Improved reactor performance and operability in the biotransformation of carveol to carvone using a solid-liquid two-phase partitioning bioreactor

In an effort to improve reactor performance and process operability, the microbial biotransformation of (-)-trans-carveol to (R)-(-)-carvone by hydrophobic Rhodococcus erythropolis DCL14 was carried out in a two phase partitioning bioreactor (TPPB) with solid polymer beads acting as the partitioning phase. Previous work had demonstrated that the substrate and product become inhibitory to the organism at elevated aqueous concentrations and the use of an immiscible second phase in the bioreactor was intended to provide a reservoir for substrates to be delivered to the aqueous phase based on the metabolic rate of the cells, while also acting as a sink to uptake the product as it is produced. The biotransformation was previously undertaken in a two liquid phase TPPB with 1-dodecene and with silicone oil as the immiscible second phase and, although improvement in the reactor performance was obtained relative to a single phase system, the hydrophobic nature of the organism caused the formation of severe emulsions leading to significant operational challenges. In the present work, eight types of polymer beads were screened for their suitability for use in a solid-liquid TPPB for this biotransformation. The use of selected solid polymer beads as the second phase completely prevented emulsion formation and therefore improved overall operability of the reactor. Three modes of solid-liquid TPPB operation were considered: the use of a single polymer bead type (styrene/butadiene copolymer) in the reactor, the use of a mixture of polymer beads in the reactor (styrene/butadiene copolymer plus Hytrel(R) 8206), and the use of one type of polymer beads in the reactor (styrene/butadiene copolymer), and another bead type (Hytrel(R) 8206) in an external column through which fermentation medium was recirculated. This last configuration achieved the best reactor performance with 7 times more substrate being added throughout the biotransformation relative to a single aqueous phase benchmark reactor and 2.7 times more substrate being added relative to the best two liquid TPPB case. Carvone was quantitatively recovered from the polymer beads via single stage extraction into methanol, allowing for bead re-use.     

A novel solid-liquid two-phase partitioning bioreactor for the enhanced bioproduction of 3-methylcatechol

The bioproduction of 3-methylcatechol from toluene via Pseudomonas putida MC2 was performed in a solid-liquid two-phase partitioning bioreactor with the intent of increasing yield and productivity over a single-phase system. The solid phase consisted of HYTREL, a thermoplastic polymer that was shown to possess superior affinity for the inhibitory 3-methylcatechol compared to other candidate polymers as well as a number of immiscible organic solvents. Operation of a solid-liquid biotransformation utilizing a 10% (w/w) solid (polymer beads) to liquid phase ratio resulted in the bioproduction of 3-methylcatechol at a rate of 350 mg/L-h, which compares favorably to the single phase productivity of 128 mg/L-h. . HYTREL polymer beads were also reconstituted into polymer sheets, which were placed around the interior circumference of the bioreactor and successfully removed 3-methylcatechol from solution resulting in a rate of 3-methylcatechol production of 343 mg/L-h. Finally, a continuous biotransformation was performed in which culture medium was circulated upwards through an external extraction column containing HYTREL beads. The design maintained sub lethal concentrations of 3-methylcatechol within the bioreactor by absorbing produced 3-methylcatechol into the polymer beads. As 3-methylcatechol concentrations in the aqueous phase approached 500 mg/L the extraction column was replaced (twice) with a fresh column and the process was continued representing a simple and effective approach for the continuous bioproduction of 3-methylcatechol. Recovery of 3-methylcatechol from HYTREL was also achieved by bead desorption into methanol.     

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

A 6K-Deletion Variant of Salmonid Alphavirus Is Non-Viable but Can Be Rescued through RNA Recombination

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.     

Enhanced Tethered-Particle Motion Analysis Reveals Viscous Effects

Tethered-particle motion experiments do not require expensive or technically complex hardware, and increasing numbers of researchers are adopting this methodology to investigate the topological effects of agents that act on the tethering polymer or the characteristics of the polymer itself. These investigations depend on accurate measurement and interpretation of changes in the effective length of the tethering polymer (often DNA). However, the bead size, tether length, and buffer affect the confined diffusion of the bead in this experimental system. To evaluate the effects of these factors, improved measurements to calibrate the two-dimensional range of motion (excursion) versus DNA length were carried out. Microspheres of 160 or 240 nm in radius were tethered by DNA molecules ranging from 225 to 3477 basepairs in length in aqueous buffers containing 100 mM potassium glutamate and 8 mM MgCl₂ or 10 mM Tris-HCl and 200 mM KCl, with or without 0.5% Tween added to the buffer, and the motion was recorded. Different buffers altered the excursion of beads on identical DNA tethers. Buffer with only 10 mM NaCl and >5 mM magnesium greatly reduced excursion. Glycerol added to increase viscosity slowed confined diffusion of the tethered beads but did not change excursion. The confined-diffusion coefficients for all tethered beads were smaller than those expected for freely diffusing beads and decreased for shorter tethers. Tethered-particle motion is a sensitive framework for diffusion experiments in which small beads on long leashes most closely resemble freely diffusing, untethered beads.     

Development of a 3D‐printed single‐use separation chamber for use in mRNA‐based vaccine production with magnetic microparticles

Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)‐functionalized magnetic beads after cell lysis. The mRNA‐loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high‐gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow‐through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)‐functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of c_B = 1.6–2.1 g·L^–1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min^–1 with the developed SU‐HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min^–1, the retained particle mass is even more than 99.99%.     

Transient performance of two-phase partitioning bioreactors treating a toluene contaminated gas stream

Two-phase partitioning bioreactors (TPPBs) consist of a cell-containing aqueous phase and an immiscible organic phase that sequesters and delivers toxic substrates to cells based on equilibrium partitioning. The immiscible organic phase, which acts as a buffer for inhibitory substrate loadings, makes it possible for TPPBs to handle high volatile organic compound (VOC) loadings, and in this study the performance of liquid n-hexadecane and solid styrene butadiene (SB) polymer beads used as partitioning phases were compared to a single aqueous phase system while treating transient loadings of a toluene contaminated air stream by Achromobacter xylosoxidans Y234. The TPPBs operated as well-mixed stirred tanks, with total working volumes of 3 L (3 L aqueous for the single-phase system, 2 L aqueous and 1 L n-hexadecane for the solvent system, and 2.518 L aqueous volume and 500 g of SB beads for the polymer system). Two 60-min step changes (7 and 17 times the nominal loading rates, termed "small" and "large" steps, respectively) were imposed on the systems and the performance was characterized by the overall removal efficiencies, instantaneous removal efficiency recovery times (above 95% removal), and dissolved oxygen recovery times. For the small steps, with a nominal loading of 343 g/m3/h increasing to 2,400 g/m3/h, the TPPB system using n-hexadecane as the second phase performed best, removing 97% of the toluene fed to the system compared with 90% for the polymer beads system and only 69% for the single-phase system. The imposed large transient gave similar results, although the impact of the presence of a second sequestering phase was more pronounced, with the n-hexadecane system maintaining much reduced aqueous toluene concentrations leading to significantly improved performance. This investigation also showed that the presence of both n-hexadecane and SB beads improved the oxygen transfer within the systems.     

Biodegradation of a phenolic mixture in a solid–liquid two-phase partitioning bioreactor

A solid–liquid two-phase partitioning bioreactor (TPPB) in which the non-aqueous phase consisted of polymer (HYTREL) beads was used to degrade a model mixture of phenols [phenol, o-cresol, and 4-chlorophenol (4CP)] by a microbial consortium. In one set of experiments, high concentrations (850 mg l⁻¹ of each of the three substrates) were reduced to sub-inhibitory levels within 45 min by the addition of the polymer beads, followed by inoculation and rapid (8 h) consumption of the total phenolics loading. In a second set of experiments, the beneficial effect of using polymer beads to launch a fermentation inhibited by high substrate concentrations was demonstrated by adding 1,300 and 2,000 mg l⁻¹ total substrates (equal concentrations of each phenolic) to a pre-inoculated bioreactor. At these levels, no cell growth and no degradation were observed; however, after adding polymer beads to the systems, the ensuing reduced substrate concentrations permitted complete destruction of the target molecules, demonstrating the essential role played by the polymer sequestering phase when applied to systems facing inhibitory substrate concentrations. In addition to establishing alternative modes of TPPB operation, the present work has demonstrated the differential partitioning of phenols in a mixture between the aqueous and polymeric phases. The polymeric phase was also observed to absorb a degradation intermediate (arising from the incomplete biodegradation of 4CP), which opens the possibility of using solid–liquid TPPBs during biosynthetic transformation to sequester metabolic byproducts.     

Novel metal-chelate affinity adsorbent for purification of immunoglobulin-G from human plasma

Metal-chelating ligand and/or comonomer 2-methacrylolyamidohistidine (MAH) was synthesized by using methacryloyl chloride and L-histidine methyl ester. MAH was characterized by NMR and FTIR. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of methylmethacrylate (MMA) and MAH carried out in an aqueous dispersion medium. Poly(MMA-MAH) beads had a specific surface area of 37.5 m(2)/g. Poly(MMA-MAH) beads were characterized by water uptake studies, FTIR, SEM and elemental analysis. Elemental analysis of MAH for nitrogen was estimated as 34.7 microM/g of polymer. Then, Cu(2+) ions were chelated on the beads. Cu(2+)-chelated beads with a swelling ratio of 38% were used in the adsorption of human-immunoglobulin G (HIgG) from both aqueous solutions and human plasma. The maximum adsorption capacities of the Cu(2+)-chelated beads were found to be 12.2 mg/g at pH 6.5 in phosphate buffer and 15.7 mg/g at pH 7.0 in MOPS. Higher adsorption value was obtained from human plasma (up to 54.3 mg/g) with a purity of 90.7%. The metal-chelate affinity beads allowed one-step separation of HIgG from human plasma. The adsorption-desorption cycle was repeated 10 times using the same beads without noticeable loss in their HIgG adsorption capacity.     

Ionotropically emulsion gelled polysaccharides beads: Preparation,in vitro and in vivo evaluation

Floating famotidine loaded mineral oil-entrapped emulsion gel (MOEG) beads were prepared by the emulsion–gelation method. Different polysaccharides (sodium alginate and pectin), oil concentrations (10%, 20% and 30% w/w) and drug:polymer (D:P) ratios (1:1, 2:1 and 3:1) were used and their influence on beads uniformity, drug entrapment efficiency (DEE) and in vitro drug release, was studied. The results clearly indicated that retardation of drug release for 4 h was achieved by the oil hydrophobic diffusional barrier, especially in the presence of the compact network of alginate beads. Calcium alginate beads containing 20% oil and 2:1 D:P ratio, showed an optimum DEE of 88.32%. When evaluated in vivo, this formula displayed superior antiulcer activity (>2) over drug suspension or marketed conventional tablets.     

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads

For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.     

Mathematical analysis of two-phase mass transfer in a batch reactor for the chemical transformation of a steroid

A reactor is described for the conversion of the slightly water-soluble steroid testosterone (T) to 4-androstene-3, 17-dione (4-AD) by enzyme in the presence of excess cofactor. Since the enzyme is subject to substrate inhibition, reaction rates are strong functions of aqueous substrate concentration. High concentrations of the substrate, testosterone, per unit reactor volume are maintained within poly(dimethylsiloxane) beads that are suspended in the aqueous enzyme solution. Mass transfer (controlled by bead size, polymer to water volume ratio, enzyme loading) is used to control the degree and rate of conversion. The reactor dynamics are predicted over a wide range of reaction conditions. The product steroid is recovered in the polymeric beads from the enzyme solution.     

A Water-Soluble Inclusion Complex of Pedunculoside with the Polymer β-Cyclodextrin: A Novel Anti-Inflammation Agent with Low Toxicity

More than 50% of new drug candidates in drug discovery are lipophilic and exhibit poor aqueous solubility, which results in poor bioavailability and a lack of dose proportionality. Here, we improved the solubility of pedunculoside (PE) by generating a water-soluble inclusion complex composed of PE and the polymer β-cyclodextrin (CDP). We characterized this novel complex by ¹H NMR, FT-IR, UV-vis spectroscopy, powder X-ray diffractometry and thermogravimetric analysis. The ratio of β-cyclodextrin (β-CD) units in CDP to PE was determined to be 2∶1. The K _D value of the inclusion complex was determined to be 4.29×10⁻³ mol•L⁻¹. In contrast to the low solubility of PE, the water-solubility of the PE–CDP complex was greatly enhanced. A preclinical toxicological study indicated that PE–CDP was well tolerated for a single administration. Importantly, the anti-inflammation potency of the PE–CDP complex was higher than that of PE. As a result, the formation of inclusion complexes by water-soluble CDP opens up possible aqueous applications of insoluble drug candidates in drug delivery.     

Lipase in biphasic alginate beads as a biocatalyst for esterification of butyric acid and butanol in aqueous media

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp.     

Salmonid alphavirus replication in mosquito cells: towards a novel vaccine production system

Salmonid alphavirus (SAV) causes pancreas disease and sleeping disease in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) and confers a major burden to the aquaculture industry. A commercial inactivated whole virus vaccine propagated in a salmon cell line at low temperature provides effective protection against SAV infections. Alphaviruses (family Togaviridae) are generally transmitted between vertebrate hosts via blood-sucking arthropod vectors, typically mosquitoes. SAV is unique in this respect because it can be transmitted directly from fish to fish and has no known invertebrate vector. Here, we show for the first time that SAV is able to complete a full infectious cycle within arthropod cells derived from the Asian tiger mosquito Aedes albopictus. Progeny virus is produced in C6/36 and U4.4. cells in a temperature-dependent manner (at 15°C but not at 18°C), can be serially passaged and remains infectious to salmonid Chinook salmon embryo cells. This suggests that SAV is not a vertebrate-restricted alphavirus after all and may have the potential to replicate in invertebrates. The current study also shows the ability of SAV to be propagated in mosquito cells, thereby possibly providing an alternative SAV production system for vaccine applications.     

Dual Phase Change Thermal Diodes for Enhanced Rectification Ratios: Theory and Experiment

Thermal diodes are materials that allow for the preferential directional transport of heat and are highly promising devices for energy conservation, energy harvesting, and information processing applications. One form of a thermal diode consists of the junction between a phase change and phase invariant material, with rectification ratios that scale with the square root of the ratio of thermal conductivities of the two phases. In this work, the authors introduce and analyse the concept of a Dual Phase Change Thermal Diode (DPCTD) as the junction of two phase change materials with similar phase boundary temperatures but opposite temperature coefficients of thermal conductivity. Such systems possess a significantly enhanced optimal scaling of the rectification ratio as the square root of the product of the thermal conductivity ratios. Furthermore, the authors experimentally design and fabricate an ambient DPCTD enabled by the junction of an octadecane‐impregnated polystyrene foam, polymerized using a high internal phase emulsion template (PFH‐O) and a poly(N‐isopropylacrylamide) (PNIPAM) aqueous solution. The DPCTD shows a significantly enhanced thermal rectification ratio both experimentally (2.6) and theoretically (2.6) as compared with ideal thermal diodes composed only of the constituent materials.     

Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

Cell–cell interactions, mediated by members of the cadherin family of Ca²⁺-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.     

Degradation of xenobiotics in a partitioning bioreactor in which the partitioning phase is a polymer

Two-phase partitioning bioreactors (TPPBs) are characterized by a cell-containing aqueous phase and a second immiscible phase that contains toxic and/or hydrophobic substrates that partition to the cells at subinhibitory levels in response to the metabolic demand of the organisms. To date, the delivery phase in TPPBs has been a hydrophobic solvent that traditionally needed to possess a variety of important properties including biocompatibility, nonbioavailability, low volatility, and low cost, among others. In the present work we have shown that the organic solvent phase can be replaced by inexpensive polymer beads that function in a similar fashion as organic solvents, delivering a toxic substrate to cells based on equilibrium considerations. Specifically, 3.4 mm diameter beads of poly(ethylene-co-vinyl acetate) (EVA) were used to reduce the aqueous concentration of phenol in a bioreactor from toxic levels ( approximately 2,000 mg/L) to subinhibitory levels ( approximately 750 mg/L), after which Pseudomonas putida ATCC 11172 was added to the system and allowed to consume the total phenol loading. Thus, the beads absorbed the toxic substrate and released it to the cells on demand. The EVA beads, which could be reused, were able to absorb 14 mg phenol/g EVA. This work has opened the possibility of using widely mixed cultures in TPPB systems without concern for degradation of the delivery material and without concern of contamination.