic acid is not need to removal.

Chemical constituents from roots of Machilus yaoshansis / 中国中药杂志

To study chemical constituents contained in ethanol extracts from roots of Machilus yaoshansis. Fifteen compounds were separated from the roots of M. yaoshansis by using various chromatographic techniques. Their structures were identified on the basis of their physicochemical properties and spectral data as twelve lignans(+)-guaiacin (1), kadsuralignan C (2), (+)-isolariciresinol (3), 5'-methoxy-(+)-isolariciresinol (4), (7'S, 8R, 8'R)-lyoniresinol (5), meso-secoisolariciresinol (6), isolariciresinol-9'-O-beta-D-xylopyranoside (7), 5'-methoxy-isolariciresinol-9'-O-beta-D-xylopyranoside (8), lyoniresinol-9'-O-beta-D-xylopyranoside (9), (2R, 3R) -2, 3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-3-methyl-5-(E)-propenylbenzofuran (10), 3, 5'-dimethoxy-4', 7-epoxy-8, 3'-neolignan-4, 9, 9'-triol (11), nectandrin B (12), and three flavanes(+)-catechin (13), (-)-epicatechin (14), and bis-8, 8'-catechinylmethane (15). All of the compounds 1-15 were separated from M. yaoshansis for the first time.

Isolation and Identification of Antifungal Compounds from Bacillus subtilis C9 Inhibiting the Growth of Plant Pathogenic Fungi

Antagonistic microorganisms against Rhizoctonia solani were isolated and their antifungal activities were investigated. Two hundred sixteen bacterial isolates were isolated from various soil samples and 19 isolates were found to antagonize the selected plant pathogenic fungi with varying degrees. Among them, isolate C9 was selected as an antagonistic microorganism with potential for use in further studies. Treatment with the selected isolate C9 resulted in significantly reduced incidence of stem-segment colonization by R. solani AG2-2(IV) in Zoysia grass and enhanced growth of grass. Through its biochemical, physiological, and 16S rDNA characteristics, the selected bacterium was identified as Bacillus subtilis subsp. subtilis. Mannitol (1%) and soytone (1%) were found to be the best carbon and nitrogen sources, respectively, for use in antibiotic production. An antibiotic compound, designated as DG4, was separated and purified from ethyl acetate extract of the culture broth of isolate C9. On the basis of spectral data, including proton nuclear magneric resonance (1H NMR), carbon nuclear magneric resonance (13C NMR), and mass analyses, its chemical structure was established as a stereoisomer of acetylbutanediol. Application of the ethyl acetate extract of isolate C9 to several plant pathogens resulted in dose-dependent inhibition. Treatment with the purified compound (an isomer of acetylbuanediol) resulted in significantly inhibited growth of tested pathogens. The cell free culture supernatant of isolate C9 showed a chitinase effect on chitin medium. Results from the present study demonstrated the significant potential of the purified compound from isolate C9 for use as a biocontrol agent as well as a plant growth promoter with the ability to trigger induced systemic resistance of plants.

Comparison of 2,3-butanediol production by several strains and optimization of the fermentation medium / 生物工程学报

Five Klebsiella pneumonia strains (including two strains whose genes for lactic acid were knocked out) were used to produce 2,3-butanediol, in which K. pneumonia HR521 LDH (gene for lactic acid was knocked out) was the best for the production, and then the fermentation medium was optimized by orthogonal design. The optimum compositions were as follows: glucose 90 g/L, (NH4)2HPO4 3 g/L, CLSP 6 g/L, sodium acetate 5 g/L, KCl 0.4 g/L, MgSO4 0.1 g/L, FeSO4 x 7H2O 0.02 g/L, MnSO4 0.01 g/L. Under the above conditions, final concentration of acetone and 2,3-butanediol could reach 37.46 g/L, 10 g/L higher than that under the initial conditions, the yield was 90.53% of the theory, and the productivity was 1.5 g/(L-h), and no lactic acid was detected, which could be benefit for the downstream processing and industrial application.

Production of 2, 3-butanediol and succinic acid by Salinivibrio YS / 生物工程学报

The production of 2, 3-butanediol and succinic acid by a moderate halophile under anaerobic condition was investigated. This halophile, termed Salinivibrio YS, was isolated from the solid samples collected from Aydingkol Lake. Based on the single factor experiment, the parameters and their values for the production were obtained. Then, the optimum values of these parameters by the orthogonal experiments were obtained: temperature, 33 degrees C; initial pH of fermentation, 8.0; the pH during fermentation, 7.0; the concentration of acetic acid was 3 g/L and NaC1 was 10 g/L. Finally, a 3-L fermentation based on these conditions was carried out. After 108 h of fermentation under anaerobic condition, 35.05 g/L of 2, 3-butanediol and 22.46 g/L of succinic acid were obtained. About 50% of total glucose conversion was achieved. The study on 2, 3-butanediol and succinic acid by a halophile under anaerobic condition will expand the applications of halophiles and open a new area of production of 2, 3-butanediol and succinic acid.

Enzymes related with NAD synthesis promote conversion of 1,4-butanediol to 4-hydroxybutyrate / 生物工程学报

Besides medical application, 4-hydroxybutyrate (4-HB) is a precursor of P3HB4HB, a bioplastic showing excellent physical properties and degradability. Escherichia coli S17-1 (pZL-dhaT-aldD) can transform 1, 4-butanediol (1,4-BD) into 4HB with participation of cofactor NAD. To enhance productivity, nicotinic acid phosphoribosyltransferase (PncB) and nicotinamide adenine dinucleotide synthetase (NadE) were overexpressed to increase intracellular nicotinamide adenine dinucleotide concentration and promote reaction process. The shake flask fermentation result showed that the conversion rate increased by 13.03% with help of PncB-NadE, leading to 4.87 g/L 4HB from 10 g/L 1,4-BD, and productivity was increased by 40.91% to 1.86 g/g. These results demonstrated that expression of PncB and NadE is beneficial for conversion of 1,4-BD to 4HB.

A new 8-O-4' neolignan from Glehnia littoralis / 药学学报

To study the chemical constituents from Glehnia littoralis, macroreticular resin column chromatography, repeated column chromatography on Sephadex LH-20 and reverse phase ODS were used to isolate the compounds whose structures were elucidated based on spectroscopic data (ESI-MS, 1D and 2DNMR). From EtOAc-soluble fraction of the ethanol extracts, a new 8-O-4' neolignan was isolated and named as glehlinoside D (1), along with five known compounds: (-)-secoisolariciresinol (2), ferulic acid (3), caffeic acid (4), vanillic acid (5) and syringin (6). Compound 1 is a new 8-O-4' neolignan.

Effects of Cross-Linking Agents on the Stability of Human Acellular Dermal Matrix

PURPOSE: Human acellular dermal matrix(ADM) is widely used in the treatment of congenital anomalies and soft tissue deficiencies. But it is rapidly degraded in the body and does not provide satisfactory results. There is a need to improve collagen fiber stability through various methods and ultimately regulate the speed of degradation. METHODS: The ADMs were added with various cross- linking agents called glutaraldehyde, dimethyl 3,3'-dithiobispropionimidate to produce cross-linked acellular dermal matrices. 1,4-butanediol diglycidyl ether solution was applied with a pH of 4.5 and 9.0, respectively. The stability of cross-linked dermal matrix was observed by measuring the shrinkage temperature and the degradation rates. The cross- and non-cross linked dermis were placed in the rat abdomen and obtained after 8, 12 and 16 weeks. RESULTS: The shrinkage temperature significantly increased and the degradation rate significantly decreased, compared to the control(p<0.05). All of cross- linked dermises were observed grossly in 16 weeks, but most of non-cross linked dermis were absorbed in 8 weeks. Histologically, the control group ADM was found to have been infiltrated with fibroblasts and most of dermal stroma were transformed into the host collagen fibers. However, infiltration of fibroblasts in the experiment was insignificant and the original collagen structure was intact. CONCLUSION: Collagen cross-linking increases the structural stability and decreases degradation of acellular dermis. Therefore, decrease in body absorption and increase in duration can be expected.

The evaluation of cytocompatibility of mesenchymal stem cells on random biopolyster scaffolds / 生物医学工程学杂志

Mesenchymal stem cells (MSCs) were isolated from the bone marrow of rabbits and inoculated respectively on 3D scaffolds of poly (3-Hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV), poly (butylenes succinate) (PBS) and the blends for evaluation of their cytocompatibilities in vitro. It was found that the blends of less microporous possessed the best performance on attachment and proliferation of MSCs in the six configurations. The toluiding blue staining showed the best morphology of mensenchymal stem cells (MSCs) on the blends of less microporous blends. The scanning electronic microscopy (SEM) results showed that the blends of less microporous blends had the appropriate roughness for MSCs to attach and proliferate, which can be used as a suitable biomaterial to create small caliber vascular grafts.

Synthesis, characterization and blood compatibility studies of waterproof breathable polyurethanes / 生物医学工程学杂志

Adopting the two-step method and changing the proportion between PEG (Polyethylene glycol) and PTMG (poly (tetrahydrofuran), we used the MDI (4,4'-diphenylmethane diisocyanate) and short chain extender BDO (1,4-butanediol) as hard segment, the PTMG and PEG as soft segment, and hence prepared a series of polyether-based thermoplastic polyurethanes. FTIR showed the structure character of these polyurethanes. The determination of mechanics property and water contact angles revealed their good mechanics properties and hydrophilicity. Blood compatibility was evaluated by hemolysis test and platelet adhesion test, which revealed their good hemocompatibility. So those polyurethanes may be of wide application in the future.

Separation and identification of cis and trans isomers of 2-butene-1,4-diol and lafutidine by HPLC and LC-MS / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The cis and trans isomers separation of 2-butene-1,4-diol and lafutidine were studied by HPLC on two kinds of chiral columns: (S,S)-Whelk-O 1 and ChiraSpher. The isomers of 2-butene-1,4-diol can be separated on both chiral columns while the isomers of lafutidine can only be resolved on ChiraSpher column. The influence of different type and amount of mobile phase modifier on the isomers separation was extensively studied. The resolution of cis and trans isomers of 2-butene-1,4-diol was 2.61 on (S,S)-Whelk-O 1 column with hexane-ethanol (97:3, v/v) as the mobile phase. The resolution of lafutidine was 1.89 on ChiraSpher column with hexane-ethanol-THF-diethylamine (92:3:5:0.1, v/v/v/v) as the mobile phase. LC-MS methods were developed to identify the isomer peaks.

Microespuma exoperimental aplicada en venas de animales de laboratorio / Experimental Microfoam applied to veins of laboratory animals

La vedette actual de la escleroterapia es la Microespuma. Logramos inocular una "mousse" de esclerosante en dos especies animales-conejos y ovinos-obteniendo resultados sorprendentes. La fórmula detallada de la microespuma es no tóxica, de muy fácil manejo y por sobre todo económica. Se utilizaron estas dos especies animales por las siguientes razones:-La estructura histológica del endotelio vascular del conejo es la más semejante al humano.-Los conejos fueron inoculados para evaluar venas de pequeño calibre,0.4-1.5mm, mientras que los ovinos para evaluar venas de calibre medianoi, 1.5-3.0 mm aproximadamente

Biochemical studies on the effect of S-1,3-butanediol of diabetes induced rats.

The biochemical effect of S-1,3-butanediol on streptozotocin induced diabetic rats was studied. Rats were made diabetic by the intraperitoneal injection of 40 mg/kg body weight streptozotocin in sodium citrate buffer. A dosage of 25 mmol/kg body weight of S-1,3-butanediol was injected intraperitoneally for treatment. The streptozotocin induced diabetic rats showed a marked increase in blood glucose level, and significant increase in the level of cholesterol, triglycerides and free fatty acids. The glycogen levels in liver and kidney were greatly decreased in diabetic rats. Treatment with butanediol normalised the glucose and glycogen level but had no significant effect on protein and lipid levels.