Polymer–solute interactions in solid–liquid two‐phase partitioning bioreactors

BACKGROUND: Biphasic systems with immiscible solvents have been studied for in situ product removal, and have shown improvements in bioreactor performance, however, problems associated with solvent biocompatibility, bioavailability and operation have been identified. One alternative is the solid–liquid system in which polymer beads are used, absorbing and removing target compounds from the aqueous phase while maintaining equilibrium conditions. This work aims to identify polymer properties that may be important in polymer selection for selected biotransformation molecules including 2‐phenylethanol, cis‐1,3‐indandiol, iso‐butanol, succinic acid and 3‐hydroxybutyrolactone. RESULTS: Relatively hydrophobic compounds (e.g. 2‐phenylethanol) tend to be absorbed by polymers better than hydrophilic ones (e.g. iso‐butanol) based on partition coefficient tests; values as high as 80 were obtained for the former and < 3 for the latter. Owing to the presence of polar functional groups on these compounds, polar polymers such as Hytrel® performed better than non‐polar ones such as Kraton®. Crystallinity and intermolecular hydrogen‐bonding were also found to be important polymer properties. CONCLUSION: Polymers showed excellent results in absorbing hydrophobic compounds such as aromatic alcohols, and positive results in absorbing hydrophilic compounds but to a lesser extent. Grafting hydrophilic functional groups onto polymers may be a promising approach for extending polymer uptake capabilities and is currently being investigated. Copyright © 2009 Society of Chemical Industry     

Bioproduction of cis‐(1S,2R)‐indandiol,a chiral pharmaceutical intermediate,using a solid–liquid two‐phase partitioning bioreactor for enhanced removal of inhibitors

BACKGROUND: A solid‐liquid two‐phase partitioning bioreactor (TPPB) was used in the biotransformation of indene to cis‐(1S,2R)‐indandiol by Pseudomonas putida 421‐5 (ATCC 55687). Metered substrate feeding in single‐phase operation, or delivery from an immiscible liquid, have previously been employed to regulate the exposure of the biocatalyst to inhibitory concentrations of the substrate. In contrast, the solid‐liquid platform provided in situ substrate addition (ISSA) as well as simultaneous it in situ product removal (ISPR) as a means of overcoming substrate and product toxicity. Three different modes of operation were compared for their effects on the performance of this biotransformation: single‐phase, fed‐batch operation was carried out as a benchmark in 2.75 L aqueous medium, and subsequently with the inclusion of either 700 g liquid silicone oil or 700 g solid polymer beads. RESULTS: Biphasic modes achieved a 3‐fold productivity improvement with respect to single‐phase (30 to 90 mg L^?1 h^?1), and solid‐liquid productivity was similar to liquid‐liquid operation while achieving more extensive removal of inhibitory compounds resulting in a slightly higher product titer (1.29 vs 1.16 g L^?1). The operability of the reactor was improved by the phase stability of the solid polymer beads relative to immiscible organic solvents, preventing emulsion formation and facilitating analytics. CONCLUSION: Solid polymer beads replaced the immiscible liquid auxiliary phase for substrate delivery while performing simultaneous inhibitory molecule sequestration. Copyright © 2011 Society of Chemical Industry     

Siloxane‐based copolymers for use in two‐phase partitioning bioreactors

Two‐phase partitioning bioreactors (TPPBs) have been recently demonstrated to be capable of using solid polymers as the absorbing/desorbing phase. In this paper, we examine the possibility of using a siloxane‐based copolymer as this phase. Copolymers were prepared using vinyl terminated oligo(dimethylsiloxane‐co‐diphenylsiloxane), copolymerized with varying amounts of the comonomers ethylene glycol dimethacrylate (EGDMA), N‐vinyl pyrrolidone (NVP), or acrylic acid (AA), via free radical polymerization. The absorption rate of phenol as a representative pollutant into these polymers from water was measured. The inclusion of a water‐soluble comonomer resulted in swelling of the polymers in water, enhanced total phenol absorption, but reduced phenol diffusivity.     

Model for a solid‐liquid airlift two‐phase partitioning bioscrubber for the treatment of BTEX

BACKGROUND: Airlift solid–liquid two‐phase partitioning bioreactors (SL‐TPPBs) have been shown to be effective for the treatment of gas streams containing benzene, toluene, ethylbenzene and o‐xylene (BTEX). The airlift SL‐TPPB is a low‐energy system that utilizes a sequestering phase of solid silicone rubber beads (10%v/v) that will uptake and release large amounts of BTEX in order to maintain equilibrium conditions within the system. This increases mass transfer from the gas phase during dynamic loading periods and improves degradation performance. This study discusses the development and analysis of a steady‐state, tanks‐in‐series mathematical model, arising from mass balances on BTEX and oxygen in the gas, aqueous and polymer phases to predict the performance of the airlift SL‐TPPB over various gas flow rates and BTEX loadings. RESULTS: An estimability analysis on model parameters determined that the parameters to which model output is most sensitive are those that affect biological activity, which were targeted for estimation. The developed tanks‐in‐series model was able to predict the removal of BTEX components and dissolved oxygen concentrations over various inlet loadings (20, 60 and 100 mg L^?1 h^?1) and gas flow rates (2,3 and 4 L min^?1) that resulted in a range of system performance from effective BTEX treatment to oxygen limiting conditions. CONCLUSIONS: The model developed, with estimated parameters, provides a valuable tool to determine operating conditions that will result in favourable performance of the airlift SL‐TPPB. Copyright © 2009 Society of Chemical Industry     

KLa measurement in two‐phase partitioning bioreactors: new insights on potential errors at low power input

BACKGROUND: Two‐phase partitioning bioreactors (TPPBs) are based on the addition of a non‐aqueous phase (NAP) to a biological process in order to overcome a limited delivery of gaseous substrates to the microorganisms in the case of compounds with low affinity for water. However, the high power input (P_g/V) required to disperse the NAP is often the major limitation for TPPB applications at full scale. Therefore, the accurate determination of the overall mass transfer coefficient (K_La) at low P_g/V values is a critical issue as these operational conditions are more attractive from a scale‐up point of view. RESULTS: NAP addition altered the typical shape of the dissolved oxygen curves used for K_La determination at the lowest P_g/V values tested (70–80 W m^?3). Below a threshold P_g/V value of 600 W m^?3, the presence of the NAP increased the error in K_La measurements up to 115% relative to controls deprived of NAP. CONCLUSIONS: The error in K_La measurements at low P_g/V values might be related to failures in the fundamental assumption regarding liquid phase homogeneity in the mass transfer model used. Copyright © 2010 Society of Chemical Industry     

Performance evaluation of a water/silicone oil two‐phase partitioning bioreactor using Rhodococcus erythropolis T902.1 to remove volatile organic compounds from gaseous effluents

BACKGROUND: In the framework of biological processes used for waste gas treatment, the impact of the inoculum size on the start‐up performance needs to be better evaluated. Moreover, only a few studies have investigated the behaviour of elimination capacity and biomass viability in a two‐phase partitioning bioreactor (TPPB) used for waste gas treatment. Lastly, the impact of ethanol as a co‐substrate remains misunderstood. RESULTS: Firstly, no benefit of inoculation with a high cellular density (>1.5 g L^?1) was observed in terms of start‐up performance. Secondly, the TPPB was monitored for 38 days to characterise its behaviour under several operational conditions. The removal efficiency remained above 63% for an inlet concentration of 7 g isopropylbenzene (IPB) m^?3 and at some time points reached 92% during an intermittent loading phase (10 h day^?1), corresponding to a mean elimination capacity of 4 × 10^?3 g L^?1 min^?1 (240 g m^?3 h^?1) for a mean IPB inlet load of 6.19 × 10^?3 g L^?1 min^?1 (390 g m^?3 h^?1). Under continuous IPB loading, the performance of the TPPB declined, but the period of biomass acclimatisation to this operational condition was shorter than 5 days. The biomass grew to approximately 10 g L^?1 but the cellular viability changed greatly during the experiment, suggesting an endorespiration phenomenon in the bioreactor. It was also shown that simultaneous degradation of IPB and ethanol occurred, suggesting that ethanol improves the biodegradation process without causing oxygen depletion. CONCLUSION: A water/silicone oil TPPB with ethanol as co‐substrate allowed the removal of a high inlet load of IPB during an experiment lasting 38 days. Copyright © 2008 Society of Chemical Industry     

Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system

Developing a novel Ionic‐liquid (IL) based aqueous two‐phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1‐butyl‐3‐methylimidazolium acetate+ potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of α‐amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three‐variable central composite design was employed to understand the effect of phase forming components on extraction studies of α‐amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting‐out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. © 2015 American Institute of Chemical Engineers AIChE J, 62: 264–274, 2016     

Practical implementation of aqueous two‐phase processes for protein recovery from yeast

A two‐stage extraction process for the recovery of intracellular proteins from brewers' yeast was selected as a practical model system to study the implementation of polyethylene glycol (PEG)–phosphate aqueous two‐phase systems (ATPS). Disrupted all suspensions generated by homogenisation and bead milling were used to study the impact of cell debris upon the partition behaviour of the intracellular products (bulk protein, fumarase and pyruvate kinase). Regardless of their origin debris particles did not significantly influence the partition behaviour of the intracellular products in selected ATPS distant from the binodal and at volume ratios greater than one. Recycling of used PEG into the initial extraction stage did not significantly influence the protein partition behaviour in batch ATPS. In the polymer recycling studies in continuous ATPS using spray columns, the addition of fresh materials to make up the deficits of phase‐forming chemicals compensate any negative effect of the continuous recycling of the top PEG‐rich phase. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. © 2000 Society of Chemical Industry     

The use of CO2 for reversible pH shifting,and the removal of succinic acid in a polymer‐based two‐phase partitioning bioreactor

BACKGROUND: Succinic acid (SA) is an intermediate in the production of commodity chemicals, but SA bioproduction has not yet been commercialized due to end product inhibition and high product separation costs. Two‐phase partitioning bioreactors (TPPBs) can increase volumetric productivity through in situ product removal, although SA uptake by polymers requires a pH below the pK_A2 of SA (4.2). It was proposed to reversibly reduce the pH with CO₂ sparging for absorption of SA, followed by nitrogen stripping to allow continued bioproduction after returning to metabolic pH levels. RESULTS: At 1 atm CO₂ sparging lowered the pH of RO water to 3.8 but only to 4.75 in medium, requiring acid/base pH adjustment in subsequent experiments. Actinobacillus succinogenes was temporarily exposed to pH 4.2 for between 5 min and 4 h to observe the effect on subsequent growth; cells could grow after up to 4 h of low pH exposure, sufficient time for SA uptake. Because atmospheric CO₂ could not adequately lower the pH of medium, a TPPB was operated with the pH being shifted using strong acid/base; SA was recovered in situ, however, the accumulation of salts hindered further cell growth. CONCLUSION: Several key elements of this novel processing strategy were successfully demonstrated, and work is continuing with high pressure CO₂ to achieve the desired pH adjustment levels. Copyright © 2011 Society of Chemical Industry     

Chemical modification of chicken egg white proteins with polyethyleneoxide–maleic anhydride copolymer and their partitioning in aqueous two‐phase systems

Aqueous two‐phase partitioning is one of the excellent bioseparation systems for proteins and other biomolecules in water‐rich solutions containing polymers or salts. To improve the partition properties of model proteins, chicken egg white (CEW) proteins were chemically modified with polyethyleneoxide–maleic anhydride (PEOMA) copolymer. The modified proteins were partitioned in polyethyleneoxide–maleic acid/potassium phosphate aqueous two‐phase systems. Firstly, the contribution of the chemical modification of proteins to the partition coefficient was investigated by using modified proteins with different degrees of modification (DM). The partition coefficients of modified proteins increased with increasing DM. The results suggested that the surface of the modified proteins had a greater hydrophobic property, similar to the PEOMA copolymer. Secondly, the effect of the pH on the partitioning of the modified proteins was studied. The partitioning of the modified proteins was found to be more dependent on pH in the alkaline systems. The increasing partition coefficient with pH at alkaline pH can be explained by the electrostatic repulsion between the phosphate anions and the maleic groups attached to the proteins. These results suggested that chemical modification of proteins with PEOMA could be a useful tool for separation and purification of crude protein mixtures. Copyright © 2005 Society of Chemical Industry     

A first principles approach to identifying polymers for use in two‐phase partitioning bioreactors

BACKGROUND: Two‐phase partitioning bioreactors (TPPBs) employ an immiscible phase to partition toxic substrates/products to/away from cells to reduce cytotoxicity and improve bioprocess performance. Initially, immiscible organic solvents were used as the sequestering phase, and their selection included consideration of solute–solvent affinity, which can be predicted through first principles consideration of solute activity and phase equilibrium thermodynamics. Polymers can replace organic solvents in such systems, however, their selection has largely been via heuristic means, and a more fundamental approach is necessary for future success in rational polymer identification. RESULTS: Material properties (polymer crystallinity, solubility parameter, and glass transition temperature T_g) were examined across several polymers and polyaromatic hydrocarbons as target solutes. All were shown to influence solute–polymer affinity. CONCLUSION: This first attempt at identifying physical/chemical properties that affect solute–polymer partitioning has been able to demonstrate some clear trends, and has allowed us to formulate a polymer selection guide, based on first principles, to facilitate the selection of solute–polymer pairs for solid–liquid TPPB applications. Copyright © 2012 Society of Chemical Industry     

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

BACKGROUND: Aqueous two‐phase extraction (ATPE) has many advantages as an efficient, inexpensive large‐scale liquid–liquid extraction technique for protein separation. However, the realization of ATPE as a protein separation technology at industrial scales is rather limited due to the large, multidimensional design space and the paucity of design approaches to predict phase and product behavior in an integrated fashion with overall system performance. This paper describes a framework designed to calculate suitable flowsheets for the extraction of a target protein from a complex protein feed using ATPE. The framework incorporated a routine to set up flowsheets according to target protein partitioning behavior in specific ATPE systems and a calculation of the amounts of phase‐forming components needed to extract the target protein. The thermodynamics of phase formation and partitioning were modeled using Flory‐Huggins theory and calculated using a Gibbs energy difference minimization approach. RESULTS: As a case study, suitable flowsheets to recover phosphofructokinase from a simple model feedstock using poly(ethylene glycol)‐dextran (PEG6000‐DxT500) and poly(ethylene glycol)‐salt (PEG6000‐Na₃PO₄) two‐phase systems were designed and the existence of feasible solutions was demonstrated. The flowsheets were compared in terms of product yield, product purity, phase settling rate and scaled process cost. The effect of the mass flowrates of phase‐forming components on product yield and purity was also determined. CONCLUSION: This framework is proposed as a basis for flowsheet optimization for protein purification using ATPE systems. Copyright © 2010 Society of Chemical Industry     

Recovery of value‐added globular proteins from tannery wastewaters using PEG – salt aqueous two‐phase systems

Leather processing involves discharge of high‐value soluble globular proteins in the wastewater. The recovery of value‐added products from the wastewaters is gaining more importance in the context of recovery of wealth from waste. The recovery of these globular proteins from tannery wastewater was selected as a practical model system to study the implementation of polyethylene glycol (PEG)‐sulfate aqueous two‐phase systems (ATPS). The partition coefficient of bovine serum albumin is comparable to that of soluble proteins from tannery wastewaters. The influence of concentration of polymer, salt, pH and temperature on the partitioning of soluble proteins from tannery wastewaters has been studied. The PEG6000 + sodium sulfate + water system provide better partitioning of these soluble proteins as compared to PEG6000 + ammonium sulfate system. The maximum protein recovery yield for PEG6000 + sodium sulfate + water system at 20 °C is 92.75%. The influence of temperature indicates the recovery of proteins from tannery wastewater to be better at lower temperature. The findings of these studies raise the potential application of ATPS processes for protein recovery from complex biological systems. Copyright © 2006 Society of Chemical Industry     

Prediction of two‐phase pressure drop and liquid holdup in co‐current gas–liquid downflow of air–Newtonian systems through packed beds

The dependency of pressure drop and liquid holdup on phase velocities, geometry of the column and packing materials as well as on the physical properties have been analyzed. Our experimental data (825 data points obtained using four liquid systems and three different particles) along with those of the available literature (776 data point from five different sources) were used for the analysis. The applicability and the limitations of the literature correlations were evaluated using the available data. Based on the analysis, new correlations for the estimation of pressure drop and liquid holdup, valid for low and high interaction regimes have been developed using the available data, with a wide range of variables. Copyright © 2005 Society of Chemical Industry