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关键词： adipose tissue engineering   adipose-derived stromal cells   angiogenesis   bioreactors   bioscaffolds   decellularized adipose tissue   hypoxia   preconditioning  

摘要： Tissue-engineering approaches hold promise to address the need in plastic and reconstructive surgery for new therapies that promote stable adipose tissue regeneration. Previous studies have demonstrated the potential of combining decellularized adipose tissue (DAT) scaffolds with adipose-derived stromal cells (ASCs) for volume augmentation applications. With the goal of enhancing in vivo angiogenesis and adipogenesis, this study evaluated the effects of culturing human ASCs on DAT scaffolds within a perfusion bioreactor. Using this system, the impact of both dynamic culture and hypoxic preconditioning were explored in vitro and in vivo. Initial studies compared the effects of 14 days of culture within the perfusion bioreactor under hypoxia (2% O-2) or normoxia (similar to 20% O-2) on human ASC expansion and expression of hypoxia inducible factor-1 alpha (HIF-1 alpha) in vitro relative to static cultured controls. The findings indicated that culturing within the bioreactor under hypoxia significantly increased ASC proliferation on the DAT, with a higher cell density observed in the scaffold periphery. Subsequent characterization in a subcutaneous implant model in athymic nude mice revealed that in vivo angiogenesis and adipogenesis were markedly enhanced when the ASCs were cultured on the DAT within the perfusion bioreactor under hypoxia for 14 days prior to implantation relative to the other culture conditions, as well as freshly seeded and unseeded DAT control groups. Overall, dynamic culture within the perfusion bioreactor system under hypoxia represents a promising approach for preconditioning ASCs on DAT scaffolds to enhance their capacity to stimulate angiogenesis and host-derived adipose tissue regeneration.

关键词： Human connective tissue growth factor   Transdermal peptide   Silkworm silk gland bioreactor   Genetic engineering  

摘要： Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designedCTGF-8htandpepCTGF-8htgenes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression;moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems.

关键词： Anaerobic membrane bioreactor   Municipal wastewater treatment   Anaerobic digestion   Microfiltration   Biogas  

摘要： Pore size is one of the most important properties in the successful operation of membrane-based bioprocesses for the treatment of municipal wastewater. The characteristics of two anaerobic membrane bioreactors (AnMBRs), one with a hollow fiber membrane of 0.4 mu m pore size (AnMBR1), and the other with a membrane of 0.05 mu m pore size (AnMBR2) were investigated for the treatment of real municipal wastewater at room temperature (25 degrees C) under varied hydraulic retention times (HRTs). Process performance was evaluated in terms of organic removal efficiency, biogas production and membrane filtration behaviours during a long-term continuous operation. Both AnMBRs showed good organic removal performance with COD and BOD removal efficiencies of around 89% and 93%, respectively. High energy recovery potential was achieved, with the biogas yield ranging between 0.20 and 0.26 L-gas/g-CODrem and a methane content of approximately 75%. The differences in the membrane filtration behaviours in the two AnMBRs included different permeate flux and total filtration resistance (R-t). In the AnMBR with a 0.4 mu m pore size membrane, an average R-t of 1.08 x 10 boolean AND 12 m(-1) was obtained even when the permeate flux was a high 0.274 m/day, while a higher average R-t of 1.51 x 10 boolean AND 12 m(-1) was observed in the AnMBR with 0.05 mu m pore size membrane even when the flux was a low 0.148 m/day. The off-line membrane cleaning strategy used for AnMBR1 indicated that the membrane restoration efficiency was 90.2%. (C) 2020 Elsevier B.V. All rights reserved.

关键词： Cryogel bioreactor   Immobilization   Hydrophobic interaction   IgG   Fab fragment   Papain  

摘要： The continuous bed bioreactor systems have been used for the production of protein therapeutics, such as IgG, using immobilized enzyme in biopharmaceutical applications. We developed macroporous poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) cryogel-based bioreactor matrix using sodium dodecyl sulfate as surfactans in the presence of ethylene glycol dimethacrylate as cross linking agent by bulk polymerization. The developed polyGMA immobilized bioreactor with papain enzyme was used for specific fragmentation of immunoglobulin G. The catalysis efficiency for immobilized enzyme were investigated in comparison with free enzyme. The immobilized papain displayed broad catalytic activity over a variety of conditions, with maximal activity around pH 7.0 and 70 degrees C. The Michaelis-Menten kinetic constant (K-m), the maximum reaction velocity (V-max), and the catalytic efficiency (k(cat)) for free enzyme were 0.1097 mg/mL, 29.9 mg/mL/min, and 92.01 1/min, respectively, whereas for immobilized enzyme, K-m, V-max, and k(cat) values were 0.1078 mg/mL, 30.53 mg/mL/min, and 94.3 1/min, respectively. In a further step, after digestion, remarkable digestion products of bioreactor, Fab and Fc fragments, produced with immobilized papain bioreactors were analyzed in two ways by SDS-PAGE and reversed-phase HPLC;it was demonstrated that papain immobilized bioreactor successfully used for the digestion of human IgG with high activity. Therefore, the polyGMA cryogel immobilized with papain exhibited a very effective matrix for the bioreactor which can be considered as an alternative bioreactor matrix with great promise in biopharmaceutical applications.

关键词： Membrane fouling   Biofouling   Support layer   Forward osmosis   Osmotic membrane bioreactor   Wastewater treatment  

摘要： Anaerobic osmotic membrane bioreactor (AnOMBR) holds promise for simultaneous wastewater purification and biogas production, allowing for an energy and carbon-neutral treatment facility. In a typical AnOMBR, reverse osmosis (RO) is employed for re-concentrating draw solution for continuous operation and cost saving. We compared membrane fouling behaviors between AnOMBR-RO hybrid system and AnOMBR without RO unit. We concluded that the porous support layer was susceptible to both inorganic scaling and biofouling in the closed-loop AnOMBR-RO system. We also explored two cleaning approaches to mitigate inorganic scaling and biofouling. Specifically, ethylenediaminetetraacetic acid (EDTA) was introduced into draw solution for minimizing inorganic scaling, but biofouling was deteriorated as EDTA provided extra nutrients for bacterial proliferation and biofouling. On the other hand, chemical cleaning of membrane support layer was performed using NaClO solution for biofouling control, but such cleaning efficacy attenuated after several cleaning cycles, because inorganic minerals accumulated and grew within membrane porous layer which could not be flushed by NaClO cleaning. Our finding highlighted the complexity and courter intuitive perspective to membrane fouling and cleaning in AnOMBR-RO hybrid system for inorganic scaling and biofouling management.

关键词： Oligosaccharide   Ceramic membrane   Ultrafiltration   Enzyme membrane bioreactor   FOS purification  

摘要： Fructo-oligosaccharides (FOS) are low-calorie sweeteners that can be synthesized by the transfructosylation of sucrose using enzymes known as fructosyltransferases. However, enzymatic conversion is inhibited by the accumulation of glucose as a byproduct, which limits the conversion rate and yield. We therefore developed a semi-continuous production process in an enzyme membrane bioreactor (EMBR) system followed by fermentation with the probiotic bacterium Bacillus coagulans. Filtration experiments were conducted in total recycle mode to evaluate membrane fouling using the resistance-in-series model. We found that fouling was predominantly caused by the accumulation of proteins at the membrane surface, which accounted for 29.6-95.5% of the total filtration resistance depending on the conditions. Using these data, we were able to achieve a stable filtration flux that fulfilled the requirements of the EMBR system by regulating the filtration parameters. The average concentration of total FOS in the products of EMBR reached 270 g.L-1, which was 4.6% higher than the batch process. Subsequently, the crude FOS preparation was treated by fed-batch fermentation with B. coagulans. The monosaccharides in the reaction mix (glucose and fructose) were completely removed, increasing the concentration of FOS to 195.9 g.L-1 and the purity to 96.6%.

关键词： Bioprocess   Dynamic model   Escherichia coli   Modeling analogy   Phenomenology  

摘要： In the bioprocess modeling field, a descriptive methodology that explicity considers the interactions between the environment and cells is lacking. This relationship directly affects all kinetics, which have so far been formulated through empirical relationship. In this research, additional steps were added to a known bioprocess modeling methodology to relate environment and cells to each other. The quantitative validation of the proposed phenomenological-based, semi-physical model was developed for a glucose culture bioprocess carried out by the bacterium Escherichia coll. strain BW25113 (a derivative of E. coll. K-12 strain BD792). The results indicated that the model obtained through the proposed methodology resulted in more accurate predictions than those in the literature using empirical functions for environment-cell relationship modeling. Therefore, predictability of the model can be improved by linking descriptive and explanatory mathematical models of mass transfer to environment-cellular material interaction in bioprocess modeling. Therefore, environment and cells can be explicitly connected with mathematical expressions from chemical process analogies, mass transfer, and phase equilibrium thermodynamic fundamentals. Thus, valuable model information and total knowledge of the bioprocesses phenomena is obtained. This information is necessary for understanding the integral operation of a bioreactor and, thus, for improving the design, optimization, automatic control, and state estimation of the bioprocess.

关键词： Butyrylcholinesterase   Plant cell suspension culture   Transgenic rice cell culture   Rice alpha-amylase 3D   Inducible promoter   Bioreactor operation  

摘要： Metabolically regulated transgenic rice cell suspension culture has been developed aiming to be an efficient and cost-effective platform for production of rice-recombinant butyrylcholinesterase (rrBChE) that could be used as a bioscavenger enzyme against organophosphate nerve agents. In two-stage batch cultures, switching between growth and induction phases by exchanging media, the maximum active rrBChE levels were comparable among the bioreactor runs with controlled dissolved oxygen (DO) concentrations from 10-40% DO and without controlled DO at constant aeration rate. For a single-stage batch operation with no media exchange at 40% DO, production of rrBChE is triggered simply through sugar depletion due to the uptake of sugar by the cells, showing similar maximum active cell-associated rrBChE level compared to two-stage batch operations. However, the bioreactor process using uncontrolled DO, no media exchange, and half-concentration of sucrose resulted in a 1.4-fold increase in the maximum total active rrBChE (77 mu g/g fresh weight) and 1.6-fold increase of total active rrBChE specific productivity (86 mu g (g dry weight)(-1) day(-1)) compared to the two-stage batch cultures. We found that monitoring the culture pH is a simple non-invasive technique that can help identify the start and progress of the induction phase in a single-stage culture. Altogether, we demonstrate a simplified, efficient, economical, and scalable bioreactor process for rrBChE production in metabolically regulated transgenic rice cell cultures.

关键词： Microfluidics   Biocatalysis   L-DOPA   Dopamine   Cascade reactions   Packed-bed reactor  

摘要： Enzymatic synthesis of biochemical commodities is of upmost importance as it represents a greener alternative to traditional chemical synthesis and provides easier downstream processing strategies compared to fermentation-based processes. A microfluidic system used to optimize the enzymatic production of both levodopa (L-DOPA) and dopamine in both single-step and multistep-reaction sequences with yield of approximately 30 % for L-DOPA production and 70 % for dopamine production is presented. The system for L-DOPA production was then up-scaled (780-fold increase) to a milliliter scale system by maintaining similar mass transport properties resulting in the same yield, space-time yield and biocatalyst yield as its microscale counterpart. The results obtained for yield and biocatalyst yield (351.7 mg(L-DOPA) mg(Tyr)(-1) h(-1)) were similar to what is reported in the literature for similar systems, however the space-time yield (0.806 mg(L-DOPA) L-1 h(-1)) was smaller. This work demonstrates a microfluidic bioreactor that can be used for complex optimizations that can be performed rapidly while reducing the consumption of reagents by immobilizing the catalyst on a carrier which can then be used in a packed-bed reactor, thus extending the enzyme life span.

关键词： AGMBR   Membrane fouling   EPS   Microbial community   XDLVO theory  

摘要： Aerobic granular sludge membrane bioreactor (AGMBR) has been widely studied in recent years owing to its excellent wastewater treatment performance and antifouling property. However, the mechanisms of membrane fouling are lack of systematic and comprehensive elaboration. Here, the mechanisms of and distinctions between fouling of polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE) membranes in an AGMBR were investigated. The nutrient-removal performances were excellent in both cases. Compared with the PVDF membrane, the PTFE membrane exhibited a better antifouling property, with a lower transmembrane-pressure growth rate (0.407 kPa/d). Moreover, the PTFE membrane had lower contents of extracellular polymeric substances (EPS) and soluble microbial products, probably owing to the lower relative abundance of the class Gammaproteobacteria, which is the producer of EPS. Additionally, a thermomechanical analysis revealed that the energy barrier of the PTFE membrane-efoulant (71.20 kT) was higher than that of the PVDF membrane-efoulant (51.77 kT), confirming that the PTFE membrane had better antifouling performance. Overall, this study enhances the understanding of mechanisms of membrane fouling in an AGMBR and is conducive motivate large-scale applications of AGMBR technology. (c) 2020 Elsevier Ltd. All rights reserved.