Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Continuous Cultures.

Rhodococcus erythropolis N1-36, a desulfurization strain, was grown in continuous culture at 10 different dilution rates with 50 (mu)M dibenzothiophene sulfone (DBTO(inf2)) as the growth-limiting nutrient. The steady-state biomass, concentrations of substrate (DBTO(inf2)) and product (monohydroxybiphenyl), saturation constant (0.39 (mu)M DBTO(inf2)), and cell yield coefficient (9 mg of biomass(middot)(mu)M(sup-1) DBTO(inf2)) were measured. Continuous cultures at five temperatures allowed calculation of activation energy (0.84 kcal(middot)mol(sup-1) [ca. 3.5 kJ(middot)mol(sup-1)]) near the optimal temperature (30(deg)C) for growth. A washout technique was used to calculate the maximum specific growth rate (0.235 h(sup-1)), a value equivalent to a minimum generation time of 2.95 h.

Optimal design of the tubular microporous membrane aerator for shear-sensitive cell cultures.

In this paper, a theoretical analysis of oxygen transport across the tubular microporous membrane is described. This analysis has provided some insight into the optimal design of the membrane aerator. It was found in this study, at fixed inlet pressure, that the overall membrane oxygen transfer rate increases with increased tubing length only up to a certain length, i.e., the "critical length". When a large membrane surface area is required, the fiber should be divided into parallel segments to increase the overall oxygen transfer rate. A manifold or a gas distributor can then be used to distribute gas into segments of tubing. The length of each segment cannot exceed the critical length. In addition, shorter tube segments should give a higher oxygen transfer rate per unit tube length; however, this advantage is counterbalanced by the fact that gas distribution into huge numbers of parallel tubings may not be uniform.

Use of fluorometry for monitoring and control of a bioreactor.

A new fluorescent bioreactor monitoring probe-multiple excitation fluorometric system (MEFS)-has been developed. This probe was compared to the commercially available BioChem Technology FluroMeasure system (NADH probe). In this task the fluorescence behavior of three model fermentation systems, ethanol fermentation by Candida utilis, phenol fermentation by Pseudomonas putida, and glucose fermentation by Saccharomyces cerevisiae, were examined. The results indicated that the fluorescence intensity and behavior of various cellular fluorophors vary significantly between the different fermentation systems. Monitoring a fermentation process using only NAD(P)H fluorescence provided limited information. The NAD(P)H fluorescence was found not to be the best fluorescence signal for monitoring cell concentrations. The best way of monitoring a bioreactor by fluorometry may be to monitor several fluorophors in the whole culture broth simultaneously and to relate these fluorescence signals to various biological parameters.

Monitoring cell concentration and activity by multiple excitation fluorometry.

Four key cellular metabolic fluorophores--tryptophan, pyridoxine, NAD(P)H, and riboflavin--were monitored on-line by a multiple excitation fluorometric system (MEFS) and a modified SLM 8000C scanning spectrofluorometer in three model yeast fermentation systems--bakers' yeast growing on glucose, Candida utilis growing on ethanol, and Saccharomyces cerevisiae RTY110/pRB58 growing on glucose. The measured fluorescence signals were compared with cell concentration, protein concentration, and cellular activity. The results indicate that the behavior and fluorescence intensity of various fluorophores differ in the various fermentation systems. Tryptophan fluorescence is the best signal for the monitoring of cell concentration in bakers' yeast and C. utilis fermentations. Pyridoxine fluoresce is the best signal for the monitoring of cell concentration in the S. cerevisiae RTY110/pRB58 fermentation. In bakers' yeast fermentations the pyridoxine fluorescence signal can be used to monitor cellular activity. The NAD(P)H fluorescence signal is a good indicator of cellular activity in the C. utilis fermentation. For this fermentation NAD(P)H fluorescence can be used to control ethanol feeding in a fed-batch process.

Energy requirements for the size reduction of poplar and aspen wood.

The energy requirements associated with conventional mechanical size reduction of poplar and aspen wood are compared to a new method of size reduction employing a wood planer. Although the planer requires about 2.3 times less energy to achieve the same size reduction as conventional methods, large-scale equipment to implement this approach does not currently exist. Explosive depressurization was also compared to conventional mechanical size reduction. The conventional mechanical methods require roughly 70% more energy to achieve the same size reduction as explosive depressurization. Thus, explosive depressurization appears to be the preferred method and has the added benefit of altering the chemical structure of the wood to enhance the enzymatic hydrolysis of the cellulose fraction.

Acetate inhibition of Pseudomonas putida.

To study the effect of acetate inhibition on the parameters of yield and maintenance for bacterial growth, Pseudomonas putida ATCC 23467 was grown in a minimal salts medium with acetate as the sole carbon source with limiting and with excess quantities of urea in the feed medium. The behavior of the chemostat cultures under sole acetate limitation results in low residual acetate present in the fermentation broth. These cultures can be described satisfactorily using the equation q(s) = D/Y(g) + m, i.e., the acetate is consumed only for growth and maintenance,. Those cultures in which urea was limiting or where urea was present in large excess contained significant amounts of residual acetate in the broth. For these cultures it was necessary to add a third term for acetate inhibition to the above expression.

Kinetic characterization of the extracellular xylanases of Thermomonospora sp.

This study deals with characterizing the extracellular xylanases produced by a strain of the thermophilic bacterial genus Thermomonospora. Supernatant from centrifuged fermentation broth was used as a crude enzyme preparation. From pH 5.5 to pH 7.7 the temperature optimum based on a 10-min assay of activity was 80 degrees C. The crude enzyme had a half-life of approximately 1 month when stored at 55 degrees C at pH 6.5. The enzyme produced a mixture of xylose oligomers from xylan, with xylobiose occuring in greatest quantity on a molar basis. Only trace quantities of xylose were produced by this hydrolysis.

The beta-xylosidase of Thermomonospora.

This study is concerned with characterizing cell-bound inducible beta-xylosidase produced by a strain of the thermophilic bacterial genus Thermomonospora. A crude preparation of this enzyme recovered from sonicated cells of this organism displayed high activity against paranitrophenyl-beta-xylopyranoside over a pH range of 5.5-7.7. The temperature optimum, based on a 30-min assay of activity, at pH 6.5 was 70 degrees C. The crude enzyme had a thermal half-life of approximately 1 week at 55 degrees C and pH 6.5. Xylose inhibited the enzyme. Values of K(m) and V(max) are estimated from the reaction rate data as 0.82 mM and 8 U/L, respectively.

A comparison of two empirical models for the enzymatic hydrolysis of pretreated poplar wood.

Two empirical models for the enzymatic hydrolysis of cellulose are used to analyze the same set of experimental data to determine if one model is superior to the other. Both models adequately describe the data. The parameters in both models may be correlated to the hydrolysis conditions of enzyme, substrate, and product inhibitor concentrations. Both empirical models have features which are consistent with theoretical models of cellulose hydrolysis.

The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar.

The use of alcohol/water/catalyst mixtures to delignify wood allows the lignin to be recovered in a usable form while leaving the carbohydrate fraction relatively intact. The effects of temperature, reaction time, and the type of solvent and catalyst on the delignification of milled poplar wood were investigated. The lignin, cellulose, and hemicellulose composition of the pretreated material was measured for each treatment condition. In addition, the pretreated samples were subjected to enzymatic hydrolysis using the cellulases produced by the thermophilic bacterium Thermomonospora sp. YX. The extent of enzymatic hydrolysis was characterized using an empirical model, and the results were used to examine the effectiveness of the pretreatment.

Determining the inhibition constants in the HCH-1 model of cellulose hydrolysis.

The products of cellulose hydrolysis, glucose and cellobiose, caused noncompetitive inhibition patterns to be exhibited when Thermomonospora sp. YX cellulase hydrolyzed dyed cellulose. The glucose binding constant, beta(1), was 0.00683 +/- 0.00031 L/g and the cellobiose binding constant, beta(2), was 0.095 +/- 0.0058 L/g. Thus, cellobiose was about 14 times more inhibitory than glucose.

The HCH-1 model of enzymatic cellulose hydrolysis.

Solka Floc BW200 was enzymatically hydrolyzed in a batch reactor using a commercial cellulase preparation. A total of 50 different hydrolysis conditions were run within a 10-fold range in enzyme concentration and a 30-fold range in cellulose concentration. The data were evaluated in three ways using five different models. Previous literature models were not as successful in correlating the data as the HCH-1 Model derived in this work.

Estimation of fermentation biomass concentration by measuring culture fluorescence.

The fluorescence of a fermentation culture was studied for its application as an estimator of biomass concentration. The measurement was obtained by irradiating the culture with ultraviolet light (366 nm) through a glass window and detecting fluorescent light at the window surface at 460 nm. It was estimated that over one-half of the fluorescent material was intercellular reduced nicotinamide adenine dinucleotide, with the remainder being reduced nicotinamide adenine dinucleotide phosphate and other unidentified intercellular and extracellular fluorophores. The culture fluorescence was found to be a function of biomass concentration, together with environmental factors, which presumably act at the cellular metabolic level to modify intercellular reduced nicotinamide adenine dinucleotide pools (e.g., dissolved oxygen tension, energy substrate concentration, and inhibitors). When these environmental conditions were controlled, a linear relationship was obtained between the log of the biomass concentration and the log of the fluorescence. Under these conditions, this relationship has considerable potential as a method to provide real-time biomass concentration estimates for process control and optimization since the fluorescence data is obtained on line. When environmental conditions are variable, the fluorescence data may be a sensitive index of overall culture activity because of its dependence on intercellular reduced nicotinamide adenine dinucleotide reserves and metabolic rates. This index may provide information about the period of maximum specific productivity for a specific microbial product.

Galactose oxidase: applications of the covalently immobilized enzyme in a packed bed configuration.

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.

Pore diffusion model for a two-substrate enzymatic reaction: application to galactose oxidase immobilized on porous glass particles.

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.

Dynamic and steady state studies of phenol biodegradation in pure and mixed cultures.

The microbial degradation of phenol by pure and mixed cultures of Pseudomonas putida was studied in batch, phenol-stat, and continuous culture systems. In the continuous culture runs, both steady state and transient experiments were performed. From these experiments, a model for the kinetic behavior of the organisms was evolved and an analysis performed on the stability and dynamic behavior of pure and mixed cultures. The results indicate that it should be possible to achieve phenol removal from wastewaters down to levels of 1-2 ppm in a single state system. However, because of the effect of substrate inhibition on kinetic behavior of the microorganisms, long lasting transients can occur. The transient behavior of such systems cannot be solely determined from mumax or Ks parameters, but must include a consideration of the transient size and response characteristic of the organism.