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.     

Inhibition Kinetics of Phenol Degradation by Pseudomonas aeruginosa from Continuous Culture and Washout Data

Steady states of a continuous culture with an inhibitory substrate were used to estimate kinetic parameters under substrate limitation (chemostat operation). Pure cultures of an indigenous Pseudomonas aeruginosa were grown in continuous culture on phenol, the sole source of carbon and energy, at dilution rates of 0.010 to 0.20 h^{- 1}. Using different dilution rates, several steady states were investigated and the specific phenol consumption rates were calculated. In addition, phenol degradation was investigated by increasing the dilution rate above the critical dilution rate (washout cultivation). The results showed that the specific phenol consumption rate increased with increased dilution rate at steady state and that the degradation by Pseudomonas aeruginosa can be described by simple substrate inhibition kinetics under substrate limitation but cannot be described by simple substrate inhibition kinetics under washout cultivation. Fitting of the steady-state data from continuous cultivation to various inhibition models resulted in the best fit for the Yano and Koga kinetic inhibition model. The r_{s max} value of 0.278 mg/mg/h obtained from the Yano and Koga equation was comparable to the experimentally calculated r_{s max} value of 0.283 mg/mg/h obtained under washout cultivation.     

Uptake rate of phenol by Pseudomonas putida grown in unsteady state

The uptake rate of phenol by washed cells of Pseudomonas putidagrown on phenol in fermenter in an un steady state, caused by the step increase of dilution rate and/or phenol concentration in the feed, was studied. The Monod-Haldane type equation was applied to fit the data and the best kinetic parameters were calculated by nonlinear least-square techniques. It was found that the minimum period of unsteady state required for induction of the phenol metabolic pathway was approximately 30 min. The values of kinetic parameters in an unsteady state varied according to each parameter. The values of u(m) first monotonically increased to reach their highest value after about 120 min and then monotonically decreased to equal the u(m) in new steady state after about five residence times. No regularity in changing of K(s) and K(i), in unsteady state was observed. However, the greatest change in the values of K(i), was 45% while the change in values of K(s) was as much as two times compared to K(i) and K(s) in steady state prior to disturbance.     

Inhibition kinetics of phenol degradation from unstable steady-state data

Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady-state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation).The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well-known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady-state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567-576, 1997.     

Dynamics of phenol degradation by Pseudomonas putida

Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h(-1) and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l(-1). These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. (c) 1993 John Wiley & Sons, Inc.     

Sand administration as an instrument for biofilm control of Pseudomonas putida ATCC 11172 in chemostat cultures

Pseudomonas putida ATCC 11172 was grown in chemostat on L-asparagine or phenol as the sole, limiting carbon and energy source. The growth characteristics of a culture where a biofilm was present, were compared with one where the biofilm was strongly reduced by the grinding and shearing effect of sand suspended in the culture. In the presence of the intact biofilm, the curve of steady-state biomass versus dilution rate diverged greatly from the theoretical pattern predicted by conventional chemostat models. The sand strongly retarded the biofilm formation and to a high degree restored the shape of the biomass versus dilution rate curve to a more conventional pattern. The maximum specific growth rate (mu(max)) could not be calculated from the biofilm cultures. However using the sand cultures, mu(max) was determined to 0.64 h(-1) with L-asparagine as the carbon source and 0.49 h(-1) with phenol which compare favorably with the respective mu(max) values calculated from batch cultures.Incorporation of sand into strongly agitated cultures is recommended as an efficient and simple means of controlling biofilm formation in continuous cultures. The method may enable the gathering of basic kinetic data difficult to obtain in the presence of biofilm.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Temperature dependence of inorganic nitrogen uptake: reduced affinity for nitrate at suboptimal temperatures in both algae and bacteria.

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 approximately 3, where Q10 is the proportional change with a 10 degrees C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures.     

Microbial Ecophysiology of Whey Biomethanation: Comparison of Carbon Transformation Parameters, Species Composition, and Starter Culture Performance in Continuous Culture

Changes in lactose concentration and feed rate altered bacterial growth and population levels in a whey-processing chemostat. The bacterial population and methane production levels increased in relation to increased lactose concentrations comparable to those in raw whey (6%) and converted over 96% of the substrate to methane, carbon dioxide, and cells. Sequential increases in the chemostat dilution rate demonstrated excellent biomethanation performance at retention times as low as 25 h. Retention times shorter than 25 h caused prevalent bacterial populations and methane production to decrease, and intermediary carbon metabolites accumulated in the following order: acetate, butyrate, propionate, lactate, ethanol, and lactose. Bacterial species dominated in the chemostat as a function of their enhanced substrate uptake and growth kinetic properties. The substrate uptake kinetic properties displayed by the mixed chemostat population were equivalent to those of individual species measured in pure culture, whereas the growth kinetic properties of species in mixed culture were better than those measured in pure culture. A designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum displayed biomethanation performance, which was similar to that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Preserved starter cultures were developed and used as inocula for the start-up of a continuous anaerobic digestion process that was effective for biomethanation of raw whey at a retention time of 100 h.     

Adsorption and bio-degradation of phenol by chitosan-immobilized Pseudomonas putida (NICM 2174)

Biodegradation of phenol by Pseudomonas putida (NICM 2174), a potential biodegradent of phenol has been investigated for its degrading potential under different conditions. Pseudomonas putida (NICM 2174) cells immobilized in chitosan were used to degrade phenol. Adsorption of phenol by the chitosan immobilized matrix played an important role in reducing the toxicity of phenol. In the present work, results of the batch equilibrium adsorption of phenol on chitosan from its aqueous solution at different particle sizes (0.177 mm, 0.384 mm, 1.651 mm) and initial concentration of phenol (20, 40, 60, 80, 100, 120, 140, 160, 180, 200 mg/l) have been reported. The adsorption isotherms are described by Langmuir, Freundlich and Redlich-Peterson types of equations. These indicate favourable adsorption with chitosan. From the adsorption isotherms, the adsorption capacity, energy of adsorption, number of layers and the rate constants were evaluated. In batch kinetic studies the factors affecting the rate of biodegradation of phenol, were initial phenol concentration (0.100 g/l, 0.200 g/l, 0.300 g/l), temperature (30v°C, 34v°C, 38v°C) and pH (7.0, 8.0, 9.0). Biodegradation kinetic data indicated the applicability of Lagergren equation. The process followed first order rate kinetics. The biodegradation data generally fit the Lagergren equation and the intraparticle diffusion rate equation from which adsorption rate constants, diffusion rate constants and diffusion coefficients were determined. Intraparticle diffusion was found to be the rate-limiting step. Cell growth contributed significantly to phenol removal rates especially when the degradation medium was supplemented with a utilizable carbon source.     

Analysis of oxygenation reactions in a multi-substrate system-A new approach for estimating substrate-specific true yields

A series of experiments was performed in an aerobic chemostat reactor using a multi-substrate system consisting of acetate, phenol, and 2,4-dichlorophenol (DCP). The phenolic compounds require initial oxygenation reactions, while acetate is oxidized without oxygenations. The biomass completely dechlorinated DCP and utilized all of the substrates simultaneously as electron donors and carbon sources. However, DCP removal was less than for phenol and depended on the solids retention time. A novel substrate-specific yield analysis indicated that true yield values were approximated well by the number of electrons removed in non-oxygenation reactions. Experiments for estimating the kinetic parameters for utilization of the phenolic compounds were designed to eliminate the effects of the key cosubstrates of oxygenation reactions, O2, and the reduced intracellular electron carrier, NADH + H+. The maximum specific rate of substrate utilization, qmax, and the half-maximum rate concentration, K, for phenol and DCP were estimated. The kinetics for DCP were much slower than those for phenol, and the largest effect was a half-maximum rate concentration, which was 19 times larger for DCP. The larger K for DCP explains why DCP removal was low and sensitive to the solids retention time.     

Unique kinetic properties of phenol-degrading variovorax strains responsible for efficient trichloroethylene degradation in a chemostat enrichment culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities. To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter(-1) day(-1)) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter(-1) day(-1) (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.     

Measurement of minimum substrate concentration (Smin) in a recycling fermentor and its prediction from the kinetic parameters of Pseudomonas strain B13 from batch and chemostat cultures.

The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.     

Pseudo-Second-Order Kinetic Model for Biosorption of Methylene Blue onto Tamarind Fruit Shell: Comparison of Linear and Nonlinear Methods

In this study, the sorption of methylene blue, a basic dye, onto tamarind fruit shell was studied by performing batch kinetic sorption experiments. The equilibrium kinetic data were analyzed using the pseudo-second-order kinetic model. A comparison between linear least squares method and nonlinear regression method of estimating the kinetic parameters was examined. Four pseudo-second-order kinetic linear equations were discussed. The coefficient of determination (r ²), and the chi-square (χ²) test were employed as error analysis methods to determine the best-fitting equation. Kinetic parameters obtained from four kinetic linear equations using the linear method differed but they were the same when nonlinear method was used. Present investigation showed that by linear method a Type 1 expression very well represent the kinetic uptake of methylene blue onto tamarind fruit shell. Linear method was found to check only the hypothesis instead of verifying the kinetic model. Nonlinear regression method was found to be the more appropriate method to determine the rate kinetic parameters.     

Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor

Phenol biodegradation in a batch reactor using a pure culture of Pseudomonas putida DSM 548 was studied. The purpose of the experiments was to determine the kinetics of biodegradation by measuring biomass growth rates and phenol concentration as a function of time in a batch reactor. The Haldane equation μ=μ(m)S/((K(s)+S+S(2))/K(i)) adequately describes cell growth with kinetic constants μ(m)=0.436h(-1), K(s)=6.19mgl(-1), K(i)=54.1mgl(-1). These values are in the range of those published in literature for pure or mixed cultures degrading phenol.     

Biodegradation by immobilized bacteria in an airlift-loop reactor-influence of biofilm diffusion limitation

Naphthalene-2-sulfonate was degraded by submerse growing Pseudomonads in a chemostat culture. The kinetic parameters for the Monod equation, including Pirts maintenance energy, were calculated from these experiments regarding naphthalene-2-sulfonate as substrate and oxygene as cosubstrate. By immobilizing the bacteria on sand particles, the degradation of naphthalene-2-sulfonate was carried out in a specialy designed three-phase airlift-loop reactor in a completely fluidized state. From these experiments, the influence of biofilm diffusion limitation on reaction kinetics and criteria for stable biofilm formation on sand particles were obtained.     

Coexistence of competing microbial species in a chemostat where one population feeds on another

In this paper, we consider a model for a chemostat in which two microbial species compete for a single rate-limiting nutrient, while one of the species feeds on another. Under certain simplifying hypotheses, such a chemostat can be described by a system of three nonlinear ordinary differential equations. A theoretical study is conducted to characterize the possible types of solutions. A limit cycle solution was obtained for some parametric values of the system indicating that coexistence of the two species is possible in a significant range of the operating parameters.     

Growth of Trichosporon cutaneum under oxygen limitation: kinetics of oxygen uptake

The relationship between oxygen concentration and growth rate in the yeast Trichosporon cutaneum was studied. In order to establish the conditions for purely oxygen-limited growth, the cells were first grown in a carbon-limited chemostat, and kinetic parameters determined. The cells were then grown in an oxygen-limited chemostat at different dilution rates yielding different oxygen uptake rates. The steady-state dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen tension was found at each dilution rate and the corresponding equilibrium dissolved oxygen concentration determined in the effluent medium. The relationship between oxygen concentration and growth rate followed Monod-type kinetics with an apparent K(O) of 4.38 x 10(-6)M.