Acetophenone Concentration Research Articles

Two anti‐Prelog NAD‐Dependent Alcohol Dehydrogenases with Broad Substrate Scope and Excellent Enantioselectivity

AbstractEnantiomerically pure alcohols are important to produce active pharmaceutical ingredients, agrochemicals and fine chemicals. Herein, we explored the substrate scope and chemo‐ and enantioselectivity of two NAD‐dependent anti‐Prelog alcohol dehydrogenases from Candida maris (Cm‐ADH) and Pichia finlandica (Pf‐ADH) in the asymmetric reduction of ketones. The ADHs were tested for the reduction of acetophenone with NADH that was recycled using a formate dehydrogenase and sodium formate. Cm‐ADH and Pf‐ADH performed best at 30 °C and at around pH 7 and pH 6, respectively. Pf‐ADH operated well at 50 mM acetophenone concentration, while Cm‐ADH was limited to 10 mM. Regarding the substrate scope, linear‐chain alkyl ketones were efficiently reduced (up to 98 % conversion), while branched and cyclic ketones gave lower conversions (up to 60 %). Aryl‐aliphatic ketones showed variable levels of conversion (<1–79 %), while α,β‐unsaturated and heteroaromatic ketones exhibited good to excellent conversions. In most of the cases, the enantiomeric excess was >99 %. Aliphatic and aryl‐aliphatic aldehydes were converted with up to >99 % conversion. A scale‐up experiment with Pf‐ADH using acetophenone as substrate led to 73 % isolated yield and >99 % ee (R).This work contributes to filling the gap in biocatalytic asymmetric synthesis of chiral alcohols by introducing two NAD‐dependent ADHs with anti‐Prelog selectivity.

Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution

The short-chain dehydrogenase/reductase (SDR) from Empedobacter brevis ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (R)-1-phenylethanol, but its industrial application is restricted by its insufficient tolerance to acetophenone. In this paper, we developed a chromogenic reaction-based high-throughput screening method and employed directed evolution to enhance the acetophenone tolerance of EbSDR8. The resulting variant, M190V, showed 74.8% improvement over the wild-type in specific activity when catalyzing the reduction of 200 mM acetophenone. Kinetic analysis revealed a 70% enhancement in its catalytic efficiency (kcat/Km). Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance, and the result implied that the M190V mutation is conducive to the binding and release of coenzyme. Aside from the improved catalytic performance when dealing with a high concentration of acetophenone, other features of M190V, such as a broad pH range (6.0 to 10.5), low optimal cosubstrate concentration (1% isopropanol), and a temperature optimum close to that of E. coli cells (35 °C), also contribute to its practical application as a whole-cell catalyst. In this study, we first designed a directed evolution means to engineer the enzyme and obtained the positive variant which has a high activity under high concentrations of acetophenone. After that, we optimized the catalytic performance of the variant to adapt to industrial applications.

Substrate selectivity and kinetic studies of (S)-specific alcohol dehydrogenase purified from Candida parapsilosis ATCC 7330

Biocatalytic reduction catalysed by alcohol dehydrogenases is a valuable tool for asymmetric synthesis of chiral alcohols. This study reports the broad substrate specificity of NADH dependent (S) - specific alcohol dehydrogenase (S-ADH) purified from Candida parapsilosis ATCC 7330. The substrates for this enzyme include aliphatic and aromatic ketones, cyclic and diketones, aldehydes, ketoesters, primary and secondary alcohols. The kinetic studies of different substrates indicate that ketones and secondary alcohols are the most preferred substrates of S-ADH with highest catalytic efficiencies reported for reduction of acetone (4153 s−1mM−1) and oxidation of 2-propanol (1358 s−1mM−1). The double reciprocal plots obtained for varied concentrations of acetophenone (0.2–16 mM) at a fixed concentration of NADH (0.05, 0.1 and 0.2 mM) and with varied concentrations of NADH (0.01–0.2 mM) at a fixed concentration of acetophenone (1, 4, 8 and 16 mM) showed intersecting lines indicating sequential kinetic mechanism. S-ADH follows Prelog's stereopreference in reducing prochiral carbonyl substrates to yield (S)-alcohols with >99% enantiomeric excess using a simple coupled substrate approach for cofactor recycling.

Influence of dielectric, Electro-Optic Kerr Effect and spectroscopic characterisation on polar–polar binary liquid mixture

ABSTRACT Complex dielectric spectra of the binary mixture of benzonitrile (BNZ) with acetophenone (ACPH) have been carried out at 11 different concentrations in the frequency range of 10 MHz to 40 GHz at four different temperatures (10°C, 15°C, 20°C and 25°C) using Time Domain Reflectometry technique. Static dielectric constant (ε0), relaxation time (τ) are obtained for above mentioned temperatures using nonlinear least squares fit method and density and refractive index are obtained at room temperature (25°C). These parameters are used to calculate the excess static dielectric constant (ε0 E) and excess inverse relaxation time (1/τ)E, excess molar volume (Vm E), effective Kirkwood correlation factor (geff), Bruggeman factor (fB) and thermodynamic parameters such as enthalpy and entropy of activation of BNZ-ACPH mixture. Static dielectric constant decreases with increasing temperature and increasing concentration of ACPH in BNZ. The deviance in the excess parameters shows the strength of the molecular interactions and the presence of intermolecular interaction between unlike molecules. The measurement of Kerr constant (B) of the binary mixture of BNZ-ACPH have been reported for the wavelengths 632.8 nm and 512 nm for 5 different concentrations at room temperature using Electro-Optic Kerr Effect method. The change in the Kerr constant was observed for the different concentrations associated with the pure liquids. This deviation shows the non-additive behaviour and which is in good agreement with the dielectric parameters of the system under study. Also, the FTIR spectra have been obtained of BNZ-ACPH mixture to have a good agreement with the dielectric study.

Kinetic study of immobilized ω-transaminase for the asymmetric synthesis of chiral amine

Biocatalysis is a powerful tool for the synthesis of high value products such as, chiral molecules and intermediates. Chiral amines can be synthesized by kinetic resolution of racemic amines or by the asymmetric synthesis from pro-chiral ketones. Kinetic parameter estimation for biocatalytic reaction is useful to evaluate process and technology options. In this work, the Michaelis–Menten kinetic parameters of immobilized ω-transaminase on methacrylate beads for the asymmetric synthesis of (R)-1-phenylethylamine (PEA) from acetophenone (ACP) and alanine (ALA) were determined. The parameters such as, MichaelisMenten constant (Km) and maximum rate of reaction (Vmax) were measured from initial rate experiments by varying ACP concentrations (2 mM to 10 mM) at a fixed 100 mM ALA under mild reaction conditions (pH 7.5 and temperature 30 °C). The concentration of ACP and PEA were measured by gas chromatography. Lineweaver-burk plot was used to estimate the Vmax and Km from the initial rate data. The results yielded a Vmax of 6.87 mM/min and a Km of 2.52 mM. The Michaelis-Menten model is useful for understanding the kinetic properties of immobilized enzymes.

Optimization of the asymmetric synthesis of (S)-1-phenylethanol using Ispir bean as whole-cell biocatalyst

Abstract In this study, enantiomerically pure (S)-1-phenylethanol was produced via asymmetric bioreduction of acetophenone. Ispir bean (Phaseolus vulgaris) was used as an alcohol dehydrogenase (ADH) source since whole cells are cheaper than isolated enzymes. Acetone powder methodology was applied for biocatalyst. Glucose was used as a cosubstrate in-order to regenerate cofactor (NADPH). The reactions were carried out in an orbital shaker whose temperature and agitation rate can be controlled. (S)-1-phenylethanol concentration was analyzed by HPLC using a Chiralcel OB column. Effects of the reaction time, substrate concentration, cosubstrate concentration and biocatalyst concentration on the (S)-1-phenylethanol production were investigated using Response Surface Methodology (RSM). 36 h bioreduction time, 6 mM acetophenone concentration, 25.15 mM glucose concentration, and 175 mg/mL biocatalyst concentration were determined as optimum values. In these conditions, 2.4 mM (S)-1-phenylethanol was obtained in phosphate buffer (pH=7.0) at 30°C with >99% enantiomeric excess.

Selective catalytic hydrogenation of acetophenone to1-phenylethanoloverCo/mordenite in water

The selective catalytic hydrogenation of acetophenone (AP)to 1-phenylethanol (PhE) has been studied in water, ethanol and n-hexanesolvent over Co/mordenite. It was shown that the highest yield of PhE (99.9%) was obtained in water solvent. The adsorption of AP hindered the adsorption of PhE in water, which in turn reduced the rate of hydrogenolysis of PhE to ethylbenzene (EB).By increasing the reaction temperature the hydrogenolysis of PhE to EB is promoted. The water phase was collected after reaction and reused successfully. The yield of PhE was 96.1% when the AP concentration in solution and the catalyst loading increased to three times. The neutral and alkaline conditions were beneficial to the hydrogenation of AP to PhE in water.

Fertilization Changes Chemical Defense in Needles of Mature Norway Spruce (Picea abies).

Nitrogen availability limits growth in most boreal forests. However, parts of the boreal zone receive significant levels of nitrogen deposition. At the same time, forests are fertilized to increase volume growth and carbon sequestration. No matter the source, increasing nitrogen in the boreal forest ecosystem will influence the resource situation for its primary producers, the plants, with possible implications for their defensive chemistry. In general, fertilization reduces phenolic compound concentrations in trees, but existing evidence mainly comes from studies on young plants. Given the role of the phenolic compounds in protection against herbivores and other forest pests, it is important to know if phenolics are reduced with fertilization also in mature trees. The evergreen Norway spruce is long-lived, and it is reasonable that defensive strategies could change from the juvenile to the reproductive and mature phases. In addition, as the needles are kept for several years, defense could also change with needle age. We sampled current and previous year needles from an N fertilization experiment in a Norway spruce forest landscape in south-central Norway to which N had been added annually for 13 years. We analyzed total nitrogen (N) and carbon (C), as well as low-molecular phenolics and condensed tannins. Needles from fertilized trees had higher N than those from controls plots, and fertilization decreased concentrations of many flavonoids, as well as condensed tannins in current year needles. In previous year needles, some stilbenes and condensed tannins were higher in fertilized trees. In control trees, the total phenolic concentration was almost five times as high in previous year needles compared with those from the current year, and there were great compositional differences. Previous year needles contained highest concentrations of acetophenone and stilbenes, while in the current year needles the flavonoids, and especially coumaroyl-astragalins dominated. Condensed tannins did not differ between current and previous year needles from control trees. In conclusion, the phenolic defense of current year needles of mature P.abies trees was strongly changed upon fertilization. This may imply that nitrogen deposition and forest fertilization leave forests less robust in a time when pests may take advantages of a changing climate.

Fungi isolated from food samples for an efficient stereoselective production of phenylethanols

Abstract Twenty strains of fungi isolated from food samples were screened for the asymmetric reduction of acetophenone. A dimorphic fungus denominated initially as GZ1 and isolated from a carrot sour rot showed very high reduction activities, and based on phenotypic and genetic characteristics was identified as Galactomyces candidus. The only product of the reduction was confirmed to be (R)-1-phenylethanol. The bioreduction conditions mediated by Gal. candidus GZ1 were investigated, and the optimal conditions in terms of acetophenone concentration, temperature, pH media, co-solvent, the charge of inoculum and shaking speed were established. Under the optimal conditions of 1.875 g/L of acetophenone dissolved in DMSO (1.25% v/v), 112.5 g/L of microorganism cells in 100 mM sterile phosphate buffer solution at pH 7.0, 25 °C and 150 rpm, the conversion and enantiomeric excess values after 48 h reaction were 99% and > 99.9%, respectively. A preparative scale reaction of (R)-1-phenylethanol was made and the isolated yield was 82% with > 99.9 e.e.%. Additionally, a study using several substituted acetophenones was carried out, and the advantages and scope of GZ1 in the production of chiral 1-phenylethanols were established. In this sense, good results were obtained with 3′-hydroxyacetophenone and a preparative scale reaction was performed which give (R)-1-(3′-hydroxyphenyl)ethanol (key intermediate of Rivastigmine) with 53% of isolated yield and > 99.9 e.e.%.

Fragaria cascadensis K.E. Hummer: first investigation of volatile organic compounds of fruit

In 2012, Fragaria cascadensis was described as a decaploid strawberry species from the Oregon Cascade Mountains. The objective of this study was to examine the aroma patterns of fruits of this F. cascadensis in contrast to those of F. vesca ssp. bracteata (A. Heller) Staudt and F. virginiana ssp. platypetala (Rydberg) Staudt. These profiles were investigated by immersion stir bar sorptive extraction-gas chromatography-quadrupol mass spectrometry (Imm-SBSE-GC-qMS). The profile of F. cascadensis fruits is clearly separated from that of either F. vesca ssp. bracteata or F. virginiana ssp. platypetala considering 53 detected volatile organic compounds (VOC). Several compounds from different groups were identified: esters, ketones, terpenoids, lactones, furanones, aldehydes, alcohols, acids, and carotenoid derived compounds. Compounds, such as 2-undecanone, phenylmethyl acetate, and 13-damascenone, were found in F. vesca ssp. bracteata, but not in F. virginiana ssp. platypetala or F. cascadensis. In contrast, the compound verbenone was detected only in F. cascadensis, though it did not appear in either the diploid F. vesca ssp. bracteata or in the octoploid F. virginiana ssp. platypetala. Linalool, an important aroma compound, is detectable with low amounts in fruit of F. vesca ssp. bracteata and F. virginiana ssp. platypetala, but with six- to ten-fold times those values in F. cascadensis. A distinctive feature of F. cascadensis is the occurrence of high concentrations of acetophenone.

Optimization of the Production of 1-Phenylethanol Using Enzymes from Flowers of Tea (Camellia sinensis) Plants.

1-Phenylethanol (1PE) can be used as a fragrance in food flavoring and cosmetic industries and as an intermediate in the pharmaceutical industry. 1PE can be synthesized from acetophenone, and the cost of 1PE is higher than the cost of acetophenone. Therefore, it is important to establish an effective and low-cost approach for producing 1PE. Our previous studies found that tea (Camellia sinensis) flowers, which are an abundant and waste resource, contained enzymes that could transform acetophenone to 1PE. In the present study, we extracted crude enzymes from tea flowers and optimized the production conditions of 1PE using response surface methodology. The optimized conditions were an extraction pH of 7.0, a reaction pH of 5.3, a reaction temperature of 55 °C, a reaction time of 100 min, a coenzyme NADPH concentration of 3.75 μmol/mL in the reaction assay, and a substrate acetophenone concentration of 1.25 μmol/mL in the reaction assay. The results provide essential information for future industrial 1PE production using plant-derived enzymes.

Selective hydrogenation of acetophenone to 1-phenyl ethanol over nanofibrous Ag-OMS-2 catalysts

Hydrogenation of acetophenone is interesting and poses a challenge in selective conversion to 1-phenyl ethanol which is a perfumery chemical. It is a complex network of series and parallel reactions. Silver incorporated octahedral molecular sieve catalysts (Ag-OMS-2) were synthesized by precipitation method and employed in the hydrogenation of acetophenone to 1-phenyl ethanol (1-PEA). The catalysts were fully characterized by TGA, XRD, SEM, EDX, TPD, TPR and nitrogen adsorption. They show a nano fibrous structure with very high activity toward hydrogenation. Acetophenone conversion increased with an increase in silver loading from 5 to 15% by wt. 15% Ag-OMS-2 showed the highest activity and 100% selectivity to 1-PEA at hydrogen pressure of 10atm and 160°C. Effects of various parameters such as speed of agitation, type of solvent, catalyst loading, acetophenone concentration, and temperature were studied. A kinetic model was built using Langmuir–Hinshelwood–Hougen–Watson mechanism with weak adsorption of all species leading to a power law model. The activation energy was found to be 12.38kcal/mol.

The Mechanism of Efficient Asymmetric Transfer Hydrogenation of Acetophenone Using an Iron(II) Complex Containing an (S,S)-Ph2PCH2CH═NCHPhCHPhN═CHCH2PPh2 Ligand: Partial Ligand Reduction Is the Key

On the basis of a kinetic study and other evidence, we propose a mechanism of activation and operation of a highly active system generated from the precatalyst trans-[Fe(CO)(Br)(Ph(2)PCH(2)CH═N-((S,S)-C(Ph)H-C(Ph)H)-N═CHCH(2)PPh(2))][BPh(4)] (2) for the asymmetric transfer hydrogenation of acetophenone in basic isopropanol. An induction period for catalyst activation is observed before the catalytic production of 1-phenethanol. The activation step is proposed to involve a rapid reaction of 2 with excess base to give an ene-amido complex [Fe(CO)(Ph(2)PCH(2)CH═N-((S,S)-C(Ph)H-C(Ph)H)-NCH═CHPPh(2))](+) (Fe(p)) and a bis(enamido) complex Fe(CO)(Ph(2)PCH═CH-N-(S,S-CH(Ph)CH(Ph))-N-CH═CHPPh(2)) (5); 5 was partially characterized. The slow step in the catalyst activation is thought to be the reaction of Fe(p) with isopropoxide to give the catalytically active amido-(ene-amido) complex Fe(a) with a half-reduced, deprotonated PNNP ligand. This can be trapped by reaction with HCl in ether to give, after isolation with NaBPh(4), [Fe(CO)(Cl)(Ph(2)PCH(2)CH(2)N(H)-((S,S)-CH(Ph)CH(Ph))-N═CHCH(2)PPh(2))][BPh(4)] (7) which was characterized using multinuclear NMR and high-resolution mass spectrometry. When compound 7 is treated with base, it directly enters the catalytic cycle with no induction period. A precatalyst with the fully reduced P-NH-NH-P ligand was prepared and characterized by single crystal X-ray diffraction. It was found to be much less active than 2 or 7. Reaction profiles obtained by varying the initial concentrations of acetophenone, precatalyst, base, and acetone and by varying the temperature were fit to the kinetic model corresponding to the proposed mechanism by numerical simulation to obtain a unique set of rate constants and thermodynamic parameters.

Experimental Evaluation of a Micromesh Gas/Liquid Reactor for Catalytic Asymmetric Transfer Hydrogenation

AbstractAsymmetric transfer hydrogenation of acetophenone with isopropanol as a hydrogen donor in the presence of a homogeneous catalyst and simultaneous acetone removal by stripping has been performed in a microstructured mesh contactor that consists of parallel metal plates, gaskets and a microstructured mesh so that passages for gas and liquid phases are formed. The function of the micromesh is to stabilize the gas/liquid interface in its pores. The gas and liquid layers are 200 μm thick, while the stainless steel mesh is 50 μm thick, has 76 μm holes and 23 % open area. The removal of the acetone by‐product from the reaction system shortens the required reaction time to achieve high conversion and reduces the enantiomeric excess erosion, as compared to a batch reactor with nitrogen bubbling as well as a continuous reactor without acetone stripping at 30 °C. Lower acetophenone concentration leads to higher enantiomeric excess for both the batch and the mesh reactor, with the latter providing superior performance in all cases. The final enantioselectivity is in the range 80–86 % for the mesh reactor and 70–85 % for the batch reactor. In the mesh reactor, the reaction reaches completion within 15 min. In the batch reactor, it takes more than 3 h to complete the reaction. At 15 °C, the mesh reactor reduces the required time to achieve a certain conversion, but does not affect reaction enantioselectivity, as compared to the batch reactor.

A complex process of the asymmetric reduction of prochiral aromatic ketone by yeast cell with the introduction of an organic solvent as the separation medium

AbstractEnantiomerically pure aromatic alcohols are important chiral building blocks for many single enantiomer pharmaceuticals. The asymmetric reduction of the corresponding prochiral ketone by active yeast cells is one of the most promising synthesis routes for chiral alcohols. But the space‐time productivity is low. To promote the reaction, a complex process of the asymmetric reduction reaction with the introduction of an organic solvent as a separation factor was studied in this work. The asymmetric reduction of acetophenone (ACP) to chiral α‐phenylethyl alcohol (PEA) by Saccharomyces cerevisiae was chosen as the model reaction. It was found that organic solvents with more than 3.5 of logP, such as n‐octane, were favorable for the asymmetric reduction reaction. The yield of the reaction could be remarkably promoted with this technique. The reaction could further be improved by adding ethanol as cosolvent. The initial ACP concentration could be as high as 60 mmol/l, which is about six times larger than that of reacting in a single aqueous phase. Copyright © 2008 Curtin University of Technology and John Wiley & Sons, Ltd.

Electrocarboxylation of Acetophenone to 2‐Hydroxy‐2‐phenylpropionic Acid in the Presence of CO2

AbstractElectrocarboxylation of acetophenone with CO2 to obtain 2‐hydroxy‐2‐phenylpropionic acid was carried out in acetonitrile solution containing 0.1 mol·L−1 tetraethylammonium bromide. Influences of the nature of the electrodes, the working potential, the passed charge and the concentration of acetophenone on the electrocarboxylation were studied. After optimizing the synthetic parameters, the maximal isolated yield reached 73.0% on Mg‐stainless steel couple electrodes under potentiostatic electrolysis until 2.2 F·mol−1 of charge was passed at 25 °C. The reduction of acetophenone was studied by cyclic voltammetry and the mechanism has been proposed on the basis of the results.

Asymmetric Reduction of Acetophenone to α-Phenylethanol by Candida valida under Compressed CO2

The asymmetric reduction of acetophenone to optically active α-phenylethanol catalyzed by cells of Candida valida CICC1444 under compressed CO2 without agitation was investigated. The reaction by the unimmobilized whole cells under compressed CO2 was feasible, and the R-enantiomer of α-phenylethanol was excessive. The yield of α-phenylethanol was greatly influenced by operating parameters such as concentration of acetophenone, cell mass, temperature, and pressure. Compared with the reaction at ambient pressure without agitation, the conversion of acetophenone and yield of α-phenylethanol under compressed CO2 were increased by 20% and 40%, respectively, and the enantiomeric excess of (R)-α-phenylethanol was increased by 5%–10%.

Kinetic characteristic of photooxidation of cypermethrin by organic peroxy radical

The kinetic characteristic of photolysis of cypermethrin (CPM) sensitized by acetophenone (AP) and the effect of probe substance 2,6-Di-tert-butyl-4-methylphenol and tetralin is studied in this report. It showed that the photolysis rate of CPM increases slightly with the increase of AP concentration; photolysis rate of CPM has no relationship with [CPM] itself; The more dipolar moment the solvent has, the more the interaction between radical and solvent has, and the slower the photolysis of CPM is. The addition of radical probe substance confirms that ROO·is more than RO·. The steady-state concentration of ROO·is about 10−8 mol·L−1.

Transmembrane distribution of substrate and product during the bioreduction of acetophenone with resting cells of Saccharomyces cerevisiae.

The average volumetric intracellular concentrations of acetophenone and phenethyl alcohol were determined during the bioreduction of acetophenone using resting cells of Saccharomyces cerevisiae in aqueous solutions at 30 degrees C. The behavior of their distribution coefficients (ratio of intracellular to extracellular concentrations) during the bioreduction process was evaluated with different cell preparation and extracellular conditions. The distribution coefficient of acetophenone was found to be in the range of 2.3-4.0. The distribution coefficient of phenethyl alcohol was found to be in the range of 1.3-1.8. Both the distribution coefficients were correlated significantly only with the physiological state of the resting cells as reflected by the relative cell mass (0.65-1.09). The correlation is approximately linear with the largest slope for the toxic reagent, acetophenone. No significant effects on the distribution coefficients were experimentally observed or were present in a regression analysis for the concentrations of acetophenone (0-0.30% v/v), phenethyl alcohol (0-0.20% v/v), ethanol (1.60-2.25% v/v), the extracellular pH (pH 2-7), or the presence of the salts: KCl, KH2PO4, MgSO4, NaCl, and CaCl2 (each 0-0.1 M) in the medium. Different cell initialization times (0-6 days) and initialization conditions were also included.

Acetophenone tolerance, chemical adaptation, and residual bioreductive capacity of non-fermenting baker's yeast ( Saccharomyces cerevisiae ) during sequential reactor cycles

Bioreduction of acetophenone (ACP) to phenethyl alcohol (PEA) by baker’s yeast (Saccharomyces cerevisiae), which is highly enantioselective, can be carried out entirely in a resting state using stored carbohydrate, suggesting that a high degree of chemical tolerance might be possible. However, viability and catalytic activity of precultured cells decline steeply within 24 h at initial ACP concentrations >0.2% (17 mM). Viability of cells at 0.4% ACP was 1/4 the viability at 0.2% ACP as determined by vital staining, and <1% based on colony-forming ability. this sensitivity was observed in suspensions with a cell content of nearly 30% (w/v). longterm pea production is strongly dependent on viability, indicating that the cumulative yield per batch of cells is maximized by maintaining a very low concentration of substrate (0.2%). however, nonviable cells (cfu ml−1 <1% cells ml−1) can achieve PEA yields up to 1/3 the maximum, an amount representing initial absorption of ACP without further uptake. Regarding population adaptability, when cells surviving the most selective (toxic) concentration of ACP (0.6%) were subcultured in an ACP-free medium and re-reacted, the 24-h percent viabilities (vital staining) and colony-forming frequencies exceeded those of non-selected cells. However, the surviving cells represented only a small fraction (1%) of the recultured progeny. Even at ACP concentrations as low as 0.25% (w/v), surviving cells were unreliable in transmitting and maintaining ACP-tolerance. In addition, there was no evidence that the chemical yield of recultured ACP-tolerant cells (amount of PEA relative to initial amount of ACP) can consistently exceed the maximum yield of an equivalent density of previously unreacted (non-selected) cells. These results indicate that over a broad range of substrate concentrations, rapid replacement of cells may be more cost-effective than maintenance or reuse of viable cells.