Prediction of Substrate Removal Rates of Attached Microorganisms and of Relative Contributions of Attached and Suspended Communities at Field Sites

A mathematical model composed of a direct proportionality relationship between bulk water velocities and field-determined second-order microbial transformation rate coefficients, and the relative rate coefficient of a benchmark chemical, was developed for estimating the substrate removal rates of rapidly degraded chemicals by attached organisms in shallow (<1 m deep) aquatic ecosystems. Data from 31 field experiments involving the addition of 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) in nine field areas were used to determine a field-derived second-order rate coefficient for microbial transformation of the ester. By using 2,4-DME as a benchmark chemical, the model was used to predict microbial transformation rates of the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE) at five other field sites. The predicted half-lives of 2,4-DBE varied 1,500-fold and were within about a threefold range or less of the measured half-lives. Under conditions of mass transport limitation, the contributions of attached microorganisms relative to total microbial activities at various field sites were related to the ratio of water velocity, U, and depth, D, showing that historical definitions of ecosystems according to flow and depth characteristics are also valid for describing the process-related structure of ecosystems. An equation was developed for predicting the relative contributions of attached and suspended communities with values of U and D for lotic and lentic ecosystems. On the basis of this equation, attached microorganisms were expected to be insignificant in deep lentic ecosystems and suspended microorganisms were expected to be insignificant in shallow lotic systems for the same process carried out by both populations. Neglecting epiphytic microorganisms, both suspended and attached organisms were expected to be significant in wetlands.     

Initial Test of the Benchmark Chemical Approach for Predicting Microbial Transformation Rates in Aquatic Environments

Using 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) as a benchmark chemical, we determined relative pseudo-first-order rate coefficients for the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE), methyl parathion, and methyl-3-chlorobenzoate in a diversity of microbial samples, including water, sediment, biofilm, and floating microbial mats collected from a laboratory mesocosm as well as from streams, lakes, and wetlands in Georgia and Florida. The decreasing order of reactivity for relative microbial transformation rates was 2,4-DBE > 2,4-DME > methyl-3-chlorobenzoate > methyl parathion. Half-lives of the chemicals varied about 60-fold depending on the chemical and microbial sample. Relative rate coefficients, however, typically varied only about threefold for field-collected samples. Relative rate coefficients determined with samples from a laboratory mesocosm were consistently low compared with the field sample data. Overall, the data indicated that microbial transformation rates of a chemical can be satisfactorily inferred for a wide variety of microbial habitats—such as water, biofilm, or a sediment—on the basis of its transformation rate relative to that of an appropriate benchmark chemical by using a single type of microbial sample.     

Rates of Transformation of Methyl Parathion and Diethyl Phthalate by Aufwuchs Microorganisms

Using batch cultures, we determined transformation rates for low concentrations of two toxicants—an insecticide, methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate), and a plasticizer, diethyl phthalate—by aufwuchs, aquatic microbial growth attached to submerged surfaces or suspended in streamers or mats. Aufwuchs samples were collected from field sites, an indoor channel, and a continuous-flow fermentor. Aufwuchs fungi, protozoa, and algae did not transform methyl parathion or diethyl phthalate, but bacteria rapidly transformed both chemicals. Second-order transformation rate coefficients, K_b, based on total plate counts of bacteria in aufwuchs, were determined for potential use in a mathematical model capable of predicting the transport and fate of chemicals in aquatic systems. K_b for both methyl parathion and diethyl phthalate decreased as the concentration of total bacteria, [B], increased in aufwuchs. This effect resulted from the proportion of nontransformer to transformer bacteria increasing as [B] increased and from the rate of transformation per transformer cell decreasing as [B] increased. First-order transformation rate coefficients, K₁, were relatively stable per unit of surface area colonized by aufwuchs, because K_b decreased as [B] increased (K₁ = K_b × [B]).     

Intramolecular general base catalyzed transesterification: The cyclization of ethyl 2-hydroxymethylbenzoate and ethyl 2-hydroxymethyl 4-nitrobenzoate to phthalide and 5-nitrophthalide

The rates of cyclization of ethyl 2-hydroxymethylbenzoate to phthalide have been measured in H₂O at 30°C with μ = 0.5. There is pronounced general base catalysis in the reaction with β = 0.87. The second-order rate constant for imidazole general base catalysis is decreased in D₂O as compared with H₂O by a factor of 3.46. The pH-rate constant profile obtained by extrapolation to zero buffer concentration shows hydronium ion and apparent hydroxide ion catalysis. The value of the second-order rate constant k_OH is 10⁵ greater than k_OH for hydrolysis of ethyl benzoate. Ethyl 2-hydroxymethyl 4-nitrobenzoate cyclizes to 5-nitrophthalide in a similar manner. The Brönsted coefficient β for general base catalysis is 0.97, within error of unity. Thus, it is probable that general base catalysis involves rate-determining proton transfer.     

Kinetic studies on the binding of Streptomyces subtilisin inhibitor with subtilisin BPN′

The binding mechanism of Streptomyces subtilisin inhibitor and subtilisin BPN′ was studied kinetically with the stopped-flow method by monitoring the protein fluorescence increase due to complex formation. In the lower concentration range of proteins, the reaction followed the second-order kinetics. The pH dependence of the apparent second-order rate constant, k_on, suggested the involvement of the two ionizable groups of pK_a of 7.8 and 10 in the binding. The activation parameters were calculated from the temperature dependence of the apparent second-order rate constants. The value of the apparent activation energy (E_A = 39.7 kJ · mol^?1, 9.50 kcal · mol^?1) and insensitivity of k_on to the viscosity of the medium suggest that the binding is not a simple diffusion-controlled bimolecular association. Further studies with a much broader range of protein concentrations have revealed that the reaction tends to approach first-order kinetics as the inhibitor concentration increases. The binding reaction is, therefore, reconcilable with a two-step mechanism, in which a fast bimolecular association is followed by a slow unimolecular isomerization step; the dissociation constant of the first step, K_L, is estimated to be 1.2 × 10^?4m and the rate constant of the second step, k₊₂, to be 770 s^?1. It was also found that the increase of tryptophan fluorescence due to the complex formation occurs solely in the rate-determining unimolecular process.     

Kinetics and mechanism of malonate replacement in bis(malonato)oxovanadate(IV) by oxalate

The kinetics of malonate replacement in bis- (malonato)oxovanadate(IV), [VO(mal)₂H₂O]²⁻(hereafter water molecule will be omitted), by oxalate has been studied by the stopped-flow method. The reaction was found to consist of two consecutive steps (k₁ and k₂: first-order rate constants) passing through a mixed ligand complex, [VO(mal)(ox)]²⁻. The rates for each step depended linearly on the concentrations of free oxalate species, Hox⁻ and ox²⁻. The second-order rate constants for the replacement by ox²⁻ were much larger in the k₁ step than in the k₂ step and the activation parameters were determined as follows: ΔH^≠= 43.5 ± 5.6 kJ mol⁻¹, ΔS±-53 ± 19 J K⁻¹ mol⁻¹ and ΔH≠= 43.6 ± 0.5 kJ mol⁻¹, δS≠ = -62 ± 2 J K^−l mol⁻¹ for the k₁ and k₂ steps, respectively. The volume of activation was determined to be -0.65 ± 0.75 cm³ mol⁻¹ at 20.2 °C by the high-pressure stopped-flow method for the apparent rate constants.     

Stopped-Flow Fluorescence Kinetic Study of Protein Sliding and Intersegment Transfer in the Target DNA Search Process

Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins' sliding and intersegment transfer (also known as “direct transfer”) in the target DNA search process. Based on the analytical expression of the mean search time for the discrete-state stochastic model, we derived analytical forms of the apparent rate constant k_app for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of k_app facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of k_app, we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA-length-dependent k_app data. Using the dependence of k_app on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target.     

Microbial Transformation of Esters of Chlorinated Carboxylic Acids

Two groups of compounds were selected for microbial transformation studies. In the first group were carboxylic acid esters having a fixed aromatic moiety and an increasing length of the alkyl component. Ethyl esters of chlorine-substituted carboxylic acids were in the second group. Microorganisms from environmental waters and a pure culture of Pseudomonas putida U were used. The bacterial populations were monitored by plate counts, and disappearance of the parent compound was followed by gas-liquid chromatography as a function of time. The products of microbial hydrolysis were the respective carboxylic acids. Octanol-water partition coefficients (K_ow) for the compounds were measured. These values spanned three orders of magnitude, whereas microbial transformation rate constants (k_b) varied only 50-fold. The microbial rate constants of the carboxylic acid esters with a fixed aromatic moiety increased with an increasing length of alkyl substituents. The regression coefficient for the linear relationships between log k_b and log K_ow was high for group 1 compounds, indicating that these parameters correlated well. The regression coefficient for the linear relationships for group 2 compounds, however, was low, indicating that these parameters correlated poorly.     

Efficient feeding profile optimization for recombinant protein production using physiological information

A multivariate study was performed aiming at the optimization of a recombinant rhamnose inducible E. coli induction system with alkaline phosphatase as target product. The effects of typical factors with impact on post- as well as pre-induction feeding rates were investigated with respect to the space–time yield of the target product. The goal was increased understanding as well as quantitative characterization of these factors with respect to their physiological impact on the model system. The optical density (OD) at which the culture was induced had a strong positive effect on the space–time yield. Pre-induction growth rate (k) had a second-order effect, while induction feed rate drop (J), a factor defining the linear post-induction feed rate, was interacting with (k). However, explanation of the observed effects to acquire more understanding regarding their effect on cell metabolism was not straight forward. Hence, the original process parameters were transformed into physiological more meaningful parameters and served as the basis for a multivariate data analysis. The observed variance with respect to observed volumetric activity was fully explained by the specific substrate uptake rate (q _s) and induction OD, merging the process parameters pre-induction growth rate (k) and feed rate drop (J) into the physiological parameter specific substrate uptake rate (q _s). After transformation of the response volumetric activity (U/ml) into the biomass specific activity (U/g_biomass), the observed variance was fully explained solely by the specific substrate uptake rate (q _s). Due to physiological multivariate data analysis, the interpretation of the results was facilitated and factors were reduced. On the basis of the obtained results, it was concluded that the physiological parameter q _s rather than process parameters (k, J, induction OD) should be used for process optimization with respect to the feeding profile.     

Characterization of 1,2-dibromoethane-degrading haloalkane dehalogenase from Bradyrhizobium japonicum USDA110

Haloalkane dehalogenases (DHAs, E.C. 3.8.1.5) are very promising biocatalytic tools for the bioremediation of environmental pollutants which consists of haloalkanes. In the present work, we investigated the DHA from Bradyrhizobium japonicum USDA110 (BjDHA). The dehalogenase activity of B. japonicum USDA110 and RT-PCR analysis revealed that the BjDHA gene expression is induced by 1,2-dibromoethane (1,2-DBE) during the early exponential phase. The BjDHA gene was cloned, expressed in Escherichia coli BL21 (DE3) and characterized. The enzyme catalyzes the irreversible hydrolysis of a variety of haloalkanes to the corresponding alcohol, halide, and a hydrogen ion. The catalytic properties of the recombinant enzyme were investigated and the kinetic parameters (K_m, k_cat) for a number of substrates were determined. The results showed that the BjDHA displays wide substrate specificity towards haloalkanes and particular high activity towards 1,2-DBE. The enzyme has a different catalytic triad topology compared to the Xanthobacter haloalkane dehalogenase and is more similar to the Rhodococcus enzyme. In addition, consistent with its broad specificity, the BjDHA has a substantially larger and more polar active site cavity compared to the Xanthobacter and Rhodococcus enzymes and as a consequence, BjDHA is able to dehalogenate longer and polar compounds. These properties make this enzyme very promising bioremediation tool for environmental applications.     

Permeability of Red Cell Membranes to Small Hydrophilic and Lipophilic Solutes

The permeability coefficients of a series of amides, ureas, and diols have been measured on red cells of man and dog using the minimum volume method of Sha'afi et al. When the molecules are grouped according to their ether-water partition coefficients, k_ether, the behavior of the hydrophilic molecules, with k_ether less than water, is different from that of the lipophilic molecules, characterized by k_ether greater than water. The rate of permeation of the hydrophilic molecules through an aqueous pathway is determined by the molar volume, a parameter in which the geometrical measure of molecular volume is modified by hydrogen-bonding ability. This indicates the importance of chemical interactions within the aqueous path. The permeation of the lipophilic molecules is determined in the first instance by k_ether, taken as a measure of the ease with which the molecule can escape from its aqueous environment. Within the membrane, lipophilic permeability is modified both by steric factors and by the formation of hydrogen bonds with membrane components. These data allow one to infer that lipid-soluble molecules travel through an organized structure within the lipid membrane and come into contact with polar moieties.     

Kinetic analysis of biphasic protein modification reactions cooperative effects

A mathematical treatment of protein modification reactions is presented, and it is shown thai in these cases protein modification is described by a summation of exponential functions of reaction time, the number of exponentials being equal to the number of modified protein species. It is shown that in cases of protein modification cooperativity, there is a strict dependence of the coefficients of the multiexponential modification equation on the constants of the same equation. The conditions necessary for a reduction of a multiexponential protein modification equation to one of a summation of two exponentials only are examined. The possible formulae for the coefficients of a two-exponential-summation equation, used to describe the modification of protein models with two, three or four modifiable residues (as well as some aspects of models with five and six modifiable residues) per protein molecule are derived. It is seen that the number of such coefficients is severely limited. The most frequently obtained formula for the lower stoichiomelric coefficient of a 'wo-exponential-summation equation is Ak_a/(k_a-k_b). where k_b and k_b are the constants of the two exponentials of the equation, and A is a constant. The value most frequently arrived at for A is (n?1)/n, where n is the number of modifiable residues per protein molecule, while values such as 1/n, or a/n (where a is an integer, and also where a < n) are also possible. In most of the cooperative protein modification models worked out, k_a is identical with k_n, viz., k_a is identical with the rate constant for the first stoichiometric protein modification.     

Decay processes of electric birefringence in chemically reacting system

Analytical and numerical calculations of the decay processes of the electric birefringence in an isomerizing system have been performed. Two modes of isomerization are considered: in the first mode, the direction of the axis (of the optical anisotropy) of a molecule is conserved during isomerization; in the second mode, it is not. In the first mode, if G_Ais not equal to G_B, and that, if k_A^op_A² is not equal to k_B^op_B² (when the orienting electric field is weak), in which G_A, G_B and p_A, p_b are the optical anisotropy and permanent electric dipole moments, of the molecule in two states A and B of the isomerization, and k_A^o and k_B^o are the rate constants for the transitions A → B and B → A, respectively, and if diffusion rates are very much slower than the chemical rates, the relaxation due to the chemical reaction can be detected in the decay of the electric birefringence. In the second mode, if diffusion rates are somewhat slower than the chemical rates, the relaxation due to the chemical reaction appears in the decay, even though there are no differences in optical anisotropy and electric moments in the two states. When the rotary diffusion coefficients are different in the two states, the decay process becomes almost one-component, bringing the chemical rate about ten times higher to diffusion coefficients in both modes.     

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k_off). Since no techniques are available to estimate k_off, we report herewith a method to determine k_off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k_off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k_off value, representing the rate-limiting step, can be directly determined. The k_off value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k_on) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k_off = k_on × K).     

Stoichiometry versus coupling ration in the cotransport of Na and different neutral amino acids

The stoichiometry of Na coupling to amino acid movement across the brush border membrane of the rabbit distal ileum has been determined under initial rate conditions.The coupling ratio, defined as the amino acid-dependent Na influx/the Na-dependent amino acid influx, was equal to unity for alanine, measured over a 10-fold range of Na and alanine concentrations. Coupling ratio values determined under a single set of conditions for a number of amino acids varied from 1 for serine to 4.6 for methionine. Reducing the methionine concentration from 12.5 to 1.5 mM caused the coupling ratio value to fall from 4.6 to 1.2.These results are explained by assuming a fixed stoichiometry of 1 : 1 under all conditions, with initial binding of the amino acid (A) to the Na-dependent carrier (E) but with some amino acids being able to cross on the Na-dependent carrier in the absence of Na.The variation in coupling ratio values can be used to calculate K_A, the apparent dissociation constant of amino acid from the Na-dependent carrier in the absence of Na, and the ratio $\text{k}{}_1\text{k}{}_2$, where k₁ and k₂ are first-order rate constants for translocation of the complexes EA and EANa, respectively. This method of processing results has been defined as delta analysis. The value of K_A for methionine is 3.6 ± 1.1 mM and the $\text{k}{}_1\text{k}{}_2$ ratio is 1.01 ± 0.07. The constant coupling ratio value of 1 for alanine indicates that the value for K_A is extremely high or that the k₁ value is extremely low.     

Light dependence of the decay of the proton gradient in broken chloroplasts

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetic analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant k_L is equal to k_D + mR₀, where R₀ is the initial rate of proton uptake which is a function of light intensity, k_D is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K⁺ increases k_D without affecting m. Treatment with N,N′-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affecting k_D. Treatment with glutaraldehyde makes k_L independent of light intensity and hence m = 0. These results suggest that the light-dependent part (mR₀) of k_L is due to leakage of protons through the coupling factor (CF₁-CF₀) complex which can open or close depending on light intensity and that the light-independent part (k_D) of the decay constant k_L is due to proton leakage elsewhere.     

Distinct effects of the C-terminal octapeptide of cholecystokinin and of a cholera toxin pretreatment on the kinetics of rat pancreatic adenylate cyclase activity

(1) The kinetic parameters of rat pancreatic adenylate cyclase were evaluated, using GTP, p[NH]ppG or GTPγS as nucleotide activator, cholecystokinin as peptide hormone, and GDPβS and dibutyryl cyclic GMP as inhibitors of guanosine triphosphate and CCK-8, respectively. The time courses of activation and the degree of activation at steady state (E_A/E_TOT) were compatible with a simple two-state model of activation-deactivation based on a pseudo-monomolecular activation process (rate constant k₊₂, and a deactivation process (rate constant k_off) that included, depending on the activating nucleotide, the hydrolysis of GTP (rate constant k₂) and/or the dissociation of the intact nucleotide (rate constant k_?1), so that E_A/E_TOT = k₊₁/(k₊₁ + k₂ + k_?a). (2) The hormone CCK-8 increased the value of k₊₁ with GTP dose-dependently, from 0.2 to 10.9 min^?1. The value of k_?1 increased 0.01 to 0.3 min^?1 but the value of k₂ was unaltered at 7 min^?1, so that E_A/E_TOT increased 15-fold, from 4% to 61%. (3) A cholera toxin pretreatment at 30 μg/ml allowed also a large increase in E_A/E_TOT with GTP (up to 51%) but the underlying mechanism was different. It consisted of a 14-fold decrease in the k_off value of the GTP-activated enzyme (from 7 min^? to 0.5 min^?1) that corresponded to a reduction in GTPase activity. When testing the system with p[NH]ppG, two added effects of the cholera toxin pretreatment were observed: a 4-fold increase in the value of k₊₁ (from 0.2 to 0.8 min^?1) and the occurrence of a significant 0.3 min^?1 value for k_?1.     

Stochastic Models of Soil Denitrification

Soil denitrification is a highly variable process that appears to be lognormally distributed. This variability is manifested by large sample coefficients of variation for replicate estimates of soil core denitrification rates. Deterministic models for soil denitrification have been proposed in the past, but none of these models predicts the approximate lognormality exhibited by natural denitrification rate estimates. In this study, probabilistic (stochastic) models were developed to understand how positively skewed distributions for field denitrification rate estimates result from the combined influences of variables known to affect denitrification. Three stochastic models were developed to describe the distribution of measured soil core denitrification rates. The driving variables used for all the models were denitrification enzyme activity and CO₂ production rates. The three models were distinguished by the functional relationships combining these driving variables. The functional relationships used were (i) a second-order model (model 1), (ii) a second-order model with a threshold (model 2), and (iii) a second-order saturation model (model 3). The parameters of the models were estimated by using 12 separate data sets (24 replicates per set), and their abilities to predict denitrification rate distributions were evaluated by using three additional independent data sets of 180 replicates each. Model 2 was the best because it produced distributions of denitrification rate which were not significantly different (P > 0.1) from distributions of measured denitrification rates. The generality of this model is unknown, but it accurately predicted the mean denitrification rates and accounted for the stochastic nature of this variable at the site studied. The approach used in this study may be applicable to other areas of ecological research in which accounting for the high spatial variability of microbiological processes is of interest.     

Inhibition of phosphate uptake in barley roots by hydroxy-benzoic acids

The influence of 15 hydroxy-benzoic acids upon active inorganic phosphate absorption by barley roots was examined. For each compound an inhibition constant (k_i) was determined, i.e. the concentration of compound required to bring about a 50% inhibition of absorption. The k_i values of the benzoic acids were strongly correlated with their octanol—water partition coefficients and their pK_a values. This suggests that the inhibition of normal membrane functions, brought about by benzoic acids, results from a generalized increase in cell membrane permeability. Salicylate derivatives were generally more inhibitory than would be predicted from their partition coefficients; their pronounced toxicity probably arises from structural impediments to their detoxication.