Enantioselective liquid‐liquid extraction of DL‐mandelic acid using chiral diphosphine ligands as extractants

In order to expand the application range of chiral diphosphine ligands, (S)‐BINAP, (S)‐SEGPHOS, and (S)‐MeO‐BIPHEP were employed as extractants to recognize DL‐mandelic acid. The results indicated that (S)‐SEGPHOS‐Cu exhibited considerable ability to recognize DL‐mandelic acid with operational enantioselectivity (α) of 2.677. The process of extraction of DL‐mandelic acid using (S)‐SEGPHOS‐Cu as extractant was systematically investigated. Performance factor (pf) was adopted to comprehensively evaluate the extraction. After optimization by response surface methodology (RSM), the optimal extraction condition is temperature of 5.5°C, (S)‐SEGPHOS‐Cu concentration of 3.0 mmol/L, and pH of 8.0. And the predicted and experimental maximum values of pf were 0.26374 and 0.26839, respectively.     

Semi‐continuous in situ magnetic separation for enhanced extracellular protease production—modeling and experimental validation

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc.     

Enantioselective liquid‐liquid extraction of 3‐chloro‐phenylglycine enantiomers using (S,S)‐DIOP as extractant

(S,S)‐DIOP, a common catalyst used in asymmetric reaction, was adopted as chiral extractant to separate 3‐chloro‐phenylglycine enantiomers in liquid‐liquid extraction. The factors affecting extraction efficiency were studied, including metal precursors, organic solvents, extraction temperature, chiral extractant concentration, and pH of aqueous phase. (S,S)‐DIOP‐Pd exhibited good ability to recognize 3‐chloro‐phenylglycine enantiomers, and the operational enantioselectivity (α) is 1.836. The highest performance factor (pf) was obtained under the condition of extraction temperature of 9.1°C, (S,S)‐DIOP‐Pd concentration of 1.7 mmol/L, and pH of aqueous phase of 7.0. In addition, the possible recognition mechanism of (S,S)‐DIOP‐Pd towards 3‐chloro‐phenylglycine enantiomers was discussed.     

An innovative membrane bioreactor (MBR) system for simultaneous nitrogen and phosphorus removal

Membrane filtration was integrated with a post-denitrification process to form an innovative membrane bioreactor (MBR) system for effective organic degradation and nutrient (N and P) removal. The system comprised of an aerobic tank, an anoxic tank, an intermediate sedimentation tank, and a membrane filtration tank. The sedimentation tank functioned not only as a rough settler for sludge–water separation before membrane filtration but also as an anaerobic chamber for P release. While half of the influent flowed into the aerobic tank, the other half was fed into the anoxic tank to favor the proliferation of phosphorus accumulating organisms (PAOs). The experiment was conducted continuously for about 430 days. With a short overall treatment time of less than 10 h for municipal wastewater, the MBR-based process could achieve the total organic carbon, total nitrogen, and total phosphorus removals of around 94%, 85%, and 87%, respectively. The growth and activity of PAOs in the MBR system were evidenced by the significant P release in the anaerobic chamber followed by the luxury P uptake in the membrane tank. With the DAPI and PAO_mix probe staining, the increases of PAOs and polyhydroxybutyrate (PHB) in sludge during the experiment were well observed under the fluorescent microscope.     

In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid‐liquid extraction

Flurbiprofen is a kind of nonsteroidal anti‐inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S‐flurbiprofen shows good performance on clinic anti‐inflammatory treatment, while R‐enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S‐flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2‐dichloroethane as organic solvent, 1‐butyl‐3‐methylimidazole L‐tryptophan ([Bmim][L‐trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L^?1, and [Bmim][L‐trp] concentration was 0.02 mol L^?1. Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale‐up of the extraction process.     

Techno‐economic analysis for incorporating a liquid–liquid extraction system to remove acetic acid into a proposed commercial scale biorefinery

Mitigating the effect of fermentation inhibitors in bioethanol plants can have a great positive impact on the economy of this industry. Liquid–liquid extraction (LLE) using ethyl acetate is able to remove fermentation inhibitors—chiefly, acetic acid—from an aqueous solution used to produce bioethanol. The fermentation broth resulting from LLE has higher performance for ethanol yield and its production rate. Previous techno‐economic analyses focused on second‐generation biofuel production did not address the impact of removing the fermentation inhibitors on the economic performance of the biorefinery. A comprehensive analysis of applying a separation system to mitigate the fermentation inhibition effect and to provide an analysis on the economic impact of removal of acetic acid from corn stover hydrolysate on the overall revenue of the biorefinery is necessary. This study examines the pros and cons associated with implementing LLE column along with the solvent recovery system into a commercial scale bioethanol plant. Using details from the NREL‐developed model of corn stover biorefinery, the capital costs associated with the equipment and the operating cost for the use of solvent were estimated and the results were compared with the profit gain due to higher ethanol production. Results indicate that the additional capital will add 1% to the total capital and manufacturing cost will increase by 5.9%. The benefit arises from the higher ethanol production rate and yield as a consequence of inhibitor extraction and results in a $0.35 per gallon reduction in the minimum ethanol selling price (MESP). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:971–977, 2016     

Crystallization and liquid‐liquid phase separation of monoclonal antibodies and fc‐fusion proteins: Screening results

Crystallization holds the potential to be used for protein purification and low‐viscosity drug substance and drug product formulations. Twenty‐two different proteins (20 monoclonal antibodies and two Fc‐fusions) were examined to determine the breadth of applicability of crystallization to these therapeutic proteins. Vapor diffusion technique and an evaporative screening method were used to identify crystallization conditions using around a 100 initial conditions based on reagents that are generally regarded as safe (GRAS). Of 16 IgG₂s examined, at least four formed diffraction‐quality crystals and four others formed crystal‐like particles. At least three of the IgG₂s that crystallized well were also crystallized under the same set of operating conditions using inexpensive GRAS reagents. The crystals were formed to high‐yields in a few hours and were dissolved quickly without impacting product quality. Although only a fraction of the proteins examined crystallized, all exhibited liquid‐liquid phase separation (LLPS), which could be used for their concentration or possibly purification. One of the Fc‐fusions, for example, was concentrated by LLPS to a self‐buffering solution at 150 g/L. Crystallization and LLPS in the salting‐in region were shown to be feasible. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Technical evaluation of the potential for streamlining of equipment validation for fermentation applications

The purpose of this article is to provide a few concrete examples of the potential to acceptably reduce the scope of validation and qualification testing based on scientific justification for the specific area of microbial fermentation. The key areas explored include: autoclave operational qualification (OQ) testing, autoclave load pattern testing, vessel sterilize-in-place testing, spore strip use and failure investigation, grouping of D-values for media and concentrated nutrients, influence of temperature on D-values, and equipment clean-in-place cleaning agent/recovery studies. Suggestions are offered based on technical data and engineering analysis of the procedures involved. Methodologies are described for how to evaluate the systems being tested relative to processing requirements to determine which testing might be minimized and which testing might warrant expansion. The ultimate risk to quality then must be evaluated by the designated quality control groups within the organization for the specific process and equipment in use.     

Overcoming co‐product inhibition in the nicotinamide independent asymmetric bioreduction of activated CC‐bonds using flavin‐dependent ene‐reductases

Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation (“dismutation”) of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co‐product phenol could be circumvented via in situ co‐product removal by scavenging the phenol using the polymeric adsorbent MP‐carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a “coupled‐substrate” approach via nicotinamide‐free hydrogen transfer with >90% conversion and complete stereoselectivity. Biotechnol. Bioeng. 2013;110: 3085–3092. © 2013 The Authors. Biotechnology and Bioengineering Published by Willey Periodicals, Inc.     

Enzymatic production of cyclodextrins from milled corn starch in an ultrafiltration membrane bioreactor

Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. (c) 1993 John Wiley & Sons, Inc.     

Process strategies to enhance pyruvate production with recombinant Escherichia coli: from repetitive fed-batch to in situ product recovery with fully integrated electrodialysis

Using the pyruvate production strain Escherichia coli YYC202 ldhA::Kan different process alternatives are studied with the aim of preventing potential product inhibition by appropriate product separation. This strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. Continuous experiments with cell retention, repetitive fed-batch, and an in situ product recovery (ISPR) process with fully integrated electrodialysis were tested. Although the continuous approach achieved a high volumetric productivity (QP) of 110 g L(-1) d(-1), this approach was not pursued because of long-term production strain instabilities. The highest pyruvate/glucose molar yield of up to 1.78 mol mol(-1) together with high QP 145 g L(-1) d(-1) and high pyruvate titers was achieved by the repetitive fed-batch approach. To separate pyruvate from fermentation broth a fully integrated continuous process was developed. In this process electrodialysis was used as a separation unit. Under optimum conditions a (calculated) final pyruvate titer of >900 mmol L(-1) (79 g L(-1)) was achieved.     

Applicability of a single‐use bioreactor compared to a glass bioreactor for the fermentation of filamentous fungi and evaluation of the reproducibility of growth in pellet form

The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease‐inhibiting substance in the single‐use bioreactor were similar to those in the glass bioreactor.     

High solids fermentation of hydrolysates of wheat starch B in a continuous dynamic immobilized biocatalyst bioreactor

Summary A simple and efficient method of conversion of wheat starch B to ethanol was investigated. Employing a two-stage enzymatic saccharification process, 95% of the wheat starch was converted to fermentable sugars in 40 h. From 140 g/l total sugars in the feed solution, 63.6 g/l ethanol was produced continuously with a residence time of 3.3 h in a continuous dynamic immobilized biocatalyst bioreactor by immobilized cells ofSaccharomyces cerevisiae. The advantages and the application of this bioreactor to continuous alcoholic fermentation of industrial substrates are presented.     

Design and characterization of a modified T‐flask bioreactor for continuous monitoring of engineered tissue stiffness

Controlling environmental conditions, such as mechanical stimuli, is critical for directing cells into functional tissue. This study reports on the development of a bioreactor capable of controlling the mechanical environment and continuously measuring force‐displacement in engineered tissue. The bioreactor was built from off the shelf components, modified off the shelf components, and easily reproducible custom built parts to facilitate ease of setup, reproducibility and experimental flexibility. A T‐flask was modified to allow for four tissue samples, mechanical actuation via a LabView controlled stepper motor and transduction of force from inside the T‐flask to an external sensor. In vitro bench top testing with instrumentation springs and tissue culture experiments were performed to validate system performance. Force sensors were highly linear (R² > 0.998) and able to maintain force readings for extended periods of time. Tissue culture experiments involved cyclic loading of polyurethane scaffolds seeded with and without (control) human foreskin fibroblasts for 8 h/day for 14 days. After supplementation with TGF‐β, tissue constructs showed an increase in stiffness between consecutive days and from the acellular controls. These experiments confirmed the ability of the bioreactor to distinguish experimental groups and monitor tissue stiffness during tissue development. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010