Bioconversion Kinetics Research Articles

A combined liquid chromatography – trapped ion mobility – tandem high-resolution mass spectrometry and multivariate analysis approach for the determination of enzymatic reactivity descriptors in biomass hydrolysates

Intermediate products such as oxygenated compounds may interfere with bioconversion kinetics of lignocellulosic biomass into bioethanol. This work presents a multidimensional approach, based on liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), tandem high-resolution mass spectrometry (HRMS/MS), and multivariate analysis, for the identification of enzymatic reactivity descriptors in 22 industrial biomass samples, called hydrolysates. The first part of the study is dedicated to the improvement of the chemical diversity assessment of the hydrolysates through an original three-dimensional Van Krevelen diagram displaying the double bond equivalent (DBE) as third dimension. In a second part, the evaluation of data by multivariate data analysis allowed the discrimination of sample according to the biomass type and the level of enzymatic reactivity. In the last part, a potential descriptor of low enzymatic reactivity was selected and used in a case study. An in-depth structural analysis was performed on the feature annotated as carbohydrate derivative. Considering the intricate fragmentation spectrum exhibited by the selected feature, trapped ion mobility was employed to enhance separation prior to the HRMS/MS experiments. This final step improved data interpretation and increased the identification confidence level leading to the characterization of xylotriose, 3,5-dimethoxy-4-hydroxybenzaldehyde and 4-hydroxy-3-methoxy-cinnamaldehyde. This is the first study to present an untargeted multidimensional approach for the identification of enzymatic hydrolysis inhibitors in industrial hydrolysate samples.

Constitutively solvent-tolerant Pseudomonas taiwanensis VLB120∆ C∆ ttgV supports particularly high-styrene epoxidation activities when grown under glucose excess conditions.

Solvent-tolerant bacteria represent an interesting option to deal with the substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120Δ C. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120Δ C and VLB120Δ CΔ ttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance. Substrate inhibition kinetics was identified but was attenuated in two-liquid phase bioreactor setups. StyA fusion to the enhanced green fluorescent protein enabled precise enzyme level monitoring without affecting epoxidation activity. Glucose limitation compromised styAB expression and specific activities (30-40 U/g CDW for both strains), whereas unlimited batch cultivation enabled specific activities up to 180 U/g CDW for VLB120Δ CΔ ttgV strains, which is unrivaled for bioreactor-based whole-cell oxygenase biocatalysis. These extraordinarily high specific activities of constitutively solvent-tolerant P. taiwanensis VLB120∆ C∆ ttgV could be attributed to its high metabolic capacity, which also enabled high expression levels. This, together with the high product yields on glucose and biomass obtained qualifies the VLB120∆ ttgV strain as a highly attractive tool for the development of ecoefficient oxyfunctionalization processes and redox biocatalysis in general.

A new hybrid photobioreactor design for microalgae culture

The objective of this study was to develop a new design of photobioreactor for microalgae culture. Hydrodynamic characterization, growth kinetics, removal of carbon dioxide and oxygen release rates, carbon and energy balances, and requirements of the surface area were determined. The results showed that the hybrid photobioreactor presented high hydrodynamic performance (mixing time of 98s, circulation time of 39s, Reynolds number of 2500 and empty bed residence time of 60s). The carbon dioxide bioconversion kinetics for Scenedesmus obliquus CPCC05 showed maximum specific growth rates of 0.96 d−1, and a maximum cell biomass of 2.8 kg/m³. In parallel, an average carbon dioxide conversion and oxygen release rates of 45.32 kgCO2/m³/d and 33.98 kgO2/m³/d were obtained, respectively. The carbon balance indicated that a small fraction (1.28%) of the carbon dioxide is fixed into a biomass form. Volatile organic compounds (82.75%) were the main products formed in the photobioreactor. The energy balance provided a net energy ratio of 2.49. Finally, this photobioreactor configuration requires a volumetric surface area in order to 0.22 m²/m³.

Isolation and Characterization of a Human Intestinal Bacterium Eggerthella sp. AUH-JLD49s for the Conversion of (-)-3'-Desmethylarctigenin.

Arctiin is the most abundant bioactive compound contained in the Arctium lappa plant. In our previous study, we isolated one single bacterium capable of bioconverting arctigenin, an aglycone of arctiin, to 3'-desmethylarctigenin (3'-DMAG) solely. However, to date, a specific bacterium capable of producing other arctiin metabolites has not been reported. In this study, we isolated one single bacterium, which we named Eggerthella sp. AUH-JLD49s, capable of bioconverting 3'-DMAG under anaerobic conditions. The metabolite of 3'-DMAG by strain AUH-JLD49s was identified as 3'-desmethyl-4'-dehydroxyarctigenin (DMDH-AG) based on electrospray ionization mass spectrometry (ESI-MS) and 1H and 13C nuclear magnetic resonance spectroscopy. The bioconversion kinetics and bioconversion capacity of strain AUH-JLD49s were investigated. In addition, the metabolite DMDH-AG showed an inhibitory effect on cell growth of human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231.

Efficient bioconversion of L-glutamate to γ-aminobutyric acid by Lactobacillus brevis resting cells.

This work investigated the efficient bioconversion process of L-glutamate to GABA by Lactobacillus brevis TCCC 13007 resting cells. The optimal bioconversion system was composed of 50g/L 48h cultivated wet resting cells, 0.1mM pyridoxal phosphate in glutamate-containing 0.6M citrate buffer (pH 4.5) and performed at 45°C and 180rpm. By 10h bioconversion at the ratio of 80g/L L-glutamic acid to 240g/L monosodium glutamate, the final titer of GABA reached 201.18g/L at the molar bioconversion ratio of 99.4%. This process presents a potential for industrial and commercial applications and also offers a promising feasibility of continuous GABA production coupled with fermentation. Besides, the built kinetics model revealed that the optimum operating conditions were 45°C and pH 4.5, and the bioconversion kinetics at low ranges of substrate concentration (0<S<80g/L) was assumed to follow the classical Michaelis-Menten equation.

Long-chain fatty acid feeding frequency in anaerobic codigestion impacts syntrophic community structure and biokinetics

This study investigated the impacts of long-chain fatty acid (LCFA) feeding frequencies on microbial community structure, bioconversion kinetics, and process stability during anaerobic codigestion. Parallel laboratory-scale anaerobic codigesters fed with dairy cattle manure were either pulse-fed every two days or continuously-fed daily, respectively, with oleate (C18:1) in incremental step increases over 200 days up to 64% of the influent chemical oxygen demand (COD). The effluent acetate concentration exceeded 3000 mg/L in the continuous-fed codigester at the highest oleate loading rate, but remained below 100 mg/L in the pulse-fed codigester at the end of its 48-hr oleate feed cycle. Maximum substrate conversion rates of oleate (qmax, oleate) and acetate (qmax, acetate) were significantly higher in the pulse-fed codigester compared to the continuous-fed codigester. 16S rRNA gene amplicon sequencing showed that Bacteria and Archaea community profiles diverged based on the codigester LCFA feeding pattern and loading rate. LCFA-degrading Syntrophomonas bacteria were significantly enriched in both LCFA codigesters relative to the control digester. The pulse-fed codigester had the highest community fraction of Syntrophomonas 16S rRNA genes by the end of the experiment with 43% of Bacteria amplicon sequences. qmax, oleate and qmax, acetate values were both significantly correlated to absolute concentrations of Syntrophomonas and Methanosaeta 16S rRNA genes, respectively. Multiple-linear regression models based on the absolute abundance of Syntrophomonas and Methanosaeta taxa provided improved predictions of oleate and acetate bioconversion kinetics, respectively. These results collectively suggest that pulse feeding rather than continuous feeding LCFA during anaerobic codigestion selected for higher microbial bioconversion kinetics and functional stability, which were related to changes in the physiological diversity and adaptive capacity of syntrophic and methanogenic communities.

Anaerobic codigester feeding pattern drives long-chain fatty acid bioconversion kinetics and syntrophic community structure

Anaerobic codigester feeding pattern drives long-chain fatty acid bioconversion kinetics and syntrophic community structure

Proteomic analysis of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis

This study investigated the impacts of long-chain fatty acid (LCFA) feeding frequencies on microbial community structure, bioconversion kinetics, and process stability during anaerobic codigestion. Parallel laboratory-scale anaerobic codigesters fed with dairy cattle manure were either pulse-fed every two days or continuously-fed daily, respectively, with oleate (C18:1) in incremental step increases over 200 days up to 64% of the influent chemical oxygen demand (COD). The effluent acetate concentration exceeded 3000 mg/L in the continuous-fed codigester at the highest oleate loading rate, but remained below 100 mg/L in the pulse-fed codigester at the end of its 48-hr oleate feed cycle. Maximum substrate conversion rates of oleate (qmax, oleate) and acetate (qmax, acetate) were significantly higher in the pulse-fed codigester compared to the continuous-fed codigester. 16S rRNA gene amplicon sequencing showed that Bacteria and Archaea community profiles diverged based on the codigester LCFA feeding pattern and loading rate. LCFA-degrading Syntrophomonas bacteria were significantly enriched in both LCFA codigesters relative to the control digester. The pulse-fed codigester had the highest community fraction of Syntrophomonas 16S rRNA genes by the end of the experiment with 43% of Bacteria amplicon sequences. qmax, oleate and qmax, acetate values were both significantly correlated to absolute concentrations of Syntrophomonas and Methanosaeta 16S rRNA genes, respectively. Multiple-linear regression models based on the absolute abundance of Syntrophomonas and Methanosaeta taxa provided improved predictions of oleate and acetate bioconversion kinetics, respectively. These results collectively suggest that pulse feeding rather than continuous feeding LCFA during anaerobic codigestion selected for higher microbial bioconversion kinetics and functional stability, which were related to changes in the physiological diversity and adaptive capacity of syntrophic and methanogenic communities.

Improved Gastrodin Production of Biotransformation Conditions by Cultured Cells Armillaria luteo-virens Sacc and the Anti-inflammatory Activity In Vivo

Gastrodin (GAS), the main bioactive component of G. elata Blume, has important pharmaceutical and functional activities. The aim of this study is to produce GAS from p-2-hydroxybenzyl alcohol (HBA) through biotransformation. The conversion of exogenous HBA into GAS compound was conducted using cell suspension cultures of Armillaria luteo-virens Sacc. The bioconversion conditions were fully optimized with response surface methodology (RSM), turning out that the optimal transformation conditions composed of 3 mg/mL HBA, 6.5 g/30 mL inoculums level, 1.5% Tween 80, pH 4.5, and transformation temperature at 23°C. Under the optimized conditions, the conversion productivity of GAS reached the highest value (5.65 ± 0.45 mg/L). Verified experiments further validated that the optimized conditions were suitable for predicting the actual process of HBA transformation in the resting-cell system. The bioconversion kinetics model was as well simulated with Michaelis–Menten equation, which showing the suitability. The present study proposed the biotransformation pathway of HBA into GAS by resting cells transformation, indicating that the biotransformation process involved glucosylation reaction. Furthermore, Imprinting Control Region (ICR) mice in vivo demonstrated that the identified gastrodin possessed a significant anti-inflammatory activity. The fundamental data in the present work provides an efficient way to produce GAS through the whole-cells biocatalysis.

Innovative two-stage mesophilic/thermophilic anaerobic degradation of sonicated sludge: performances and energy balance.

This study investigates for the first time, on laboratory scale, the possible application of an innovative enhanced stabilization process based on sequential mesophilic/thermophilic anaerobic digestion of waste-activated sludge, with low-energy sonication pretreatment. The first mesophilic digestion step was conducted at short hydraulic retention time (3-5 days), in order to favor volatile fatty acid production, followed by a longer thermophilic step of 10 days to enhance the bioconversion kinetics, assuring a complete pathogen removal. The high volatile solid removals, up to 55%, noticeably higher compared to the performances of a single-stage process carried out in same conditions, can guarantee the stability of the final digestate for land application. The ultrasonic pretreatment influenced significantly the fatty acid formation and composition during the first mesophilic step, improving consequently the thermophilic conversion of these compounds into methane. Methane yield from sonicated sludge digestion reached values up to 0.2 Nm(3)/kgVSfed. Positive energy balances highlighted the possible exploitation of this innovative two-stage digestion in place of conventional single-stage processes.

Kinetics of CO conversion into H2 by Carboxydothermus hydrogenoformans

The objective of this study was to improve the biological water-gas shift reaction for producing hydrogen (H(2)) by conversion of carbon monoxide (CO) using an anaerobic thermophilic pure strain, Carboxydothermus hydrogenoformans. Specific hydrogen production rates and yields were investigated at initial biomass densities varying from 5 to 20 mg volatile suspended solid (VSS) L(-1). Results showed that the gas-liquid mass transfer limits the CO conversion rate at high biomass concentrations. At 100-rpm agitation and at CO partial pressure of 1 atm, the optimal substrate/biomass ratio must exceed 5 mol CO g(-1) biomass VSS in order to avoid gas-liquid substrate transfer limitation. An average H(2) yield of 94 ± 3% and a specific hydrogen production rate of ca. 3 mol g(-1) VSS day(-1) were obtained at initial biomass densities between 5 and 8 mg VSS(-1). In addition, CO bioconversion kinetics was assessed at CO partial pressure from 0.16 to 2 atm, corresponding to a dissolved CO concentration at 70°C from 0.09 to 1.1 mM. Specific bioactivity was maximal at 3.5 mol CO g(-1) VSS day(-1) for a dissolved CO concentration of 0.55 mM in the culture. This optimal concentration is higher than with most other hydrogenogenic carboxydotrophic species.

Kinetic analysis of 11α-hydroxylation of steroids by Rhizopus nigricans

Optimization of bioconversion of 16,17α-epoxyprogesterone by Rhizopus nigricans TJ 108 was investigated by means of uniform design. Batch cell growth and bioconversion kinetics were simulated under optimal conditions. Contois equation was used in the kinetics study of fungal growth on glucose. Simulation of bioconversion process was done at a constant value of substrate concentration according to the reaction mechanism. It was demonstrated that the above model satisfactorily described the kinetic behaviors of cell growth and bioconversion of the filamentous fungi.

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 micromol per min per g cells (dry weight), the absence of limitations by O(2) and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.

Bioisomerization kinetics of γ-HCH and biokinetics of Pseudomonas aeruginosa degrading technical HCH

Bioconversion kinetics of γ-HCH and biological growth kinetics of Pseudomonas aeruginosa degrading technical HCH was investigated in flask experiments under aerobic condition. Degradation studies for various isomers of technical HCH were also performed. The biodegradation of the most persistent β-HCH and δ-HCH isomers was also observed. At lower technical HCH concentrations (1–10 mg/l), above 99% of the degradation was observed whereas at higher concentration (20–50 mg/l), the degradation efficiency was dropped. This may be due to inhibitory effect of technical HCH at higher concentrations. The technical HCH inhibitory behavior and bacterial growth kinetics could be correlated well by simple Haldane's growth kinetic model. The value of decay and yield coefficient was also determined. Further experimental results also revealed that degradation of the γ-HCH isomer is faster (after incubation of 5 days) in the presence of other isomers of technical HCH, whereas γ-HCH alone does not degrade even after a long period of incubation (14 days), but isomerizes to α-HCH isomer by the P. aeruginosa. The rate constant for the bioisomerization was found to be 0.0474 day −1. The above estimated parameters (maximum specific growth rate, half saturation coefficient, inhibition constant, decay coefficient, yield coefficient and bioisomerization rate) are invariably required for the design and simulation of batch and continuous bioreactors treating HCH containing wastewaters.

Analysis of Two-Liquid-Phase Multistep Biooxidation Based on a Process Model: Indications for Biological Energy Shortage

A process model for whole-cell biocatalysis in a two-liquid-phase system including cell growth and bioconversion kinetics was developed. The reaction considered is the kinetically controlled multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde catalyzed by recombinant Escherichia coli expressing the Pseudomonas putida genes encoding xylene monooxygenase (XMO). XMO catalyzes the successive oxygenation of one methyl group of xylenes to corresponding alcohols, aldehydes, and acids. The biocatalytic process includes cells growing in fed-batch mode and a two-liquid-phase system consisting of bis(2-ethylhexyl)phthalate as organic carrier solvent and an aqueous minimal medium in a phase ratio of 1:1. The process model comprises a description of the bioconversion kinetics, mass transfer kinetics, cell growth, and mass balances for both the aqueous and the organic phase. Bioconversion kinetics and consistent process simulation indicated the occurrence of direct substrate uptake from the organic phase and provided evidence for a pH-influenced competition for NADH between XMO and the respiratory chain with its consequential impact on bioconversion and cell growth. For the simulation of such differential NADH limitation, a pH-dependent feedback inhibition of the NADH consuming bioconversions was introduced as a modeling tool, which allowed good simulations of biotransformation experiments performed at varying pH, scale, and initial substrate concentration. Moreover, modeling indicated a product inhibition in the second oxidation step, which could be confirmed experimentally. A sensitivity analysis for the aqueous−organic mass transfer coefficient showed that this transfer is not critical for the process performance and emphasized the efficient substrate-cell transfer in the investigated two-liquid-phase process. Based on a process model, this study provides an in-depth analysis of a biooxidation process based on growing cells with indications for biological energy shortage as a limiting factor.

Metal Speciation Dynamics and Bioavailability. 2. Radial Diffusion Effects in the Microorganism Range

The free ion activity model for the biouptake of metals from complex media is limited to cases where mass transfer is not flux determining. This paper follows a previous paper (Van Leeuwen, H. P. Environ. Sci. Technol. 1999, 33, 3743) where speciation dynamics and bioavailability of metals are analyzed in terms of bioconversion kinetics and simultaneous metal transport in the medium coupled with dissociation kinetics. Such analysis shows under what conditions labile complex species contribute to the biouptake process or, equivalently, under what conditions the free ion activity model is not obeyed. The present work addresses the theoretical extension of the expressions for the metal flux in the medium by a radial diffusion term so that these are also applicable in the microorganism size range. The transition from macroscopic to microscopic surfaces affects not only the nature of the flux but also the extent of lability of complex species (Van Leeuwen, H. P.; Pinheiro, J. P. J. Electroanal. Chem. 1999, 471, 55), and this can have a dramatic influence on the rate of biouptake of metal ions. Labilities of metal complexes and the ensuing limiting metal fluxes are therefore systematically analyzed for various dimensions of the uptaking surface. Different conditions of bioaffinities and bioconversion capacities are considered, and a number of examples of metal complexes with specified kinetic features are discussed.

Metal Speciation Dynamics and Bioavailability: Inert and Labile Complexes

The free-ion activity model for the biouptake of metals from complex media is limited to cases where mass transfer is not flux-determining. This paper considers the simultaneous effects of bioconversion kinetics and metal transport in the medium coupled with metal complex dissociation kinetics. For the two kinetically limiting situations of inert and fully labile complexes, the bioavailabilities of bioinactive metal complexes are analyzed under conditions where (i) the actual biouptake follows a Michaelis−Menten type of steady-state flux and (ii) the supply of free metal is governed by diffusion of free metal or coupled diffusion of the different labile metal species. The resulting steady-state fluxes are given in terms of two fundamental quantities, i.e., the relative bioaffinity parameter (a) and the ratio between the limiting uptake flux and the limiting transport flux (b). For labile complexes, these variables are differentiated by a complexation parameter defined by the ratio between the free metal ion activity and the total labile metal activity. Limits of the uptake flux for extreme values of the bioaffinity parameter a and the limiting flux ratio b are easily derived from the general flux expression. The analysis precisely shows under what conditions labile complex species contribute to the biouptake process or, equivalently, under what conditions the free-ion activity model is not obeyed.

Immobilized Enzyme Three-Phase Reactors for Oxidation of Cephalosporin C

The enzymatic oxidation of Cephalosporin C (CEPHC) was catalyzed by D-aminoacid oxidase, from the red yeast Trigonopsis variabilis, immobilized on Duolite A365. The study was performed in two different three phase bioreactors, gas-liquid-solid (immobilized enzyme): the fluidized-bed batch reactor, fed continuously with oxygen and discontinuously with CEPHC, and the UF-membrane reactor continuously fed with both substrates. Only the first reactor allowed significant product yield (>70%) while the second was a very useful tool for laboratory investigation of both bioconversion kinetics and enzyme stability.Optimum reaction temperature was 15d`C for the control of CEPHC spontaneous degradation (roughly 15% in 30 h), and enzyme deactivation (half-life greater than 30 h). Immobilization improved (one order of magnitude longer half-life) enzyme resistance to mechanical stresses induced by liquid stirring and gas bubbling. Roughly 0.04g of CEPHC was adsorbed per gram of enzyme carrier. The limiting step in oxygen transfer was the gas to liquid transport. In order to attain kinetic control of the bioconversion the mildest conditions were atmospheric gas pressure and oxygen flow rate equal to 2 × 10 2NmL/s per mL of liquid phase.

The physicochemical properties, plasma enzymatic hydrolysis, and nasal absorption of acyclovir and its 2'-ester prodrugs.

A series of 2'-(O-acyl) derivatives of 9-(2-hydroxyethoxymethyl)guanine (acyclovir) was synthesized by acid anhydride esterification. Aqueous solubilities in isotonic phosphate buffer (pH 7.4), partition coefficients in 1-octanol/phosphate buffer, and hydrolysis kinetics in rat plasma were determined. The ester prodrugs showed consistent increases in lipophilicity with corresponding decreases in aqueous solubility as a function of side-chain length. The bioconversion kinetics of the prodrugs appear to depend on both the apolar and the steric nature of the acyl substituents. When perfused through the rat nasal cavity using the in situ perfusion technique, acyclovir showed no measurable loss from the perfusate. Nasal uptake of acyclovir prodrugs, on the other hand, were moderately improved. Furthermore, the extent of nasal absorption appears to depend on the lipophilicity of the prodrugs in the descending order hexanoate > valerate > pivalate > butyrate. Simultaneous prodrug cleavage by nasal carboxylesterase was also noted in the case of hexanoate.

Relative biochemical reactivity of three hexachlorocyclohexane isomers

The rate and mechanisms of degradation of three hexachlorocyclohexane (HCH) isomers were studied with rat liver cytochrome P450, which is known to react with chlorinated compounds in the presence and absence of molecular oxygen. In addition, P450 is produced from HCH metabolites which are found aerobically and anaerobically during microbial biodegradation of this compound in a complex soil system. Conversion assays with P450 were carried out with α-, β-, and γ-HCH. Hexachloroethane served as a reference compound for bioconversion kinetics. Under anaerobic conditions, γ-HCH was readily dechlorinated ( r 0 = 0.31 nmol/min · nmol cyt · P450) to tetrachlorocyclohexene and monochlorobenzene. γ-HCH was also reductively dechlorinated, but at a much slower rate ( r 0 = 0.03 nmol/min · nmol cyt.P450). Both substrates followed Michaelis-Menten kinetics (γ-HCH: V max = 1.87 nmol/min · mg and K m = 47 μmol/liter; γ-HCH: V max = 0.24 nmol/min · mg and K m = 201 μmol/liter). Under aerobic conditions, bioconversions of γ-HCH ( r 0 = 0.05 nmol/min · nmol cyt. P450) and α-HCH ( r 0 smaller than detection limit) were slower. β-HCH proved to be recalcitrant under both aerobic and anaerobic conditions. The differences in bioconversion rates between the three HCH isomers could be related to the differences in spatial chlorine configuration of the three isomers. β-HCH recalcitrance was explained by the absence of axial-orientated chlorines.