Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Experimental evaluation of a model for cometabolism: Prediction of simultaneous degradation of trichloroethylene and methane by a methanotrophic mixed culture

A model for cometabolism is verified experimentally for a defined methanotrophic mixed culture. The model includes the effects of cell growth, endogenous cell decay, product toxicity, and competitive inhibition with the assumption that cometabolic transformation rates are enhanced by reducing power obtained from oxidation of growth substrates. A theoretical transformation yield is used to quantify the enhancement resulting from growth substrate oxidation. A systematic method for evaluating model parameters independently is described. The applicability of the model is evaluated by comparing experimental data for methanotrophic cometabolism of TCE with model predictions from independently measured model parameters. Propagation of errors is used to quantify errors in parameter estimates and in the final prediction. The model successfully predicts TCE transformation and methane utilization for a wide range of concentrations of TCE (0.5 to 9 mg/L) and methane (0.05 to 6 mg/L). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 492-501, 1997.     

Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate

A kinetic model to describe the degradation of phenol and cometabolictransformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate(SG) has been developed and validated experimentally. The integrated modelaccounts for cell growth, toxicity of 4-cp, cross-inhibitions among the threesubstrates, and the different roles of the specific growth substrate (phenol)and the conventional carbon source (SG) in the cometabolism of 4-cp. In thisternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate. Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)^-1) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is notdirectly affected by the presence of SG. The competitive inhibition coefficient of 4-cp to phenol degradation (K_i,cp) and that of phenol to 4-cp transformation (K_i,ph) were determined to be 6.49 mg l^-1 and 0.193 mg l^-1, respectively, indicatingthat phenol imposes much larger competitive inhibition to 4-cp transformation than the converse. The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.     

Microbial Maintenance: A Critical Review on Its Quantification

Microbial maintenance is an important concept in microbiology. Its quantification, however, is a subject of continuous debate, which seems to be caused by (1) its definition, which includes nongrowth components other than maintenance; (2) the existence of partly overlapping concepts; (3) the evolution of variables as constants; and (4) the neglect of cell death in microbial dynamics. The two historically most important parameters describing maintenance, the specific maintenance rate and the maintenance coefficient, are based on partly different nongrowth components. There is thus no constant relation between these parameters and previous equations on this subject are wrong. In addition, the partial overlap between these parameters does not allow the use of a simple combination of these parameters. This also applies for combinations of a threshold concentration with one of the other estimates of maintenance. Maintenance estimates should ideally explicitly describe each nongrowth component. A conceptual model is introduced that describes their relative importance and reconciles the various concepts and definitions. The sensitivity of maintenance on underlying components was analyzed and indicated that overall maintenance depends nonlinearly on relative death rates, relative growth rates, growth yield, and endogenous metabolism. This quantitative sensitivity analysis explains the felt need to develop growth-dependent adaptations of existing maintenance parameters, and indicates the importance of distinguishing the various nongrowth components. Future experiments should verify the sensitivity of maintenance components under cellular and environmental conditions.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

Comparison of TCE Transformation Abilities of Methane- and Propane-Utilizing Microorganisms

Subsurface microorganisms from McClellan Air Force Base (AFB) were grown in batch aquifer microcosms on methane, propane, and butane to evaluate the potential for aerobic trichloroethylene (TCE) cometabolism. Microorganisms stimulated on all three substrates indicated the existence of a subsurface microbial community capable of utilizing alkanes as growth substrates. Initial growth substrate utilization lag periods of 2 weeks for methane and 3 weeks for propane and butane were observed. Methane- and propane-utilizers were active toward TCE cometabolism, whereas butane-utilizers showed no ability to transform TCE. Gradually increasing TCE concentrations were effectively transformed with uniform additions of methane and propane for up to 1 year. TCE was transformed most rapidly during active methane utilization, and continued at a slower rate for approximately 1 week after methane was consumed. Propane microcosms maintained first-order TCE transformation for up to 4 weeks after propane was consumed. The microbial communities remained active toward primary substrate utilization as the TCE concentration was gradually increased. Both methane- and propane-utilizers showed positive correlations between TCE transformation rates and primary substrate utilization rates. Observed maximum TCE transformation yields were 0.068 g TCE/g methane and 0.048 g TCE/g propane. The methane-utilizers also transformed chloroform (CF) but not 1,1,1-trichloroethane (1,1,1-TCA). Propane-utilizers transformed both CF and 1,1,1-TCA, indicating they were better suited for cometabolizing chlorinated aliphatic hydrocarbon mixtures in the McClellan AFB subsurface.     

Macrokinetic model for methylotrophic Pichia pastoris based on stoichiometric balance

A macrokinetic model for Pichia pastoris expressing recombinant human serum albumin is proposed. The model describes the balances of some key metabolites, ATP and NADH, during glycerol and methanol metabolism. In the glycerol growth phase, the metabolic pathways mainly include phosphorylation, glycolysis, tricarboxylic acid cycle, and respiratory chain. In the methanol growth phase, methanol is oxidized to formaldehyde at first. Then, while a part of formaldehyde is oxidized to formate, the rest is condensed with xylulose-5-monophosphate to form glyceraldehyde-3-phosphate, and further assimilated to form cell constituents. The metabolic pathways following glyceraldehyde-3-phosphate were assumed to be similar to those in the glycerol growth phase. Based on the model, the macrokinetic bioreaction rates such as the specific substrate consumption rate, the specific growth rate, the specific acetyl-CoA formation rate as well as the specific oxygen uptake rate are obtained. The specific substrate consumption rate and the specific growth rate are then coupled into a bioreactor model such that the relationship between substrate feeding rates and the main state variables, i.e., the medium volume, the concentrations of the biomass, the substrate, and the product, is set up. Experimental results demonstrate that the model can describe the cell growth and the protein production with reasonable accuracy.     

From a descriptive toward an explicative growth-based model on immature Oulema duftschmidi (Coleoptera: Chrysomelidae) development at different temperatures

The developmental rates of Oulema duftschmidi (Redtenbacher) eggs, larvae, and pupae were studied at different constant temperatures. A linear regression model was fitted to the data obtained in this and in a previous study within a temperature range where the rate proportionally increases with temperature. Ratios of SEs to the mean thermal constant and to the mean developmental threshold indicated that reliable estimates have been obtained for the three life stages. Within the framework provided by the metabolic theory of ecology, a growth-based model was evaluated to explain developmental rates in the entire temperature range permissive of development of the three life stages. The model is based on component functions describing growth patterns through time, temperature-dependent consumption rates of biomass, transformation of consumed food into body biomass change, and respiration rates with respect to temperature. Experimental data were used for the selection and validation of models and for the estimation of the parameters of different regression models. Limitations and opportunities for using the growth-based model to explain developmental rates are discussed. An empirical function was used to describe the variability of developmental rates.     

Laboratory evaluation of two bioenergetics models applied to yellow perch: identification of a major source of systematic error

Laboratory growth and food consumption data for two size classes of age 2 year yellow perch Perca flavescens , each fed on two distinct feeding schedules at 21° C, were used to evaluate the abilities of the Wisconsin (WI) and Karas–Thoresson (KT) bioenergetics models to predict fish growth and cumulative consumption. Neither model exhibited consistently better performance for predicting fish body masses across all four fish size and feeding regime combinations. Results indicated deficiencies in estimates of resting routine metabolism by both models. Both the WI and KT models exhibited errors for predicting growth rates, which were strongly correlated with food consumption rate. Consumption-dependent prediction errors may be common in bioenergetics models and are probably the result of deficiencies in parameter values or assumptions within the models for calculating energy costs of specific dynamic action, feeding activity metabolism or egestion and excretion. Inter-model differences in growth and consumption predictions were primarily the result of differences in egestion and excretion costs calculated by the two models. The results highlighted the potential importance of parameters describing egestion and excretion costs to the accuracy of bioenergetics model predictions, even though bioenergetics models are generally regarded as being insensitive to these parameters. The findings strongly emphasize the utility and necessity of performing laboratory evaluations of all bioenergetics models for assurance of model accuracy and for facilitation of model refinement.     

Cometabolism of methyl tert-butyl ether (MTBE) with alkanes

The release of methyl tert-butyl ether (MTBE) to the environment, mainly from damaged gasoline underground storage tanks or distribution systems spills, has provoked extended groundwater pollution. Biological treatments are, in general, a good alternative for bioremediation of polluted sites; however, MTBE elimination from environment has constituted a challenge because of its chemical structure and physicochemical properties. The combination of a stable ether link and the branched moiety hinder biodegradation. Initial studies found MTBE to be highly recalcitrant but, in the last decade, reports of its biodegradation have been published first under aerobic conditions and just recently under anaerobic conditions. Microbial MTBE degradation is characterized by bacteria having low growth rates (0.35 day⁻¹) and biomass yields (average value 0.24 g biomass/g MTBE). Alternatively, cometabolism (defined as the transformation of a non-growth substrate in the obligate presence of a growth substrate), has been considered since it uncouples biodegradation of the contaminant from growth, reducing the long adaptation and propagation period. This period has been reported to be of several months in systems where it is degraded as sole carbon source. Cometabolic degradation rates are between 0.3 and 61 nmol/min/mg protein (in the same range of direct aerobic metabolism). However, a major concern in MTBE cometabolism is that the accumulation of tert-butyl alcohol (TBA) may, under certain cases, result in an incomplete site cleanup. This paper reviews in detail the implicated enzymes and field treatments for the cometabolism of MTBE degradation with alkanes as growth substrates.     

Kinetic model for microbial uptake of insoluble solid-state substrate

A kinetic model for anaerobic digestion of insoluble solid-state substrates was developed. Rate equations for cell growth and substrate consumption were derived based on the assumption that the microorganisms assimilate the substrate mainly at the point of contact where they grow. The model emphasizes effects of substrate particle size, organic loading, and cell concentration on the rates of cell growth and substrate utilization. Batch digestion of a stearic acid emulsion with a mean particle size of 2.0 mum and a biological sludge was conducted at 30 and 37 degrees C to verify the proposed model. Agreement between the experimental and calculated results indicated the validity of the model for describing the microbial degradation of insoluble solid-state substrates. Further examinationof the model revealed that with low cell substrate affinity or at low cell concentration, it coincided with a Michaelis-Menten type kinetics in which the effect of particle size was taken into consideration.     

Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production

A mathematical model describing the kinetics of continuous production of single cell protein from cheese whey using Kluyveromyces fragilis was developed from the basic principles of mass balance. The model takes into account the substrate utilization for growth and maintenance and the effect of substrate concentration and cell death rate on the net cell growth and substrate utilization during the fermentation process. A lactose concentration below 1.91 g/L limited growth of yeast cells whereas a lactose concentration above 75 g/L inhibited the growth of the yeast. The model was tested using experimental data obtained from a continuous system operated at various retention times (12, 18 and 24 h), mixing speeds (200, 400 and 600 rpm) and air flow rates (1 and 3 vvm). The model was capable of predicting the effluent cell and substrate concentrations with R2 ranging from 0.95 to 0.99. The viable cell mass and lactose consumption ranged from 1.3 to 34.3 g/L and from 74.31% to 99.02%, respectively. A cell yield of 0.74 g cell/g lactose (close to the stoichiometric value of 0.79 g cell/g lactose) was achieved at the 12 h retention time-3 vvm air flow rate-600 rpm mixing speed combination. The total biomass output (viable and dead cells) at this combination was 37 g/L.     

Evidence for inhibitory substrate interactions during cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

Abstract Acinetobacter sp. strain 4-CB1 cometabolized 3,4-dichlorobenzoate (3,4-DCB), via 3-chloro-4-hydroxybenzoate (3-C-4-OHB) and 4-carboxy-1,2-benzoquinone, in the presence of 4-chlorobenzoate (4-CB) as a growth substrate. In resting cell incubations, 3,4-DCB acted competitively as an inhibitor with 4-CB metabolism, and as a substrate inhibitor of its own metabolism. The inhibitor constant K _i and the affinity constant K _m were 800 and 181 μM, respectively with a maximal rate of 3,4-DCB disappearance of 18.8 nmol 3,4-DCB min⁻¹ (mg protein)⁻¹, in resting cells incubated solely with 3,4-DCB. Resting cells were less tolerant to 3,4-DCB than growing cells, as noted from the inhibition constants ( K _i). Moreover, 3-C-4-OHB competitively inhibited 4-hydroxybenzoate monooxygenase by acting as a pseudosubstrate ( K _i= 7.3 μ M). The next sequential intermediate, 4-carboxy-1,2-benzoquinone, uncompetitively inhibited 4-CB metabolism in resting cell incubations. Thus, 3,4-DCB inhibited its own cometabolism as well as metabolism of 4-CB in Acinetobacter sp. strain 4-CB1.     

A Carbon-balance Model of Stand Growth: a Derivation Employing Pipe-model Theory and the Self-thinning Rule

A carbon-balance model of the growth of an even-aged, self-thinning,mono-specific stand of trees is derived using a structural frameworkbased on pipe-model theory. Within the pipe-model framework,dry matter arising from extension of roots and shoots is separablefrom that arising from the cross-sectional expansion of stems.This permits the derivation of models describing the growthof average stem length, total basal area, and total volume ofthe stand. Variations of these models are described for twosituations: (1) where the annual rates of substrate productionand feeder-root turnover can be assumed constant over time;and (2) where these rates are expected to change over time,such as in polluted environments. The model describing the growthof stand volume for the first situation fits published yieldtables. Growth-rate models applicable to individual trees arealso described. Carbon-balance model, dry-matter partitioning, pipe-model theory, stand growth, self-thinning     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1.

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cometabolism in biological oxidation of synthetic compounds

A significant portion of the total biodegradative activity toward synthetic compounds may involve cometabolism; thus, the latter represents an important type of microbial process. A mixed microbial population acclimated to highstrength industrial wastewater degraded about 90% of the organic carbon present in the wastewater. A reasonable agreement in the carbon balance provided clear evidence for the biodegradability of residual organic species. Based on the growth expression of Haldane to account for substrate inhibition, a substrate utilization model to quantify the role of cometabolism coupled with cellular maintenance is proposed. The kinetic parameters of the model have been estimated from experimental data. If the maintenance values are assumed negligible, as has been observed for wastewater systems, it is possible to quantify the magnitude of co-metabolism encountered in the biological oxidation of synthetic organic compounds.     

Facilitation of cometabolic degradation of 4-chlorophenol using glucose as an added growth substrate

This paper reports on the feasibility of using glucose as an added substrate for cometabolic transformation of 4-chlorophenol (4-cp). When glucose was fed as the added growth substrate, only 78% and 43% of the initial 4-cp concentrations of 100 and 200 mg l^–1, respectively, were transformed before the pH dropped to below 4.5 and stopped all reactions. By maintaining the medium pH, complete removal of 4-cp was achieved even at the high initial concentration of 200 mg l^–1. Phenol induction prior to inoculation was not a prerequisite to ensure transformation of 4-cp when glucose was the added growth substrate. Compared with phenol as the added growth substrate, cells grown on glucose displayed a longer acclimation phase and, in general, a lower specific transformation rate. The volumetric transformation rate of 4-cp, however, was greatly enhanced due to the increased cell density. The results of this work suggest that 4-cp itself induced the enzymes necessary for its cometabolism. With NADH regenerated effectively through metabolism of glucose, 4-cp was transformed in the absence of added phenol. Consequently, the competitive inhibition involved in cometabolism was avoided and the risks associated with addition of toxic growth substrates such as phenol were eliminated