A theoretical derivation of the Contois equation for kinetic modeling of the microbial degradation of insoluble substrates

Although developed as an empirical model to describe microbial growth on soluble substrates, the Contois equation has been widely accepted for kinetic modeling of insoluble substrate degradation. Yet, the mechanistic basis underlining these successful applications remains unanswered. Unlike soluble substrates that mainly cultivate suspended cultures, microbes cultivated on insoluble substrates have the populations that attach to the substrate surface or remain suspended in the bulk solution, while those attached usually grow faster than those suspended due to their proximity to food resources. This imbalanced growth provides a plausible explanation to the inverse relationship between microbial concentration and their specific growth rate as conveyed in the Contois equation. Based on a theoretical derivation, this study revealed that the Contois equation holds true only when attached microbes substantially obstruct the access of food to their suspended counterparts. On the other hand, when plentiful insoluble substrate surfaces are exposed for cell attachment, the Contois equation will be reduced back to the classic Monod equation.     

The ideal free distribution and bacterial growth on two substrates

A population dynamical model describing growth of bacteria on two substrates is analyzed. The model assumes that bacteria choose substrates in order to maximize their per capita population growth rate. For batch bacterial growth, the model predicts that as the concentration of the preferred substrate decreases there will be a time at which both substrates provide bacteria with the same fitness and both substrates will be used simultaneously thereafter. Preferences for either substrate are computed as a function of substrate concentrations. The predicted time of switching is calculated for some experimental data given in the literature and it is shown that the fit between predicted and observed values is good. For bacterial growth in the chemostat, the model predicts that at low dilution rates bacteria should feed on both substrates while at higher dilution rates bacteria should feed on the preferred substrate only. Adaptive use of substrates permits bacteria to survive in the chemostat at higher dilution rates when compared with non-adaptive bacteria.     

Influence of Particle Size and Concentration on Rheological Behaviour of Reconstituted Apple Purees

This work investigates the impact of structural parameters on the rheological behaviour of apple purees. Reconstructed apple purees from 0 g/100 g up to 2.32 g/100 g of insoluble solids content and varying in particle size were prepared. Three different particle size distributions were obtained by mechanical treatment only, to modify both size and morphology of the particles without modifying the intrinsic rigidity of the cell walls. Rheological measurements showed that the insoluble solids content have a first order effect on the rheological behaviour of the suspensions: three concentrations domains were observed in both dynamic and flow measurements. A model is proposed for each domain. The existence of a weak network between particles is clearly shown over a critical concentration of insoluble solids (cell walls) depending on particle size distribution (semi-diluted domain). In a concentrated domain, particles are on close packing conditions and their apparent volume begin to shrink. Particle size and shape also play an important role on the rheological behaviour of reconstructed apple puree. Due to their irregular shape, cell clusters clog the medium at lower concentration compared to individual cells.     

Theoretical analysis of selection-based strain improvement for microorganisms with growth dependent upon extracytoplasmic enzymes

This study analyzes selection in continuous culture as a means to improve the growth of microorganisms dependent upon the expression of extracytoplasmic enzymes. A quantitative, theoretical model was developed that considers increases in enzyme activity and/or expression due to mutation in conjunction with reaction and diffusion at the cell surface and in the surrounding boundary layer. This model was applied to a system consisting of a recombinant yeast cell growing on either soluble or insoluble substrates by virtue of extracytoplasmic enzymes either with or without tethering to the cell surface. Our results indicate that selection of faster-growing cells can be effective, arbitrarily defined as a faster-growing mutant representing 1% of the population in < or =3 months, but only under some conditions. For both soluble and insoluble substrates, tethering of enzymes to the cell surface is required for selection to be effective under the conditions examined. Significant increases in heterologous enzyme expression (2.5-fold for mutants as compared to the parent strain) are also required. In the soluble substrate/enzyme tethered case, the value of k(S) must also be low in order for selection to be effective. Cells growing on non-native substrates by virtue of extracytoplasmic enzyme production are expected to experience selective pressure in response to several additional factors, including cell shape, distance of the cell-substrate gap, properties of the gap, and perhaps mutation frequency. However, these factors exert a smaller impact on selection time and it is not clear that favorable values for these factors are required in order for selection to be effective.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.     

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.     

Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass.     

Shuttling happens: soluble flavin mediators of extracellular electron transfer in <Emphasis Type="Italic">Shewanella</Emphasis>

The genus Shewanella contains Gram negative γ-proteobacteria capable of reducing a wide range of substrates, including insoluble metals and carbon electrodes. The utilization of insoluble respiratory substrates by bacteria requires a strategy that is quite different from a traditional respiratory strategy because the cell cannot take up the substrate. Electrons generated by cellular metabolism instead must be transported outside the cell, and perhaps beyond, in order to reduce an insoluble substrate. The primary focus of research in model organisms such as Shewanella has been the mechanisms underlying respiration of insoluble substrates. Electrons travel from the menaquinone pool in the cytoplasmic membrane to the surface of the bacterial cell through a series of proteins collectively described as the Mtr pathway. This review will focus on respiratory electron transfer from the surface of the bacterial cell to extracellular substrates. Shewanella sp. secrete redox-active flavin compounds able to transfer electrons between the cell surface and substrate in a cyclic fashion—a process termed electron shuttling. The production and secretion of flavins as well as the mechanisms of cell-mediated reduction will be discussed with emphasis on the experimental evidence for a shuttle-based mechanism. The ability to reduce extracellular substrates has sparked interest in using Shewanella sp. for applications in bioremediation, bioenergy, and synthetic biology.     

Substrate consumption and biomass growth of Ralstonia eutropha at various S0/X0 levels in batch cultures

The biomass growth, substrate consumption and polyhydrobutyrate (PHB) production of Ralstonia eutropha with butyric acid and fructose as the carbon and energy sources at various ratios of initial substrate concentration (S0) to initial biomass concentration (X0) were investigated in this study. Results indicated that the PHB content increased with the increasing S0/X0 ratio. Different substrates exhibited a similar trend for cell growth and substrates consumption with the changing S0/X0 ratio. The specific consumption rates of both butyric acid and fructose increased with the increasing S0/X0 ratio. An S0/X0-dependent kinetic model was modified to describe the kinetics of biomass growth and substrate consumption for R. eutropha. This model was verified with the experimental results from this work and in literature.     

Enzyme catalyzed biotransformations in aqueous two-phase systems with precipitated substrate and/or product

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.     

Rigorous model for spherical cell-support aggregate

The activity of immobilized cell-support particle aggregates is influenced by physical and biochemical elements, mass transfer, and physiology. Accordingly, the mathematical model discussed in this study is capable of predicting the steady state and transient concentration profiles of the cell mass and substrate, plus the effects of the substrate and product inhibition in an immobilized cell-support aggregate. The overall mathematical model is comprised of material balance equations for the cell mass, major carbon source, dissolved oxygen, and non-biomass products in a bulk suspension along with a single particle model. A smaller bead size and higher substrate concentration at the surface of the particle, resulted in a higher supply of the substrate into the aggregate and consequently a higher biocatalyst activity.     

THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

Prevention of fungal colonization and digestion of cellulose by the addition of methylcellulose

When the attachment of cellulolytic rumen fungi to cellulose is blocked by the addition of methylcellulose, cellulose digestion is entirely inhibited. Even after these fungi have colonized and penetrated the cellulosic fibers of filter paper, the addition of methylcellulose effectively halts cellulose digestion. This effect of methylcellulose is accompanied by the complete inhibition of fungal attachment to cellulose fibers; the addition of methylcellulose does not affect the growth of these organisms on soluble substrates. We conclude that fungal cellulose digestion, like bacterial cellulose digestion, requires the spatial juxtaposition of the cellulolytic organism and its insoluble substrate. The simultaneous inhibition of both attachment and digestion by the same inhibitor suggests that these two processes are functionally linked in the fungi.     

Models for the kinetics of biodegradation of organic compounds not supporting growth.

We developed 12 models of kinetics to describe the metabolism of organic substrates that are not supporting bacterial growth. These models can be used to describe the biodegradation of organic compounds that are not supporting growth when the responsible populations are growing logistically, logarithmically, or linearly or are not increasing in numbers. Nonlinear regression analysis was used to fit patterns of mineralization by two bacteria to these kinetic models. Pseudomonas acidovorans mineralized 1 ng of phenol per ml while growing exponentially at the expense of uncharacterized organic carbon in a synthetic medium. Phenol at a concentration of 1 ng/ml did not affect the growth of P. acidovorans. These data were best fit by the model that incorporates the equation for logarithmic growth and assumes a concentration of test substrate well below its Km value. In the absence of a second substrate, glucose at concentrations below those supporting growth was mineralized by Salmonella typhimurium in a manner best described by pseudo first-order kinetics. In the presence of different concentrations of arabinose, however, the kinetics of glucose mineralization by S. typhimurium reflected linear, logistic, or logarithmic growth of the population on arabinose. We conclude that the kinetics of mineralization of organic compounds at concentrations too low to support growth are best described either by the first-order model or by models that incorporate expressions for the kinetics of growth of the metabolizing population on other substrates. When growth is at the expense of other substrates, the kinetics observed reflect such growth, as well as the concentration of the substrate of interest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Models for the kinetics of biodegradation of organic compounds not supporting growth

We developed 12 models of kinetics to describe the metabolism of organic substrates that are not supporting bacterial growth. These models can be used to describe the biodegradation of organic compounds that are not supporting growth when the responsible populations are growing logistically, logarithmically, or linearly or are not increasing in numbers. Nonlinear regression analysis was used to fit patterns of mineralization by two bacteria to these kinetic models. Pseudomonas acidovorans mineralized 1 ng of phenol per ml while growing exponentially at the expense of uncharacterized organic carbon in a synthetic medium. Phenol at a concentration of 1 ng/ml did not affect the growth of P. acidovorans. These data were best fit by the model that incorporates the equation for logarithmic growth and assumes a concentration of test substrate well below its Km value. In the absence of a second substrate, glucose at concentrations below those supporting growth was mineralized by Salmonella typhimurium in a manner best described by pseudo first-order kinetics. In the presence of different concentrations of arabinose, however, the kinetics of glucose mineralization by S. typhimurium reflected linear, logistic, or logarithmic growth of the population on arabinose. We conclude that the kinetics of mineralization of organic compounds at concentrations too low to support growth are best described either by the first-order model or by models that incorporate expressions for the kinetics of growth of the metabolizing population on other substrates. When growth is at the expense of other substrates, the kinetics observed reflect such growth, as well as the concentration of the substrate of interest.(ABSTRACT TRUNCATED AT 250 WORDS)     

嗜热真菌DSM10635生产耐热木聚糖酶的小试研究

应用嗜热真菌Thermomyces lanuginosus DSM10635,采用固体发酵的方法探索耐热木聚糖酶的优化生产条件。在研究玉米芯,玉米皮,玉米秆,麸皮,松树屑,桦树屑等不同底物,在不同温度、玉米芯颗粒大小以及料水比条件下培养比较酶产量后,发现该嗜热真菌产耐热木聚糖酶的最佳底物为玉米芯或玉米皮,最佳培养温度为50℃--55℃,在加水量为1份玉米芯：2．8份水,玉米芯的颗粒直径大约为1mm时产酶量最高。实验结果显示,嗜热真菌DSM10635在优化后的培养条件下木聚糖酶产量可达到12525．80IU／g玉米芯。     

Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur

Thiobacillus ferroxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacillus ferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed.     

Polymeric collagen fibrils. An example of substrate-mediated steric obstruction of enzymic digestion.

Polymeric collagen fibrils have been reacted with fluorescein and rhodamine isothiocyanates to produce fluorescent dye-labelled fibrils, containing seven dye substituents per molecule of tropocollagen within the polymeric collagen fibrils. Two dye-labelled peptides per molecule of tropocollagen were solubilised by trypsin (EC 3.4.21.4) from the telopeptide regions and four dye-labelled peptides were located in the helical regions solubilised by bacterial collagenase (EC 3.4.24.3). The solubilisation of dye-labelled peptides from these insoluble substrates were employed to measure the kinetics of trypsin and collagenase digestion of the telopeptide and helical regions, respectively, of the insoluble polymeric collagen fibrils. These studies demonstrated an apparent excess of enzyme for the readily available substrate under conditions when it was known that a vast excess of substrate existed in the reaction mixture calculated in terms of a molecular ratio. A point of equivalence was established for both trypsin and bacterial collagenase, approximately one enzyme molecule per 870 substrate molecules. On either side of this point the quantity of products formed was controlled by either the enzyme concentration or the substrate concentration. The results can be explained in terms of the inaccessibility of tropocollagen molecules within the molecular architecture of the polymeric collagen fibrils. The external layer of tropocollagen molecules obstruct collagenolytic enzymes penetrating to, and forming enzyme-substrate complexes with, the bulk of the substrate within the interior of the fibrils.     

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.     

Packed bed dynamics during microbial treatment of wastewater: modelling and simulation

A mathematical model consisting of mass balance equations and accounting for bioreaction and mass transfer is presented to describe both unsteady and steady-state degradation of phenol in a biofilter. The model has been validated for the steady-state situation with literature work. The model has been able to predict the dynamics of the biofiltration process with variations in system and operating conditions as inlet substrate concentration, liquid phase mass transfer coefficients, particle size, Henry's constant, inlet velocity, growth and half saturation constants and bed void fraction. The results show that inlet substrate concentration, inlet velocity, growth and half saturation constants and liquid phase mass transfer coefficients significantly control the operational dynamics. It is also shown that inhibition effects can be neglected for low concentrations (<0.5 kg m(-3)) of phenol. Thus, the model can be used as a design tool for a biofilter.