Acoustic field assisted enhanced demixing of aqueous two-phase systems

Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.     

Downstream processing of luciferase from fireflies (Photinus pyralis) using aqueous two-phase extraction

The firefly luciferase has been extensively used for sensitive detection of bacteria, gene expression and environmental toxins (biosensors). The aim of the present study was to design a simple and more efficient method for the purification and concentration of luciferase using aqueous two-phase extraction (ATPE). Downstream processing of luciferase from North American Firefly Photinus pyralis was carried out, for the first time, using polymer/salt aqueous two phase system (ATPS) at 4 °C. The enzyme was observed to preferentially partition to the polyethylene glycol (PEG) rich top phase. The best results of purification (13.69 fold) and enzyme activity recovery (118.34%) were observed in the system containing 4.0% (w/w) PEG (1500) and 20.5% (w/w) (NH₄)₂SO₄ with a phase volume ratio of 0.21.     

Fractionation and purification of the phycobiliproteins from Spirulina platensis

C-Phycocyanin and allophycocyanin of Spirulina platensis are fractionated and purified using a non-chromatographic method namely, aqueous two phase extraction for the first time. Optimized process parameters of aqueous two phase extraction (PEG 4000/potassium phosphate of tie line length 18.64% with a phase volume ratio 1.45) resulted in pure C-phycocyanin and allophycocyanin with a purity of 3.23 and 0.74, respectively, in a single extraction. Multiple extractions (two) improved the purity of C-phycocyanin from 3.23 to 4.02. Integration of aqueous two phase extraction with membrane process not only facilitated the separation of phase forming components from the products and also increased the purity of allophycocyanin from 0.74 to 1.5.     

Extraction of bovine serum albumin using nanoparticulate reverse micelles

The extraction of a relatively large molecular weight protein, bovine serum albumin (BSA), using nano-sized reverse micelles of nonionic surfactant polyoxyethylene p-t-octylphenol (Triton-X-100) is attempted for the first time. Suitability of reverse micelles of anionic surfactant sodium bis (2-ethyl hexyl) sulfosuccinate (AOT) and Triton-X-100/AOT mixture in organic solvent toluene for BSA extraction is also investigated. Although, the size of the Triton-X-100 reverse micelle in toluene is large enough to host BSA molecule in the hydraulic core, the overall extraction efficiency is found to be low, which may be due to lack of strong driving force. AOT/toluene system resulted in complete forward extraction at aqueous pH 5.5 and a surfactant concentration of 160 mM. The back extraction with aqueous phase (pH 5.5) resulted in 100% extraction of BSA from the organic phase. The addition of Triton-X-100 to AOT reduced the extraction efficiency of AOT reverse micelles, which may be attributed to reduced hydrophobic interaction. The circular dichroism (CD) spectrum of BSA extracted using AOT/toluene reverse micelles indicated the structural stability of the protein extracted.     

Field-assisted extraction of cells, particles and macromolecules

Improved bioseparation techniques are increasingly important for biotechnology because separation is often the limiting factor for the success of biological processes. Manufacturers of new enzymes and pharmaceutical products require improved methods for recovering intact cells and intracellular products. Similarly the isolation, purification and concentration of many biomolecules produced in fermentation processes is extremely important. Often such downstream processing contributes a large portion of the product cost and thus efficient and economical alternative approaches to bioseparation processes are needed to eliminate, reduce or facilitate the handling of solids. Field-assisted separations, which hold immense potential for providing a major improvement in bioseparation in the near future, are considered in this review. Special emphasis is given to multistage methods, which are cost-effective compared with competing technologies. Commercial applications of these methods are detailed, we present suggestions for future work and we analyse the scale-up and economic aspects of these processes.     

Effect of investigator bias on the significance level of the Wilcoxon rank-sum test

When using subjective ordered categorical variables to measure the efficacy of an active treatment versus placebo in a double-blind clinical trial setting, bias may be introduced into the response variables when investigators become partially or totally unblinded to treatment assignment due to characteristic side effects. The investigators may alter the classification of a patient's response to treatment based on perceived treatment assignment. The introduction of bias leads to a considerable increase in the actual significance level of the Wilcoxon rank-sum test.     

Membrane processing for purification and concentration of β-glycosidases from barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>)

Tangential flow ultrafiltration with a polyethersulfone (100 kDa) membrane was used for the purification and concentration of β-glycosidases (β-galactosidase and β-glucosidase) from aqueous extract of barley. The performances of mode 1 (concentration followed by diafiltration) and mode 2 (diafiltration followed by concentration) were compared. In mode 1 activity recoveries of 91.44 and 88% as well as purifications of 1.84 and 1.77-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 9 h. In mode 2 activity recoveries of 95.68 and 91.76% with purifications of 4.56 and 4.38-fold for β-galactosidase and β-glucosidase, respectively, were obtained in a total processing time of about 6 h. The removal of total carbohydrates and protein was 56.74 and 50.73%, respectively, in mode 1, whereas it was 81.46 and 79.04%, respectively, in mode 2. The diafiltration volume and volume concentration of 3 were maintained in both mode 1 and 2. Flux decline was severe in mode 1 and led to a long processing time of about 9 h. These results indicate that mode 2 was better than mode 1 for purification of β-glycosidases.     

Fractional Plans to Estimate Main Effects and Two Factor Interactions Inclusive of a Specific Factor

Balanced incomplete block designs are used to construct non‐geometric 2ⁿ fractional factorial plans to estimate all n main effects and n – 1, 2 factor interactions with a specific factor included in each interaction. When the balanced incomplete block design is of Family (A), the resulting fractional factorial plan has the same number of runs as a fold‐over Hadamard matrix giving same variances for the estimates; however, some new designs are shown to be non‐isomorphic to the fold‐over Hadamard matrix plans.     

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.