Triple-Mechanism Enhanced Flexible SiO2 Nanofiber Composite Hydrogel with High Stiffness and Toughness for Cartilaginous Ligaments.

Hydrogels are widely used in tissue engineering, soft robotics and wearable electronics. However, it is difficult to achieve both the required toughness and stiffness, which severely hampers their application as load-bearing materials. This study presents a strategy to develop a hard and tough composite hydrogel. Herein, flexible SiO2 nanofibers (SNF) are dispersed homogeneously in a polyvinyl alcohol (PVA) matrix using the synergistic effect of freeze-drying and annealing through the phase separation, the modulation of macromolecular chain movement and the promotion of macromolecular crystallization. When the stress is applied, the strong molecular interaction between PVA and SNF effectively disperses the load damage to the substrate. Freeze-dried and annealed-flexible SiO2 nanofibers/polyvinyl alcohol (FDA-SNF/PVA) reaches a preferred balance between enhanced stiffness (13.71 ± 0.28 MPa) and toughness (9.9 ± 0.4 MJ m-3). Besides, FDA-SNF/PVA hydrogel has a high tensile strength of 7.84 ± 0.10 MPa, super elasticity (no plastic deformation under 100 cycles of stretching), fast deformation recovery ability and excellent mechanical properties that are superior to the other tough PVA hydrogels, providing an effective way to optimize the mechanical properties of hydrogels for potential applications in artificial tendons and ligaments.

Effects of Fe2+ addition to sugarcane molasses on poly-γ-glutamic acid production in Bacillus licheniformis CGMCC NO. 23967.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA. RESULTS: Here γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO4·7H2O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO4·7H2O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle. CONCLUSIONS: These results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO4·7H2O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.

A sustainable pH shift control strategy for efficient production of ß-poly(L-malic acid) with CaCO3 addition by Aureobasidium pullulans ipe-1.

ß-poly(L-malic acid) (PMLA) has attracted industrial interest for its potential applications in medicine and other industries. For a sustainable PMLA production, it requires replacing/reducing the CaCO3 usage, since the residual CaCO3 impeded the cells' utilization, and a large amount of commercially useless gypsum was accumulated. In this study, it was found that more glucose was converted into CO2 using soluble alkalis compared with CaCO3 usage. Moreover, since the high ion strength and respiration effect of soluble alkalis also inhibited PMLA production, they could not effectively replace CaCO3. Furthermore, comparing the fermentations with different neutralizers (soluble alkali vs. CaCO3), it was found that the differential genes are mainly involved in the pathway of starch and sucrose metabolism, pentose and glucuronate interconversions, histidine metabolism, ascorbate and aldarate metabolism, and phagosome. In detail, in the case with CaCO3, 562 genes were downregulated and 262 genes were upregulated, and especially, those genes involved in energy production and conversion were downregulated by 26.7%. Therefore, the irreplaceability of CaCO3 was caused by its effect on the PMLA metabolic pathway rather than its usage as neutralizer. Finally, a combined pH shift control strategy with CaCO3 addition was developed. After the fermentation, 64.8 g/L PMLA and 38.9 g/L biomass were obtained with undetectable CaCO3 and less CO2 emission. KEY POINTS: • The effect of CaCO3 on PMLA metabolic pathway resulted in its irreplaceability. • A pH shift control strategy with CaCO3 addition was developed. • Undetectable CaCO3 and less CO2 emission were detected with the new strategy. Graphical abstract.

Green process for isolation and purification of poly(ß-L-malic acid) from Aureobasidium spp. by an integrated ion exchange and membrane separation.

Poly (ß-L-malic acid) (PMLA) is a biopolymer used in food and medical fields. However, the industrial processes are susceptible to the pollution of CaSO4 waste and organic solvent owing to the heavy use of CaCO3 in fermentation process and organic solvents in isolation process. This study developed an organic solvent and CaSO4 -free process for the industrial-scale production of PMLA. Firstly, calcium ion was removed at pH 9.2 by pH adjustment with Na2CO3, and the generated CaCO3 was reused in the fermentation process. Then, the D296 resin was selected to isolate the PMLA from the Ca2+-free broth, where the adsorption data were both primely described by the Freundlich and Langmuir equation, while Freundlich model better fit the process than Langmuir equation, indicating that it was non-monolayer adsorption of PMLA on the resin. Meanwhile, a three-step gradient elution with phosphate buffer (i.e., 0.2 mol/L, pH 7.0) containing 0.1, 0.2 and 1 mol/L NaCl was developed to recover PMLA. Finally, a PES15 membrane was selected to recover the PMLA from the elution solution, which could be reused in the next cycle. As a result, the PMLA with a purity of 98.89 % was obtained with the developed green process. In the developed process, it removed the pollution of organic solvent and calcium waste for the biosynthesis of PMLA on an industrial scale, which also offers a sustainable and green route for the biosynthesis of other carboxylic acids.

Fractionation and characterization of poly(ß-L-malic acid) produced by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries, whose functions primarily depend upon its molecular size and chemical structure. Up to now, the fractionation and characterization of PMLA produced by Aureobasidium spp. were still unclear. In this study, the product from A. melanogenum ipe-1 was effectively fractionated using 300 and 50 kDa membranes. During the filtration, the mechanisms of membrane fouling were illegible since the PMLA can both reject and permeate the membrane, while the main fouling mechanism varied between standard blocking and complete blocking during the diafiltration. After fractionation, 14.0, 8.4 and 77.6 % of the PMLAs with Mws of 75,134, 21,344 and 10,056 Da were distributed in the 300 kDa retentate after diafiltrating, 50 kDa retentate after diafiltrating, and the 50 kDa permeate, respectively. The Mw/Mns of the PMLAs were 4.12, 1.92, and 1.12 in the three fractions. Based on characteristic spectra of NMR, HPLC and FTIR, the product was not usual L-malic acid monomers, but glucose-terminated PMLA. The glucose was located at the terminal hydroxyl of PMLA. These results would serve as a valuable guide for process design and practical operation in subsequent industrial application.

Wolfberry water extract attenuates blue light-emitting diode damage to ARPE-19 cells and mouse retina by activating the NRF2 signaling pathway.

The wolfberry is believed to improve eyesight in traditional Chinese medicine. Soaking wolfberry in thermos cups has become a common health-preserving practice. The object of this paper was to research the protective effects of wolfberry water extract (WWE) on oxidative injury induced by blue light-emitting diodes (LEDs) in ARPE-19 cells and C57BL/6J mice. Wolfberry water extract significantly increased cell viability, reduced ROS production, stabilized mitochondrial membrane potential, and inhibited apoptosis in blue LED-induced cells (P < 0.05). The protective effects of WWE against blue LED-induced cytotoxicity and ROS accumulation in cells were abolished by transfection with Nrf2 siRNA. In blue LED-exposed C57BL/6J mice, WWE treatment markedly increased the amplitudes of electroretinogram (ERG) waves a and b, increased the thickness of retinal outer nuclear layer (ONL), activated endogenous antioxidant enzymes, and decreased MDA levels in the retina and lens. WWE also promoted NRF2 translocation and the expression of the downstream genes Ho-1, Nqo1, Gclc, and Gclm in the retina. The protection of WWE in ERG a and b wave amplitudes and ROS levels were abrogated in Nrf2 knockout mice. These results suggested that WWE has beneficial effects on retinal injury induced by blue LED, and mechanisms of action at least partly via the NRF2 signaling pathway.

Intensification of ß-poly(L: -malic acid) production by Aureobasidium pullulans ipe-1 in the late exponential growth phase.

ß-Poly(malic acid) (PMLA) has attracted industrial interest because this polyester can be used as a prodrug or for drug delivery systems. In PMLA production by Aureobasidium pullulans ipe-1, it was found that PLMA production was associated with cell growth in the early exponential growth phase and dissociated from cell growth in the late exponential growth phase. To enhance PMLA production in the late phase, different fermentation modes and strategies for controlling culture redox potential (CRP) were studied. The results showed that high concentrations of produced PMLA (above 40 g/l) not only inhibited PMLA production, but also was detrimental to cell growth. Moreover, when CRP increased from 57 to 100 mV in the late exponential growth phase, the lack of reducing power in the broth also decreased PMLA productivity. PMLA productivity could be enhanced by repeated-batch culture to maintain cell growth in the exponential growth phase, or by cell-recycle culture with membrane to remove the produced PMLA, or by maintaining CRP below 70 mV no matter which kind of fermentation mode was adopted. Repeated-batch culture afforded a high PMLA concentration (up to 63.2 g/l) with a productivity of 1.15 g l(-1) h(-1). Cell-recycle culture also confirmed that PMLA production by the strain ipe-1 was associated with cell growth.

Bio-based poly (γ-glutamic acid)-gelatin double-network hydrogel with high strength for wound healing.

Building bio-based hydrogels with high strength and biocompatibility is still a challenge. Herein, we successfully constructed a hybrid double-network (DN) full biological hydrogel with excellent mechanical properties and biocompatibility by introducing a physically cross-linked gelatin (GEL) network in a covalently cross-linked poly (γ-glutamic acid) (γ-PGA) network. The γ-PGA-GEL DN hydrogel demonstrated ultra-high compression performance (38 MPa), which was better than all currently reported γ-PGA-based hydrogels, and its tensile performance (0.27 MPa) was also satisfactory. Due to the unique multi-crosslinked DN structure, the γ-PGA-GEL DN hydrogel had better recovery and healing properties than those of the γ-PGA single-network (SN) hydrogel. In addition, the γ-PGA-GEL DN hydrogel exhibited good transparency, swelling and degradability. In vitro cell experiments demonstrated that the γ-PGA-GEL DN hydrogel was beneficial to cell adhesion and proliferation. The evaluation of the full-thickness skin defects model in rats exhibited that the γ-PGA-GEL DN hydrogel could significantly accelerate wound healing. These results indicated that the γ-PGA-GEL DN hydrogel was an ideal candidate material for wound dressing.

Electrolytic stimulation in aid of poly(ß-L-malic acid) production by Aureobasidium melanogenum ipe-1.

Poly (ß-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO3-) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO3- addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO3- addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production.

Bio-based poly (γ-glutamic acid) hydrogels reinforced with bacterial cellulose nanofibers exhibiting superior mechanical properties and cytocompatibility.

Natural polymer hydrogels are expected to be promising biomaterial because of its excellent biocompatibility and biodegradability, but they are soft and easily broken. Herein, the poly (γ-glutamic acid) (γ-PGA)/bacterial cellulose (BC) composite hydrogels with excellent mechanical properties were constructed by introducing bacterial cellulose. The γ-PGA/BC composite hydrogels were obtained by the covalent cross-linking of γ-PGA in the BC nanofibers suspensions. The γ-PGA/BC composite hydrogels exhibited excellent strength and toughness due to the more effective energy dissipation of hydrogen bonds network among BC nanofibers and γ-PGA hydrogel matrix and BC also acts as an enhancer. The compressive fracture strength and toughness of the γ-PGA/BC composite hydrogels could reach up to 5.72 MPa and 0.42 MJ/m3 respectively. Additionally, the tensile strength of γ-PGA/BC composite hydrogels were improved 8.16 times compared with γ-PGA single network hydrogels. More significantly, BC could disperse evenly in the γ-PGA hydrogels because of the hydrophilic nature of γ-PGA and BC nanofillers, which led to good interface compatibility. The result of cytotoxicity tests indicated that γ-PGA/BC composite hydrogels present excellent cytocompatibility, which suggested that the γ-PGA/BC composite hydrogels could serve as promising materials for many biomaterial related applications.

Effects of corn steep liquor on ß-poly(L-malic acid) production in Aureobasidium melanogenum.

ß-poly(L-malic acid) (PMLA) is a water-soluble biopolymer used in medicine, food, and other industries. However, the low level of PMLA biosynthesis in microorganisms limits its further application in the biotechnological industry. In this study, corn steep liquor (CSL), which processes high nutritional value and low-cost characteristics, was selected as a growth factor to increase the PMLA production in strain, Aureobasidium melanogenum, and its metabolomics change under the CSL addition was investigated. The results indicated that, with 3 g/L CSL, PMLA production, cell growth, and yield (Yp/x) were increased by 32.76%, 41.82%, and 47.43%, respectively. The intracellular metabolites of A. melanogenum, such as amino acids, organic acids, and key intermediates in the TCA cycle, increased after the addition of CSL, and the enrichment analysis showed that tyrosine may play a major role in the PMLA biosynthesis. The results presented in this study demonstrated that the addition of CSL would be an efficient approach to improve PMLA production.

Membrane-assisted ß-poly(L-malic acid) production from bagasse hydrolysates by Aureobasidium pullulans ipe-1.

Membrane-assisted ß-poly(L-malic acid) (PMLA) production from bagasse hydrolysates was developed. For the first time, it was found that mixing the acid and enzyme hydrolysates was unfavorable for PMLA production because too high hexose: pentose ratio and glucose concentration in the mixed sugar could inhibit the assimilation of pentose. 120 g/L sugar concentrations in the acid hydrolysate was suitable for PMLA production with 23.2 g/L PMLA and 34.7 g/L biomass. Moreover, an integrated membrane process consisting of ultrafiltration, nanofiltration and reverse osmosis membranes could concentrate sugars and adjust acetic acid concentration prior to fermentation of lignocellulosic sugars. Meanwhile, it was found that 1.46 g/L acetic acid was preferred for PMLA production from enzyme hydrolysate or sole glucose which respectively increased PMLA production and cell growth by 25.4% and 5.9% from sole glucose, while it showed no significant enhancement in PMLA production with a higher cell growth and productivity from acid hydrolysate.

Combination of multi-enzyme expression fine-tuning and co-substrates addition improves phenyllactic acid production with an Escherichia coli whole-cell biocatalyst.

The aim of this study was to develop an environmentally safe and efficient method for phenyllactic acid (PLA) production using whole-cell cascade catalysis with l-amino acid deaminase (l-AAD), lactate dehydrogenase (LDH), and formate dehydrogenase (FDH). The PPA titer was low due to relatively low expression of LDH, intermediate accumulation, and lack of cofactors. To address this issue, ribosome binding site regulation, gene duplication, and induction optimization were performed to increased the PLA titer to 43.8 g/L. Then co-substrates (glucose, yeast extract, and glycerol) were used to increase NADH concentration and cell stability, resulting that the PLA titer was increased to 54.0 g/L, which is the highest reported production by biocatalyst. Finally, glucose was replaced with wheat straw hydrolysate as co-substrate to decrease the cost. Notably, the strategies reported herein may be generally applicable to other whole-cell cascade biocatalysts.

Efficient ß-poly(l-malic acid) production from Jerusalem artichoke by Aureobasidium pullulans ipe-1 immobilized in luffa sponge matrices.

ß-poly(l-malic acid) (PMLA) production by Aureobasidium pullulans ipe-1 using Jerusalem artichoke tuber (JA) hydrolysate as a low cost carbon source was developed. The PMLA production was favored by JA pretreated with 0.06 M nitric acid without adding exogenous nitrogen sources into fermentation medium. With an initial 130 g/L total sugar of the JA hydrolysate, the highest PMLA productivity 0.52 g/L·h was achieved, which was increased by 2.0 folds compared to that with sole glucose case. To further enhance PMLA productivity, the cells were immobilized in luffa sponge matrices, and repeated batch culture was carried out for 4 cycles. The resulting PMLA productivity was further enhanced by 50% compared with the batch culture. The cost of PMLA production in the JA case was only 5.4% of that in the glucose case. The outcomes of this work provided a strategy of PMLA production on a commercial scale.

Toward understanding the key enzymes involved in ß-poly (L-malic acid) biosynthesis by Aureobasidium pullulans ipe-1.

ß-poly (L-malic acid) (PMLA) is a biopolyester which has attracted industrial interest for its potential application in medicine and other industries. A high dissolved oxygen concentration (DO) was beneficial for PMLA production, while the mechanisms of DO in PMLA biosynthesis by Aureobasidium pullulans are still poorly understood. In this work, the amount of PMLA was first compared when A. pullulans ipe-1 were cultured under a high DO level (70% saturation) and a low DO level (10% saturation). Meanwhile, the key enzymes involved in different pathways of the precursor L-malic acid biosynthesis were studied. The results revealed that the activities of glucose-6-phosphate dehydrogenase (G6PDH) and phosphoenolpyruvate carboxylase (PEPC) were positively correlated with cell growth and PMLA production, while the activities of phosphofructokinases (PFK), pyruvic carboxylase (PC) and citrate synthetase (CS) did no show such correlations. It indicated that the Pentose Phosphate Pathway (PPP) may play a vital role in cell growth and PMLA biosynthesis. Moreover, the precursor L-malic acid for PMLA biosynthesis was mainly biosynthesized through phosphoenolpyruvic acid (PEP) via oxaloacetate catalyzed by PEPC. It was also found that low concentration of sodium fluoride (NaF) might impel carbon flux flow to the oxaloacetate through PEP, but inhibit the flux to the oxaloacetate via pyruvic acid.

High molecular weight ß-poly(L-malic acid) produced by A. pullulans with Ca²âº added repeated batch culture.

ß-Poly(malic acid) (PMLA) has attracted increasing attentions because of its potential application in medicine and other industries. In this study, the variation of PMLA molecular weight (Mw) in the batch culture and the strategies to enhance PMLA Mw were studied. Adding exogenous Ca(2+) (0.1g/L CaCl2) to the medium caused a significant increase in both PMLA concentration and Mw (11.38% and 26.3%, respectively) when Na2CO3 was used as the neutralizer. The Mw of PMLA during the process of batch culture, which associated with the specific PMLA production per unit cell mass (Yp/x) before glucose was depleted, increased from 12.522 KDa to its maximum 18.693 KDa and then kept decreasing until the end of the culture. Compared with the results in batch culture, Mw increased by 84.4% (up to 19.51 kDa) with a productivity of 1.1 gh(-1)L(-1) when the cells were maintained in exponential growth phase during Ca(2+) added repeated batch culture. The present work provides an efficient approach to obtain superior quality PMLA product with high Mw.

Preparation and properties of EDC/NHS mediated crosslinking poly (gamma-glutamic acid)/epsilon-polylysine hydrogels.

In this paper, a novel pH-sensitive poly (amino acid) hydrogel based on poly γ-glutamic acid (γ-PGA) and ε-polylysine (ε-PL) was prepared by carbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated polymerization. The influence of PGA/PL molar ratio and EDC/NHS concentration on the structure and properties was studied. Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) proved that hydrogels were crosslinked through amide bond linkage, and the conversion rate of a carboxyl group could reach 96%. Scanning electron microscopy (SEM) results showed a regularly porous structure with 20 µm pore size in average. The gelation time in the crosslink process of PGA/PL hydrogels was within less than 5 min. PGA/PL hydrogels had excellent optical performance that was evaluated by a novel optotype method. Furthermore, PGA/PL hydrogels were found to be pH-sensitive, which could be adjusted to the pH of swelling media intelligently. The terminal pH of swelling medium could be controlled at 5 ± 1 after equilibrium when the initial pH was within 3-11. The swelling kinetics was found to follow a Voigt model in deionized water but a pseudo-second-order model in normal saline and phosphate buffer solution, respectively. The differential swelling degrees were attributed to the swelling theory based on the different ratio of -COOH/-NH2 and pore size in hydrogels. The results of mechanical property indicated that PGA/PL hydrogels were soft and elastic. Moreover, PGA/PL hydrogels exhibited excellent biocompatibility by cell proliferation experiment. PGA/PL hydrogels could be degraded in PBS solution and the degradation rate was decreased with the increase of the molar ratio of PL. Considering the simple preparation process and pH-sensitive property, these PGA/PL hydrogels might have high potential for use in medical and clinical fields.

Trehalose biosynthesis enhancement for six yeast strains under pressurized culture.

Six yeast strains of the commercial brewing yeasts CICC1391 and CICC1471, the commercial baker yeasts CICC1339 and CICC1447, and the commercial alcohol yeasts CICC1286 and CICC1291 have been cultured under 1.0 MPa of pressure with N(2) and CO(2) as pressure media. The concentration of intracellular trehalose and the activity of trehalose synthases complex have been measured. Also, the morphology changes of yeast cells have been observed by scanning electronic microscope. There was a positive correlation between the activity of trehalose synthase complex and the concentration of intracellular trehalose; and there was a negative correlation between the activity of trehalose synthase complex and the viability of yeast strains. Having been cultured for 3 h at high pressure of 1.0 MPa, the concentration of intracellular trehalose and the activity of trehalose synthases complex were improved by 50.1% to 116.4% and 45.2% to 219.1%, respectively, compared to those of atmospheric pressure culture. Under high pressure, many wrinkles appeared on the membrane surface of yeast cells. It has been found that yeasts are more sensitive to high pressure for having more and sharper wrinkles on their cell membranes.

Optimization of effect factors for mycelial growth and exopolysaccharide production by Schizophyllum commune.

This paper is concerned with the optimization of effect factors for mycelial growth and exopolysaccharide production by Schizophyllum commune by one-factor-at-a-time and orthogonal methods. The one-factor-at-a-time method was adopted to investigate the effects of six different compounds (sodium carboxymethylcellulose, L: -glutamic acid, V(B1), naphthalene acetic acid, oleic acid, and Tween 80) on mycelial growth and exopolysaccharide production. Among these factors, oleic acid, V(B1) and Tween 80 were identified to be the most important factors. Subsequently, the concentration of oleic acid, V(B1) and Tween 80 were optimized using the orthogonal matrix method. The effects of the factors on the mycelial growth of S. commune were in the order of oleic acid > V(B1) > Tween 80, and those on exopolysaccharide production were in the same order. The optimal concentration for mycelia and exopolysaccharide were determined as oleic acid 0.1% (v/v), V(B1) 0.5 mg/L, and Tween 80 6 mg/L. The subsequent verification experiments confirmed the validity of the models. Under this optimized conditions in shake flask culture, the mycelial yield and exo-biopolymer production were 25.93 and 2.79 g/L, respectively, which were considerably higher than those obtained in the preliminary studies. The result was further confirmed in a 7-L fermentor experiments.