Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Methylosinus trichosporium OB3b whole-cell methane monooxygenase activity in a biphasic matrix

 Reconstituted whole-cell preparations of lyophilized Methylosinus trichosporium OB3b were used to demonstrate soluble methane monooxygenase activity in a two-phase (biphasic) matrix consisting of a buffered aqueous phase and 2,2,4-trimethylpentane (isooctane). The rate of conversion of gaseous propylene to propylene oxide, a non-metabolized liquid, was used as the primary measure of enzyme activity. Appreciable soluble methane monooxygenase activity was detected when the volume of the aqueous phase represented at least 1% of the total volume, although the initial rate of product formation did increase as the volume of the aqueous phase increased. In comparison to the aqueous system, the specific rate and yields in the biphasic system were much less sensitive to increases in the concentrations of formate and protein (the methane monooxygenase). However, there was some evidence that the enzyme system was more stable in the biphasic matrix, since the rate of propylene oxide formation remained linear for an extended period of time. V _(app.) in the biphasic system decreased by a factor of 0.6 relative to the same parameter in the aqueous system. Conversely, K _m(app.) for propylene was 1.6 times greater in the biphasic system. Hence, the apparent catalytic efficiency in the aqueous system was four times that in the biphasic system, as indicated by a decrease in the corresponding ratios of V _(app.) to K _m(app.). Received: 21 July 1995/Received last revision: 1 February 1996/Accepted: 5 December 1996     

Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.     

A mathematical model for the synthesis of Gly-Phe by papain in an aqueous-organic system

The synthesis of the protected dipeptide BocGlyPheOMe, has been modellised when working in an aqueousorganic biphasic system, with papain as a catalyst. The mathematical model takes into account that one of the substrates, PheOMe, has parallel hydrolysis reactions and that the reaction only takes place in the aqueous phase while the whole reaction system is biphasic. The reaction system has been modellised when working in batch as well as when working in fed-batch mode, achieving a good prediction of the product evolution for both working strategies. When working in fed-batch mode, the extension of the undesired parallel reactions has been diminished, the model has been used for a computer aided optimisation of the addition sequence of PheOMe. The results obtained led to a process operation strategy with a compromise between yield and productivity.List of Symbols [i] concentration of any component i - [i] _aq concentration of i in the aqueous phase - [i] _bi concentration of i in the biphasic system - [E] ₀ initial concentration of enzyme - k _e, k_q first order kinetic constants - K _A, K_B equilibrium constants - r _m maximum rate of reaction This worked was financed by the Interministerial Commission for Science and Technology (CICYT)from the Spanish Government under projects number BIO/88-370 and SAF92-0261-CO2-02.     

Model for the production of L-tryptophan from L-serine and indole by immobilized cells in a three-phase liquid-impelled loop reactor

Production of L-tryptophan from L-serine and indole catalyzed by Escherichia coli, immobilized in k-carrageenan gel beads, is technically feasible in the liquidimpelled loop reactor (LLR), using an organic solvent, e.g. n-dodecane.With L-serine in large excess intrinsic reaction kinetics is approximately first order with respect to indole, with a reaction constant of 8.5×10^–5 m³ kg _dw ^–1 s^–1.The overall process kinetics is jointly controlled by intrinsic kinetics and by intraparticle mass transfer resistance, which can be quantified using an effectiveness factor.Mass transfer of indole from the organic to the aqueous phase and from the aqueous to the gel phase are relatively fast and thus have negligible influence in the overall process kinetics, under the operational conditions tested. However, they may become important if the process is intensified by increasing the cell concentration in the gel and/or the gel hold-up in the reactor.A simple model which includes indole mass balances over the aqueous and organic phases, mass transfer and reaction kinetics, with parameters experimentally determined in independent experiments, was successful in simulating L-tryptophan production in the LLR.List of Symbols a, b, c coefficients of the equilibrium curve for indole between organic and aqueous phases - A, B, C, D, E, F auxiliary variables used in liquid-liquid mass transfer studies - a _x specific interfacial area referred to the volume of the aqueous phase (m^–1) - A _x interfacial area (m²) - a _Y specific interfacial area referred to the volume of the organic phase (m^–1) - A _Y interfacial area (m²) - C _b substrate concentration in the bulk of the aqueous phase (kg m^–3) - C _e substrate concentration in exit stream (kg m^–3) - C _E biocatalyst concentration referred to the aqueous phase (kg m^–3) - C _{E s} biocatalyst concentration referred to the volume of gel (kg m^–3) - C _s substrate concentration at the gel surface (kgm^–3) - d, e, f coefficients of the equilibrium curve for indole between aqueous and organic phases - dp particle diameter (m) - K ₂ kinetic constant (s^–1) - K ₁ kinetic constant K₂/K_M (kg^–1 m³ s^–1) - K _M Michaälis-Menten constant (kgm^–3) - K _X mass transfer coefficient referred to the aqueous phase (ms^–1) - K _Xa_X volumetric mass transfer coefficient based on the volume of the aqueous phase (s^–1) - k _Y mass transfer coefficient referred to the organic phase (ms^–1) - K _Ya_Y volumetric mass transfer coefficient based on the volume of the organic phase (s^–1) - N _X mass flux of indole from organic to aqueous Phase (kg m^–2s^–1) - N _Y mass flux of indole from aqueous to organic phase (kg m^–2s^–1) - Q _e volumetric flow rate in exit stream (m³s^–1) - Q _f volumetric flow rate in feed stream (m³s^–1) - _obs observed reaction rate (kg s^–1 m^–3) - intrinsic reaction rate (kg s^–1 m^–3) - Re Reynolds number - Sc Schmidt number - Sh Sherwood number - t time (s) - u superficial velocity (m s^–1) - V _max maximum reaction rate (kg s^–1m^–3) - V _S volume of the support (m³) - V _X volume of aqueous phase (m³) - V _Y volume of the organic phase (m³) - X indole concentration in the aqueous phase (kgm^–3) - Y indole concentration in the organic phase (kg m^–3 Greek Letters overall effectiveness factor - _e external effectiveness factor - _i internal effectiveness factor - Thiele module A fellowship awarded to one of us (D.M.R.)by INICT is gratefuly acknowledged.     

Lipase-catalyzed hydrolysis of 2-naphtyl esters in biphasic system

The authors measured the rate of hydrolysis of the homologs of 2-naphtyl ester by using a Lewis cell with constant interfacial area to elucidate the kinetic mechanism of the lipase-catalyzed hydrolysis in biphasic system. On the basis of the two-film model, it was found from the analysis of experimental results that the hydrolysis of these substrates proceeds at the interface between the aqueous and organic phases. The interfacial reaction rate could be correlated by Michaelis-Menten mechanism. The values of the rate constant and the Michaelis constant were almost independent of the kinds of 2-naphtyl ester. The values of the interfacial kinetic parameters for 2-naphtyl ester were much greater than those for the hydrolysis in the aqueous phase.     

Enhancement of the gas-to-water ethene transfer coefficient by a dispersed water-immiscible solvent: effect of the cells

  For a mass-transfer-limited system, it was demonstrated that the volumetric ethene transfer coefficient (k _l a) from gas to water could be enhanced by dispersing adequate amounts of a water-immiscible organic liquid, namely the perfluorocarbon FC40, in the aqueous phase. When 26% (v/v) FC40 was dispersed in a culture of Mycobacterium parafortuitum an enhancement of k _l a, calculated on a total liquid volume basis, of 1.8 times was found. Steady-state experiments in the absence of microorganisms, however, showed a 1.2-fold enhancement of k _l a at 18.5% (v/v) FC40. At all FC40 volume fractions tested, enhancement factors with cells were higher than enhancements without cells; apparently the microorganisms or their excretion products affected the interfacial areas or characteristic phase dimensions. Received: 4 December 1995 / Received revision: 7 June 1996 / Accepted: 10 June 1996     

The kinetic behaviour of a two-enzyme system in biphasic media: coupling hydrolysis and lipoxygenation

Analysis of the kinetic behaviour of a two-enzyme-system carrying out two consecutive reactions was investigated in macroheterogeneous biphasic media (octane/buffer pH 9.6, v/v=1:1). The lipase-catalysed hydrolysis of trilinolein and the subsequent lipoxygenation of the liberated linoleic acid, were coupled in a modified Lewis cell with a well-defined liquid/liquid interfacial area. Trilinolein was dissolved in the organic phase and hydrolysed in the presence of Mucor javanicus lipase at the organic/aqueous interface. Linoleic acid, liberated after hydrolysis was transferred to the aqueous phase and reacted with lipoxygenase. This reaction consumed linoleic acid and produced hydroperoxides, which favoured the transfer of residual linoleic acid, since they possess surface active properties. Catalysis and transfer influenced each other reciprocally. At low substrate concentrations, cooperativity phenomena were observed in the experimental and also the modelled two-enzyme systems. When the initial substrate concentration was high, the kinetic behaviour of the two-enzyme system in a compartmentalised medium, seemed to be independent of the substrate concentration, unlike that observed in homogeneous monophasic enzymology. The numerical integration program used to model the two-enzyme system was based on results obtained in separate studies of the following three phenomena: (1) trilinolein hydrolysis in biphasic medium, (2) linoleic acid transfer across a liquid/liquid interface and (3) lipoxygenation in an aqueous media. Results obtained by modelling were similar to the results observed experimentally.     

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t _d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.     

Evaluation of steady-state-biofilm kinetics

Laboratory-scale biofilm reactors were used to evaluate a model of the kinetics of steady-state biofilm and the concept that there is a minimum concentration, S_min, below which no steady-state activity can occur. With acetate as the ratelimiting substrate, the steady-state concept of S_min was verified for naturally grown biofilms. Substrate removal and biofilm thickness declined rapidly as the substrate concentration approached S_min, which was 0.66 mg/liter for acetate. Using independently derived kinetic parameters, the model of steady-state-biofilm kinetics successfully predicted substrate utilization and biofilm thickness without the need for fitting factors. The results imply that organic materials may persist in water and wastewater, in part, because they are too low in concentration to supply sufficient energy to sustain the microorganisms.     

Enzymatic Synthesis of N-formyl-L-aspartyl-L-phenylalanine Methyl Ester (Aspartame Precursor) Utilizing an Extractive Reaction in Aqueous/Organic Biphasic Medium

N-Formyl-L-aspartyl-L-phenylalanine methyl ester (N-formyl aspartame, F-AspPheOMe) was synthesized enzymatically utilizing an extractive reaction in an aqueous/organic biphasic system. The N-formyl aspartame yield in a pure aqueous monophasic system was, in general, ca. 3% , however, it was over 80 % in a water/1-butanol biphasic system using a simultaneously extractive operation of an enzymatic reaction in an aqueous phase and a product separation from an aqueous to an organic phases.     

Enhancing productivity for cascade biotransformation of styrene to (S)-vicinal diol with biphasic system in hollow fiber membrane bioreactor

Biotransformation is a green and useful tool for sustainable and selective chemical synthesis. However, it often suffers from the toxicity and inhibition from organic substrates or products. Here, we established a hollow fiber membrane bioreactor (HFMB)-based aqueous/organic biphasic system, for the first time, to enhance the productivity of a cascade biotransformation with strong substrate toxicity and inhibition. The enantioselective trans-dihydroxylation of styrene to (S)-1-phenyl-1,2-ethanediol, catalyzed by Escherichia coli (SSP1) coexpressing styrene monooxygenase and an epoxide hydrolase, was performed in HFMB with organic solvent in the shell side and aqueous cell suspension in the lumen side. Various organic solvents were investigated, and n-hexadecane was found as the best for the HFMB-based biphasic system. Comparing to other reported biphasic systems assisted by HFMB, our system not only shield much of the substrate toxicity but also deflate the product recovery burden in downstream processing as the majority of styrene stayed in organic phase while the diol product mostly remained in the aqueous phase. The established HFMB-based biphasic system enhanced the production titer to 143 mM, being 16-fold higher than the aqueous system and 1.6-fold higher than the traditional dispersive partitioning biphase system. Furthermore, the combination of biphasic system with HFMB prevents the foaming and emulsification, thus reducing the burden in downstream purification. HFMB-based biphasic system could serve as a suitable platform for enhancing the productivity of single-step or cascade biotransformation with toxic substrates to produce useful and valuable chemicals.

     

Simplified Modeling of Simultaneous Reaction Kinetics of Carbon Oxidation and Nitrification in Biofilm Processes

Batch experiments with varying initial substrate concentrations and biomass volumes were performed in a three‐phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the simultaneous carbon oxidation and nitrification in the biofilm process. A simplified mass balance equation for the biofilm was proposed and five different kinetic rate equations were used to match the actual data. The kinetic parameters were obtained by nonlinear regression analysis on a set of two differential equations representing the simultaneous carbon oxidation and nitrification. The competitive inhibition model incorporating the effects of total organic carbon (TOC) concentrations on nitrification rates was the best‐suited model based on the average r². In this model, oxygen concentration and its affinity constants were not included. Instead, it was assumed that the rate of carbon oxidation is independent of the NH₄⁺‐N, while nitrification is affected by TOC. The number of parameters was successfully minimized without reducing its ability to accurately predict the bulk concentration time course, which would reduce computational complexity and possibly enhance the availability for an actual wastewater treatment process.     

Systematic assessment of the stability of benzaldehyde lyase in aqueous–organic biphasic systems and its stabilization by modification with methoxy-poly(ethylene) glycol

Benzaldehyde lyase from Pseudomonas fluorescens Biovar I [BAL; E.C.4.1.2.38] catalyzes the stereoselective formation of C–C bonds coupling aldehydes to generate alpha-hydroxy ketones. A broad range of poorly water-soluble substrates are accepted in forward and reverse reactions. In this study, the stability of BAL in aqueous–organic biphasic systems as promising reaction media was systematically investigated using methyl-tert-butylether, 2-octanone, and toluene as the organic phase. Surprisingly, a strong individual molecular toxicity of these water-immiscible solvents was observed along with the interfacial toxicity exerted by the aqueous–organic interfaces. They could be considerably reduced by covalent attachment of methoxy-poly(ethylene) glycol (mPEG₇₅₀ and mPEG₂₀₀₀) to the enzyme surface increasing the half-life by a factor of up to 18. However, under reactive conditions solvent effects were strongly superimposed by an additional deactivating effect, possibly caused by the aldehyde substrate, and no differences between unmodified and modified BAL were detectable. For technical application of the enzyme in aqueous–organic biphasic media additional strategies for stabilization will therefore be desirable.     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

Kinetics of phenol oxidation by washed cells

The uptake of phenol by pure cultures of Pseudomonas putida growing on phenol in continuous culture has been studied. The purpose of the experiments was to determine the kinetic parameters governing uptake of phenol by organisms growing on phenol in the high-conversion range by measuring uptake rates per unit biomass per unit time at various phenol concentrations. The microorganisms used were taken from a chemostat at residence times of 8, 5.25, 3.85, 3.2, 3, and 2.7h. The Monod–Haldane model and modifications of it were applied to the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The best model was a two-parameters simplification of Monod–Haldane in which μ = K₁S/(K₂ + S²). The value of K₁ was found to increase monotonically with the value of phenol concentration in the original chemostat with an apparent induction “threshold” of 0.1 mg/L.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.