Dynamic modeling and optimal fed-batch feeding strategies for a two-phase partitioning bioreactor

A dynamic model for the degradation of phenol in a two-phase partitioning bioreactor has been developed based on mechanistic balances around the bioreactor. The key process characteristics including substrate transfer between the organic and aqueous phases, substrate inhibition, oxygen limitation, and cell entrainment were incorporated into the model. The model predictions were validated against existing experimental data obtained for a 2-L bioreactor, and good correlation was observed for the time frames of the simulations, as well as for trends in cell and substrate concentrations. Optimal fed-batch, phenol feeding strategies were then developed based on two approaches: (1) maximization of phenol consumption in a fixed time interval and (2) consumption of a fixed amount of phenol in minimal time. The optimal feeding policies, determined using the Iterative Dynamic Programming algorithm, provided substantial improvements in the amount of phenol consumed when compared to a typical experimental heuristic approach. For example, 45.73 g of phenol was predicted to be consumed in 50 h (not including lag phase) using the optimal feeding profile compared to 10.26 g of phenol consumed in the simulated experimental approach. Oxygen limitation was predicted to be a recurring operational challenge in the partitioning bioreactor, and had a strong impact on the optimization results.     

Polymer development for enhanced delivery of phenol in a solid-liquid two-phase partitioning bioreactor

Two-Phase Partitioning Bioreactors (TPPBs) have traditionally been used to partition toxic concentrations of xenobiotics from a cell-containing aqueous phase by means of an immiscible organic solvent and to deliver these substrates back to the cells on the basis of metabolic demand and the maintenance of thermodynamic equilibrium between the phases. A limitation of TPPBs, which use organic liquid solvents, is the possibility that the solvent can be bioavailable, and this has therefore limited organic liquid TPPBs to the use of pure strains of microbes. Solid polymer beads have recently been introduced as a replacement for liquid organic solvents, offering similar absorption properties but with the capability to be used with mixed microbial populations. The present work was aimed at identifying a polymer with a greater capacity for and more rapid uptake and release of phenol for use as the second phase in a mixed culture TPPB. Polarity and hydrogen bonding capabilities between polymer and phenol were considered in the screening and selection process of candidate polymers. Hytrel (a copolymer of poly(butylene terephthalate) and butylene ether glycol terephthalate) polymer beads, offered improved capacity (19 mg phenol/g polymer at a fixed initial phenol concentration of 2000 mg/L) and a greater diffusivity (1.54 x 10(-7) cm2/s) when compared to the capacity and diffusivity of previously used EVA (ethylene vinyl acetate) beads (12.4 mg phenol/g polymer and 3.73 x 10(-9) cm2/s, respectively). Hytrel polymer beads were then used in a TPPB for the investigation of various substrate feeding strategies (fed-batch, bead replacement, and concentrated spikes of phenol), with rapid and complete phenol degradation shown in all cases.     

Polymer selection for biphenyl degradation in a solid-liquid two-phase partitioning bioreactor

The commercially available thermoplastic polymer Hytrel was selected as the delivery phase for the hydrophobic model compound biphenyl in a solid-liquid two-phase partitioning bioreactor (TPPB), and 2.9 g biphenyl could successfully be degraded in 1-L TPPBs by a pure culture of the biphenyl-degrading bacterium Burkholderia xenovorans LB400 in 50 h and by a mixed microbial consortium isolated from contaminated soil in 45 h. TPPBs consist of an aqueous cell-containing phase and an immiscible second phase that partitions toxic and/or poorly soluble substrates (in this case biphenyl) on the basis of maintaining a thermodynamic equilibrium. This paper illustrates a rational strategy for selecting a suitable solid polymeric substance for the delivery of the poorly water-soluble model compound biphenyl. The partitioning of biphenyl between the selected polymers and water was analogous to partitioning of solutes between two immiscible liquid phases. The partitioning coefficients varied between 180 for Nylon 6.6 and 11,000 for Desmopan, where the later numerical value is comparable to biphenyl partitioning coefficients between water and organic solvents. Employing a solid delivery phase enabled the utilization of a surfactant-producing microbial mixed culture, which could not be cultivated in liquid-liquid TPPBs and thereby extended the range of biocatalysts that can be employed in TPPBs.     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Biodegradation of pyrene by Mycobacterium frederiksbergense in a two-phase partitioning bioreactor system

Biodegradation of pyrene by Mycobacterium frederiksbergense was studied in a two-phase partitioning bioreactor (TPPB) using silicone oil as non-aqueous phase liquid (NAPL). The TPPB achieved complete biodegradation of pyrene; and during the active degradation phase, utilization rates of 270, 230, 139, 82 mg l(-1)d(-1) for initial pyrene loading concentrations (in NAPL) of 1000, 600, 400 and 200 mg l(-1), respectively, were obtained. The degradation rates achieved using M. frederiksbergense in TPPB were much higher than the literature reported values for an ex situ PAH biodegradation system operated using single and pure microbial species. The degradation data was fitted to simple Monod, logistic, logarithmic, three-half-order kinetic models. Among these models, only exponential growth form of the three-half-order kinetic model provided the best fit to the entire degradation profiles with coefficient of determination (R2) value >0.99. From the experimental findings, uptake of pyrene by the microorganism in TPPB was proposed to be a non-interfacial based mechanism.     

Phosphonium ionic liquids for degradation of phenol in a two-phase partitioning bioreactor

Six ionic liquids (ILs), which are organic salts that are liquid at room temperature, were tested for their biocompatibility with three xenobiotic-degrading bacteria, Pseudomonas putida, Achromobacter xylosoxidans, and Sphingomonas aromaticivorans. Of the 18 pairings, seven were found to demonstrate biocompatibility, with one IL (trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl) amide) being biocompatible with all three organisms. This IL was then used in a two-phase partitioning bioreactor (TPPB), consisting of 1 l aqueous phase loaded with 1,580 mg phenol and 0.25 l IL, inoculated with the phenol degrader P. putida. This initially toxic aqueous level of phenol was substantially reduced by phenol partitioning into the IL phase, allowing the cells to utilize the reduced phenol concentration. The partitioning of phenol from the IL to the aqueous phase was driven by cellular demand and thermodynamic equilibrium. All of the phenol was consumed at a rate comparable to that of previously used organic-aqueous TPPB systems, demonstrating the first successful use of an IL with a cell-based system. A quantitative ³¹P NMR spectroscopic assay for estimating the log P values of ILs is under development.     

Solvent selection for enhanced bioproduction of 3-methylcatechol in a two-phase partitioning bioreactor

The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.     

Use of a two-phase partitioning bioreactor for degrading polycyclic aromatic hydrocarbons by a Sphingomonas sp

A two-phase partitioning bioreactor (TPPB) utilizing the bacterium Sphingomonas aromaticivorans B0695 was used to degrade four low molecular weight (LMW) polycyclic aromatic hydrocarbons (PAHs). The TPPB concept is based on the use of a biocompatible, immiscible organic solvent in which high concentrations of recalcitrant substrates are dissolved. These substances partition into the cell-containing aqueous phase at rates determined by the metabolic activity of the cells. Experiments showed that the selected solvent, dodecane, could be successfully used in both solvent extraction experiments (to remove PAHs from soil) and in a TPPB application. Further testing demonstrated that solvent extraction from spiked soil was enhanced when a solvent combination (dodecane and ethanol) was used, and it was shown that the co-solvent did not significantly affect TPPB performance. The TPPB achieved complete biodegradation of naphthalene, phenanthrene, acenaphthene and anthracene at a volumetric consumption rate of 90 mg l(-1) h(-1) in approximately 30 h. Additionally, a total of 20.0 g of LMW PAHs (naphthalene and phenanthrene) were biodegraded at an overall volumetric rate of 98 mg l(-1) h(-1) in less than 75 h. Degradation rates achieved using the TPPB and S. aromaticivorans B0695 are much greater than any others previously reported for an ex situ PAH biodegradation system operating with a single species.     

Delivery of benzene to Alcaligenes xylosoxidans by solid polymers in a two-phase partitioning bioreactor

Toxic levels of benzene were decreased to sub-inhibitory levels in a bioreactor via absorption by polymer beads or cylinders (poly(ethylene-co-vinyl acetate) or poly(styrene-co-butadiene)). After inoculation with Alcaligenes xylosoxidans, the remaining benzene in the aqueous phase as well as the benzene taken up by the polymers was degraded to completion. The capacity of these polymers for benzene, and benzene diffusivity within the polymers were also determined.     

Mandelic acid chiral separation utilizing a two-phase partitioning bioreactor built by polysulfone microspheres and immobilized enzymes

A novel two-phase partitioning bioreactor (TPPB) modified by polysulfone (PSF) microspheres and immobilized enzyme (novozym-435) was formed, and the resulting TPPB was applied into mandelic acid chiral separation. The PSF microspheres containing n-hexanol (named PSF/hexanol microspheres) was prepared by using the phase inversion method, which was used as the organic phase. Meanwhile, the immobilized enzyme novozym-435 was used as a biocatalyst. The water phase was composed of the phosphate buffer solution (PBS). (R, S)-Methyl mandelate was selected as the substrate to study enzymatic properties. Different reaction factors have been researched, such as pH, reaction time, temperature and the quantity of biocatalyst and PSF/hexanol microspheres added in. Finally, (S)-mandelic acid was obtained with an 80 % optical purity after 24 h in the two-phase partitioning bioreactor. The enantiomeric excess (ee_p) values were very low in the water phase, in which the highest ee_p value was only 46 %. The ee_p of the two-phase partitioning bioreactor had been enhanced more obviously than that catalyzed in the water phase.     

Biodegradation of polycyclic aromatic hydrocarbons in a two-phase partitioning bioreactor in the presence of a bioavailable solvent

Mycobacterium PYR-1 was used in a two-phase partitioning bioreactor (TPPB) to degrade low and high molecular weight polycyclic aromatic hydrocarbons. TPPBs are characterized by a cell-containing aqueous phase, and an immiscible and biocompatible organic phase that partitions toxic substrates to the cells based on their metabolic demand and the thermodynamic equilibrium of the system. A bioavailable solvent, that is, a solvent usable as a carbon source, was used as the organic layer. Although bioavailable solvents are traditionally deemed unsuitable for use in TPPBs, bis(ethylhexyl) sebacate had superior chemical properties to other solvents examined and was cost-effective. In this system, 1 g of phenanthrene and 1 g of pyrene were completely degraded within 4 days, at rates of 168 mg l(-1) day(-1) and 138 mg l(-1 )day(-1), respectively, based on a 3-l aqueous volume. This is the highest pyrene degradation rate reported in the literature to date. Significant degradation of naphthalene and anthracene was also obtained. This work demonstrates that bioavailable solvents can be successfully used in TPPB systems, and may change the protocols commonly used to select solvents for TPPBs in the future.     

Removal and destruction of high concentrations of gaseous toluene in a two-phase partitioning bioreactor by Alcaligenes xylosoxidans

A two-phase bioreactor consisting of hexadecane dispersed in an aqueous, cell-containing medium (organic fraction = 0.33) was used to trap toluene vapours from an air stream. The affinity for toluene by the solvent resulted in high efficiency of removal and transfer to the aqueous phase based on equilibrium transfer. The system was readily able to handle a loading capacity of 748 mg l^–1 h^–1 at a toluene degradation efficiency of greater than 98%.     

High-level production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli

Reconstructing synthetic metabolic pathways in microbes holds great promise for the production of pharmaceuticals in large-scale fermentations. By recreating biosynthetic pathways in bacteria, complex molecules traditionally harvested from scarce natural resources can be produced in microbial cultures. Here we report on a strain of Escherichia coli containing a heterologous, nine-gene biosynthetic pathway for the production of the terpene amorpha-4,11-diene, a precursor to the anti-malarial drug artemisinin. Previous reports have underestimated the productivity of this strain due to the volatility of amorphadiene. Here we show that amorphadiene evaporates from a fermentor with a half-life of about 50 min. Using a condenser, we take advantage of this volatility by trapping the amorphadiene in the off-gas. Amorphadiene was positively identified using nuclear magnetic resonance spectroscopy and determined to be 89% pure as collected. We captured amorphadiene as it was produced in situ by employing a two-phase partitioning bioreactor with a dodecane organic phase. Using a previously characterized caryophyllene standard to calibrate amorphadiene production and capture, the concentration of amorphadiene produced was determined to be 0.5 g/L of culture medium. A standard of amorphadiene collected from the off-gas showed that the caryophyllene standard overestimated amorphadiene production by approximately 30%.     

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.     

Enhancement of a two-phase partitioning bioreactor system by modification of the microbial catalyst: demonstration of concept

Application of two-phase partitioning bioreactors (TPPB) to the degradation of phenol and xenobiotics has been limited by the fact that many organic compounds that would otherwise be desirable delivery solvents can be utilized by the microorganisms employed. The ability to metabolize the solvent itself could interfere with xenobiotic degradation, limiting remediation efficiency, and hence represents a microbial characteristic incompatible with process goals. To avoid the issue of bioavailability, previous TPPB applications have relied on complex and often expensive delivery solvents or suboptimal catalyst-solvent pairings. In an effort to enhance TPPB activity and applicability, a genetically engineered derivative of Pseudomonas putida ATCC 11172 mutated in its ability to utilize medium-chain-length alcohols was generated (AVP2) and applied as the catalyst within a TPPB system with decanol as the delivery solvent. Kinetic analysis verified that the genetic alteration had not negatively affected phenol degradation. The volumetric productivity of AVP2 (0.48 g/L x h(-1)) was equivalent to that seen for wild-type ATCC 11172 (0.51 g/L x h(-1)), but a comparison of initial cell concentrations and yields revealed an improved phenol-degrading efficiency for the mutant under process conditions. Yield coefficients, cell dry weight, and viable count determinations all confirmed the stability of the modified phenotype. This work illustrates the possibilities for TPPB process enhancement through a careful combination of genetic modification and solvent selection.     

Enhancement of mass transfer characteristics and phenanthrene degradation in a two-phase partitioning bioreactor equipped with internal static mixers

Oxygen and substrate supply have always been considered physical constraints for the performance and operation of two-phase partitioning bioreactors (TPPB), widely used for the degradation of hydrophobic substrates. In this regard, the potential advantages of static mixers in upgrading the oxygen transfer and liquid-liquid dispersions in TPPB have been highlighted. In the present paper, the concomitant influence of static mixers on the gas-liquid mass transfer coefficient k _L a and on substrate bioavailability was examined in TPPB. The static method based on conventional forms was developed to estimate the oxygen volumetric mass transfer coefficient. Over a broad range of liquid and air flow rates, the presence of static mixers was found to significantly enhance k _L a relative to a mixer-free mode of operation. For identical conditions, static mixers improved the k _L a threefold. In the presence of external aeration supply, the boost in the k _L a was associated with an increase of 16% in the phenanthrene biodegradation rate due to bubble break up accomplished by the static mixers. On the other hand, static mixers were efficient in enhancing substrate bioavailability by improving the liquid-liquid interfacial area. This effect was reflected by a threefold increase in the degradation rate in the bioreactors with no external supply of air when equipped with static mixers.     

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.     

Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system

The prevailing view of intra-Golgi transport is cisternal progression, which has a key prediction--that newly arrived cargo exhibits a lag or transit time before exiting the Golgi. Instead, we find that cargo molecules exit at an exponential rate proportional to their total Golgi abundance with no lag. Incoming cargo molecules rapidly mix with those already in the system and exit from partitioned domains with no cargo privileged for export based on its time of entry into the system. Given these results, we constructed a new model of intra-Golgi transport that involves rapid partitioning of enzymes and transmembrane cargo between two lipid phases combined with relatively rapid exchange among cisternae. Simulation and experimental testing of this rapid partitioning model reproduced all the key characteristics of the Golgi apparatus, including polarized lipid and protein gradients, exponential cargo export kinetics, and cargo waves.     

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.     

Fed-batch bioreactor strategies for microbial decolorization of azo dye using a Pseudomonas luteola strain

A Pseudomonas luteola strain possessing azoreductase activity was utilized to decolorize a reactive azo dye (C. I. Reactive Red 22) with fed-batch processes consisting of an aerobic cell growth stage and an anaerobic fed-batch decolorization stage. The fed-batch decolorization was conducted with different agitation and aeration rates, initial culture volumes, dye loading strategies, and yeast extract to dye (Y/D) ratios, and the effect of those operation parameters on azo dye decolorization was evaluated. Dissolved oxygen strongly inhibited the azo reduction activity; thus aeration should be avoided during decolorization but slight agitation (around 50 rpm) was needed. With the periodical feeding strategy, the specific decolorization rate (v(dye)) and overall decolorization efficiency (eta(dye)) tended to increase with increasing feeding concentrations of dye, whereas substrate inhibition seems to arise when the feeding concentration exceeded 600 mg dye/L. In the continuous feeding mode, higher initial culture volume resulted in better eta(dye) due to higher biomass loading, but lower v(dye) due to lower dye concentration in the bioreactor. With a volumetric flow rate (F) of 25 mL/h, both v(dye) and eta(dye) increased almost linearly with the increase in the loading rate of dye (F(dye)) over the range of 50-200 mg/h, while further increase in F(dye) (400 mg/h) gave rise to a decline in v(dye) and eta(dye). As the F was doubled (50 mL/h), the v(dye) and eta(dye) increased with F(dye) only for F(dye) < 80 mg/h. The best v(dye) (113.7 mg dye g cell(-)(1) h(-)(1)) and eta(dye) (86.3 mg dye L(-)(1) h(-)(1)) were achieved at F(dye) = 200 mg/h and F = 25 mL/h. The yield coefficient representing the relation between dye decolorized and yeast extract consumed was estimated as 0.8 g/g. With F(dye) = 75 mg/h, the Y/D ratio should be higher than 0.5 to ensure sufficient supply of yeast extract for stable fed-batch operations. However, performance of the fed-batch decolorization process was not appreciably improved by raising the Y/D ratio from 0.5 to 1.875 but was more sensitive to the changes in the dye loading rate.