Comparison of downstream processing methods in purification of highly active laccase.

Laccases have received the attention of researchers in the last few decades due to their ability to degrade phenolic and lignin-related compounds. This study aimed at obtaining the highest possible laccase activity and evaluating the methods of its purification. The crude laccase from bioreactor cultivation of Cerrena unicolor fungus was purified using ultrafiltration, aqueous two-phase extraction (ATPE) and foam fractionation (FF), which allowed for the assessment of these three downstream processing (DSP) methods. The repeated fed-batch cultivation mode applied for the enzyme production resulted in a high laccase specific activity in fermentation broth of 204.1 U/mg. The use of a specially constructed spin filter inside the bioreactor enabled the integration of enzyme biosynthesis and biomass filtration in one apparatus. Other methods of laccase concentration and purification, namely ATPE and FF, proved to be useful for laccase separation; however, the efficiency of FF was rather low (recovery yield of 24.9% and purification fold of 1.4). Surprisingly, the recovery yield after ATPE in a PEG 6000-phosphate system in salt phase was higher (97.4%) than after two-step ultrafiltration (73.7%). Furthermore, it was demonstrated that a simple, two-step purification procedure resulted in separation of two laccase isoforms with specific activity of 2349 and 3374 U/mg. All in all, a compact integrated system for the production, concentration and separation of fungal laccases was proposed.

Modification of chitin structure with tailored ionic liquids.

Chitin, poly N-acetylglucosamine, has a great potential for use on an industrial scale as an enzyme carrier but it has an unfavorable particle structure that can be modified using ionic liquids (ILs). Several ionic liquids were investigated that have the same substituents on the ring (methyl- and propyl-) but differed in the type of cationic ring (pyrrolidinium, piperidinium, and piperazinium). Organic acid ions (acetic and lactic) were used as counter ions. 1-ethyl-3-methyl-imidazolium acetate and 1-ethyl-3-methyl-imidazolium lactate were used as a reference. The results confirm that the chitin particle structure or size, or both, simultaneously changes if chitin is dissolved in an IL and then precipitated. Organic acid anions and short substituents on the cationic ring of ILs influenced particle modification substantially, whereas the type of ring played a minor role. Additionally, the ionic liquids [MPpyrr][OAc], [MPpip][OAc] and [DMPpz][OAc] could be reused up to at least 4 times without losing their ability to dissolve chitin.

Continuous laccase concentration in an aqueous two-phase system.

An approach to describe continuous partitioning of Cerrena unicolor laccase in a PEG 6000-phosphate aqueous two-phase system was proposed. The laccase was separated from crude supernatant of C. unicolor-submerged culture, and all the experiments were carried out in 25 °C and pH 7 conditions. Masses of both phases and their compositions at phase equilibrium, as well as laccase activity concentrations at different mixing points, were measured in batch experiments. An empirical short-cut method was developed which allows for calculation of mass and volume fractions of the phases, laccase concentration factors, and laccase recoveries. Theoretical predictions were verified by several experiments carried out in a special mixer-settler unit with automatic substrate feed and continuous collection of separated phases. Required concentration of the laccase was possible to achieve in a one-step extraction process in the mixer-settler unit. The predictions of the short-cut method were compared to the results of experimental measurements of phase compositions, phase volume fractions, concentration factors and enzymatic yields at steady-state operation of the extraction unit. The values of experimental results lay well within the 10% error range of the predicted values.

Kinetics of chitin deacetylase activation by the ionic liquid [Bmim][Br].

Chitin deacetylase is the only known enzyme that can deacetylate the N-acetyl-d-glucosamine units in chitin and chitosan to D-glucosamine. Unfortunately, this enzyme, originally obtained from fungi, usually has low activity. Here, we present that it is possible to enhance the activity of chitin deacetylase using the ionic liquid [Bmim][Br]. An increase in activity of up to 160% from the basal chitin deacetylase activity was observed. Kinetic investigations suggest that [Bmim][Br] is a non-essential activator.

Multistage aqueous two-phase extraction of a monoclonal antibody from cell supernatant.

This article presents results of continuous multistage aqueous two-phase extraction of an immunoglobulin G1 from cell supernatant in a mixer-settler unit. An aqueous two-phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three-stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step.

Investigation of gas stripping and pervaporation for improved feasibility of two-stage butanol production process.

Gas stripping and pervaporation are investigated for butanol recovery in a two-stage acetone-butanol-ethanol (ABE) fermentation process. The first stage is operated in a continuous mode and the second stage as a fed-batch. Gas stripping coupled to the second stage and operated intermittently enabled additional glucose feeding in the second stage and up to 59 g/L butanol and 73 g/L total ABE solvents in the condensate. Concentration of 167 g/L butanol and 269 g/L ABE in the permeate was measured in ex situ pervaporation experiments using a PDMS membrane at temperature of 37 °C and pressure of 10mbars. The "operating window" tool is introduced to evaluate the feasibility of the existing ABE fermentations operated as continuous with cell recycle, as two-stages, with biomass immobilization or with integrated product removal. This tool enables the identification of the most favorable process configuration, which is the combination of cell immobilization and integrated product removal.

Experimental and theoretical investigation of multistage extraction of 1,3-propanediol using the extraction system phosphate/1-butyl-3-methylimidazolium trifluoromethanesulfonate/water.

The separation of 1,3-propanediol from fermentation broth is a challenging and energy-demanding step using conventional unit operations. One alternative process is the use of an aqueous two-phase system incorporating ionic liquids to use synergy effects of both technologies. Within this manuscript, the technical feasibility of the extraction of 1,3-propanediol using the aqueous two-phase system phosphate (salt)/1-butyl-3-methylimidazolium trifluoromethanesulfonate (ionic liquid)/water in a continuously operated process in pilot-scale is presented. The extraction was performed in a multistage mixer-settler unit and successfully modeled with an equilibrium-stage model and correlations to describe the liquid-liquid equilibrium of the salt/ionic liquid/water two-phase system. The developed and validated model was used for a further investigation of the influence of different process parameters in the determined operating window. Theses parameters include the number of stages, the phase ratio, the pH, and the mass fraction of the involved components. The results prove that the phosphate and 1-butyl-3-methylimidazolium trifluoromethanesulfonate mass fraction, the phase ratio, and the number of stages have a considerable influence on the recovery of 1,3-propanediol, whereas the pH value has only a smaller impact. Those results can be used for optimization of the system as well as for targeting future research within this area.

Intensified reaction and separation systems.

Process intensification follows four main goals: to maximize the effectiveness of intra- and intermolecular events, to give each molecule the same processing experience, to optimize the driving forces/maximize specific interfacial areas, and to maximize the synergistic effects of partial processes. This paper shows how these goals can be reached in reaction and separation systems at all relevant time and length scales and is focused on the structuring of reactors and separation units, on the use of different energy forms to improve the reaction and separation, on combining and superimposing of different phenomena in one integrated unit or reactor, and on the application of oscillations for intensification of reaction and separation processes.