Showing papers in "Biotechnology and Bioengineering in 1974"
TL;DR: In this article, a variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates, and the rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities.
Abstract: Waste cellulose was a suitable carbon source for cellulose production by Trichoderma viride. The enzyme can be produced in submerged fermentation using newspaper as a growth substrate. A variety of pure and complex cellulosic materials were hydrolyzed by culture filtrates. Saccharification of 5% slurries after 48 hr ranged from 2–92%. The rate and extent of hydrolysis was controlled by degree of crystallinity, particle size, and presence of impurities. Newspaper was used to evaluate methods for the pretreatment of substrate. The best pretreatment was ball milling which gave good size reduction, maximum bulk density, and maximum susceptibility. Hammer milling, fluid energy milling, colloid milling, or alkali treatments were less satisfactory. Dissolving cellulose in cuprammonium, or carbon disulfide (Viscose) and then reprecipitating gave a susceptible, but low bulk density product. However the susceptibility was lost if the substrate was dried. Because of costs, low bulk density, necessity of keep ing the substrate wet, and generation of chemical waste streams dissolving cellulose to increase reactivity does not seem a practical approach.
Cellulose fractions separated from municipal trash or agricultural residues such as milled fibres from bovine manure are promising substrates for conversion.
445 citations
TL;DR: In this article, the authors present reaction rate constants for anaerobic fermentation of domestic refuse and evaluate the effect of temperature on the rate of methane fermentation using the Monod model.
Abstract: Anaerobic fermentation of organic solid waste can provide a significant source of fuel gas (methane). Application of this process requires a better understanding of the kinetics of the biological system. The literature is replete with kinetic studies of this process as applied to waste solids from water pollution control systems. Much of this work has been conducted in the mesophilic temperature range. Increased temperatures yield higher reaction rates that will improve the economics of the process. The rate limiting step in the fermentation of refuse is the hydrolysis of the complex organic solids, in particular cellulose. Cellulose is a major component of the refuse. A laboratory study employing domestic refuse has shown the effect of temperature on the rate of methane fermentation. The optimum mesophilic temperature was found to be 42°C, while the optimum thermophilic temperature was at least 60°C. No data was obtained beyond the 60°C temperature. Reaction rate constants are presented for anaerobic fermentation of domestic refuse. Because of the characteristics of the substrate it−was not possible to obtain the necessary measurements for evaluation of constants in the Monod model. An overall system constant was developed.
211 citations
TL;DR: A model of the continuous mixed culture under oxygen limitation has been formulated and Qualitative predictions of transient phenomena by the model have been verified experimentally.
Abstract: The growth of a mixed methane-utilizing culture in a continuous flow fermenter has been studied under both methane and oxygen limitation. Small additions of methanol have been shown to inhibit the methane-utilizing moiety in the culture and it has been shown that the Hyphomicrobium sp. in the mixed culture removes any inhibitory methanol. The interaction between the methane-utilizing Pseudomonas sp., and the Hyphomicrobium sp. has been explained and a model of the continuous mixed culture under oxygen limitation has been formulated. Qualitative predictions of transient phenomena by the model have been verified experimentally.
139 citations
TL;DR: Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable COOH.
Abstract: Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.
120 citations
TL;DR: The intrusion of diffusion in heterogeneous enzyme reactions, which follow, is described and a graphical method is described for the evaluation of the reaction rate as a function of the surface concentration of the substrate from measured data.
Abstract: The intrusion of diffusion in heterogeneous enzyme reactions, which follow. Michaelis-Menten kinetics, is quantitatively characterized by dimensionless parameters that are independent of the substrate concentration. The effects of these parameters on the overall rate of reaction is illustrated on plots commonly employed in enzyme kinetics. The departure from Michaelis-Menten kinetics due to diffusion limitations can be best assessed by using Hofstee plots which are also suitable to distinguish between internal and external transport effects. A graphical method is described for the evaluation of the reaction rate as a function of the surface concentration of the substrate from measured data.
110 citations
TL;DR: Maximal rates at which E. coli can grow on glucose as carbon source under various limiting oxygen‐supply conditions were determined in a bench‐scale fermentor in order to study aerobic cell growth in a fermentor.
Abstract: Oxygen supply is one of the main factors which influences aerobic cell growth in a fermentor. Maximal rates at which E. coli can grow on glucose as carbon source under various limiting oxygen-supply conditions were determined in a bench-scale fermentor. Culture conditions are described which gave yields of about 38 g dry cells per liter medium.
109 citations
TL;DR: In this article, an experimental investigation on the rheology of penicillin broths was undertaken in order to obtain more understanding of this important aspect of the fermentation process, which consisted in observation of the torque exerted upon a rotating turbine impeller.
Abstract: An experimental investigation on the rheology of penicillin broths was undertaken in order to obtain more understanding of this important aspect of the fermentation process. The measuring technique consisted in observation of the torque exerted upon a rotating turbine impeller. The experimental data were interpreted in terms of a model which basically is a synthesis of a known relationship for the rheological behavior of printing ink (Casson equation) and some considerations analogous to the rheological description of polymer solutions (excluded volume concept). One of the key variables in the model is a morphology factor, which can be used for a quantitative description of mycelial morphology. The value of the morphology factor can be determined experimentally by a simple viscosity measurement in combination with a mycelial dry weight determination. There are strong indications that the model may be applicable to mycelial broths other than those of penicillin.
106 citations
TL;DR: The disintegration of baker's yeast by a high pressure homogenizer, to a pressure of 25,000 psi.
Abstract: The disintegration of baker's yeast (Saccharomyces cerevisiae) by a high pressure homogenizer, to a pressure of 25,000 psi. (172.37 MNm−2) is described, together with details of the methods of measurement used to obtain information on the valve movement and pressure transients. The theory of the mechanism of cell disintegration is discussed.
102 citations
TL;DR: Thermophilic actinomyces appear to be the most effective organisms for single cell protein production from cellulosic wastes.
Abstract: Conventional sources of protein cannot meet the present or projected needs for human consumption. Single cell proteins from fermentation of petroleum and cellulosic wastes are likely sources of additional protein. The volume of cellulosic wastes is sufficient to supply all additional protein needs on a continuing basis for cellulose is a renewable resource. Both mesophilic and thermophilic microorganisms utilize cellulose at reasonable rates. Biodegradation of lignin and lignin–cellulose complexes constitutes a major obstacle to commercial utilization of cellulosic wastes. Thermophilic actinomyces appear to be the most effective organisms for single cell protein production from cellulosic wastes.
99 citations
TL;DR: The microbial cells of Pseudomonas putida ATCC 4359 were immobilized by entrapment in a polyacrylamide gel lattice and the permeability of substrate or product through the cell wall und the heat stability of the enzyme were increased by immobilization of the cells.
Abstract: The microbial cells of Pseudomonas putida ATCC 4359 were immobilized by entrapment in a polyacrylamide gel lattice. Enzymatic properties of L‐arginine deiminase of the immobilized P. putida cells were investigated and compared with those of the intact cells. The permeability of substrate or product through the cell wall und the heat stability of the enzyme were increased by immobilization of the cells. No difference was observed between pH activity curves of the intact and immobilized cells. The optimal temperature for the formation of L‐citrulline was 37°C for the intact cells and 55° C for the immobilized cells.
98 citations
TL;DR: In this paper, the enzyme β-galactosidase (lactose) obtained from several microbial sources was immobilized on zirconia-coated porous glass particles.
Abstract: The enzyme β‐galactosidase (lactose) obtained from several microbial sources was immobilized on zirconia‐coated porous glass particles. The immobilized enzymes were characterized by determining pH profiles, kinetic constants, thermal profiles, and operationalhalf‐lives in lactose and whey ultrafiltrate solutions. Studies were carried out on continuous reactor performance, and enzyme requirements for scale‐up were estimated. Lactose or whey hydrolyzed by this technique could find use commercially as a sweetener in a number of dairy products.
TL;DR: It is proposed that a high cellular lipid concentration is necessary for hydrocarbon assimilation, and this is not just a reflection of the lipophilic nature of the substrate.
Abstract: The yeast Candida tropicalis utilizes both glucose and hydrocarbons as sole carbon sources. When grown on hydrocarbons, the cells contain twice as much lipid as when grown on glucose. In transient continuous culture experiments, following a substrate change from glucose to hexadecane, an adaption phase occurred. During this phase the lipid concentration per cell increased greatly. It is proposed that a high cellular lipid concentration is necessary for hydrocarbon assimilation, and this is not just a reflection of the lipophilic nature of the substrate.
TL;DR: The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled‐pore titania has yielded a remarkably stable enzyme system.
Abstract: The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled-pore titania has yielded a remarkably stable enzyme system. Catalase apparently acts as both a stabilizer and an activator for glucose oxidase within the pores of this material. Hydrogen peroxide concentrations and flow rates have a marked effect upon the apparent activity of the immobilized enzyme system. The carrier parameters were varied to obtain optimum loading and stability information.
TL;DR: A theoretical treatment of the kinetics of reactions catalyzed by enzymes attached to the inner surface of a tube, through which the substrate solution passes, and criteria for insignificant and significant diffusional effects are proposed.
Abstract: A theoretical treatment is given of the kinetics of reactions catalyzed by enzymes attached to the inner surface of a tube, through which the substrate solution passes. A utilization factor, the ratio of the actual reaction rate to that in the absence of diffusional effects, is defined. A numerical procedure is proposed and numerical and approximate solutions for the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product, inhibition (non-competitive), and (e) product inhibition (anticompetitive). When the enzyme chemically attached to a tube obeys a Michaelis-Menten relationship, criteria for insignificant and significant diffusional effects are proposed.
TL;DR: A cultivation method is presented which permits the study of the effects of aeration intensity under conditions where the influence of catabolite repression is eliminated, and a completely synthetic medium with glucose as the only carbon and energy source is described.
Abstract: Efforts were made to eliminate the influence of other factors as far as possible in order to obtain reliable results on the effects of oxygen on the growth of baker's yeast. A cultivation method is presented which permits the study of the effects of aeration intensity under conditions where the influence of catabolite repression is eliminated. A completely synthetic medium with glucose as the only carbon and energy source is also described.
TL;DR: Several microorganisms having higher L‐histidine ammonia‐lyase activity were immobilized into polyacrylamide gel lattice and the yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667.
Abstract: Several microorganisms having higher L‐histidine ammonia‐lyase activity were immobilized into polyacrylamide gel lattice. The yield of enzyme activity by immobilization was highest in Achromobacter liquidum IAM 1667. As A. liquidum has urocanase activity, the cells were heat‐treated at 70°C for 30 min to inactivate the urocanase.
TL;DR: High protein alga was grown in effluents from the London municipal waste treatment plant and the advantages of this process in tertiary waste‐water treatment and the quality of the single cell protein were investigated.
Abstract: Spirulina maxima, a high protein alga, was grown in effluents from the London municipal waste treatment plant. Optimum growth conditions were developed, the composition of algae and the removal of nitrogen and phosphorus in effluents were studied. The advantages of this process in tertiary waste-water treatment and the quality of the single cell protein were investigated.
TL;DR: Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes.
Abstract: Numerical solutions to the equations describing simultaneous mass transfer and enzymic reaction within porous spherical particles have been used to examine the effect of enzyme content and other parameters on the kinetic behavior of immobilized enzymes. These solutions have also been compared with experimental data for enzymes immobilized to DEAE-cellulose particles. The influence of particle size and enzyme content on catalyst design is discussed.
TL;DR: Maltase, phosphatase, malate dehydrogenase, glucose‐6‐phosphate dehydrogensase, and 6-phosphogluconate dehydration activities were determined in the supernatant of brewer's yeast cells from samples taken at various intervals during a 90 min treatment in a Dyno‐Mill disintegrator.
Abstract: Maltase, phosphatase, malate dehydrogenase, glucose‐6‐phosphate dehydrogenase, and 6‐phosphogluconate dehydrogenase activities were determined in the supernatant of brewer's yeast cells from samples taken at various intervals during a 90 min treatment in a Dyno‐Mill disintegrator.
TL;DR: Viscosity profiles of polysaccharide isolated from various stages of the fermentation showed a change from Newtonian to pseudoplastic behavior depending on the concentration (0–2%) of poly Saccharide, which was found to be a sensitive factor for the indication of the non‐Newtonian behavior.
Abstract: Polysaccharide was synthesized by Aureobasidium pullulans (or Pullularia pullulans) 2552 in a sucrose medium. The field apparent viscosity of the culture medium from shake flask experiments rose to 24,500 cP and then dropped toward its initial value as the fermentation progressed. The magnitude of the maximum apparent viscosity depended on the initial pH of the fermentation broth. The inoculum age influenced the cultivation period before which the maximum viscosity was reached. Rheograms of the fermentation broths showed a change in viscosity behavior from Newtonian to pseudoplastic, and then toward Newtonian characteristics during the fermentation. The calculated non-Newtonian index was found to be a sensitive factor for the indication of the non-Newtonian behavior. Such behavior could not be detected from rheograms. Viscosity profiles of polysaccharide isolated from various stages of the fermentation showed a change from Newtonian to pseudoplastic behavior depending on the concentration (0–2%) of polysaccharide.
TL;DR: The immobilized enzymes could be easily recovered, from solutions or suspensions, magnetically, and was less active than invertase when acting on oligosaccharides of the raffinose family.
Abstract: Trypsin (EC 3.4.4.4) was immobilized in low yield on aminoalkylsilylated magnetite (Fe3O4). Better results were obtained when trypsin was immobilized by crosslinking with glutaraldehyde on magnetite. The preparation contained 36 mg protein/g magnetite and the enzyme retained 46% and 11% of esterase and proteolytic activity. Immobilized trypsin was more heat stable than trypsin. Invertase (β‐D‐fructofuranoside fructohydrolase, EC 3.2.1.26) was cross‐linked on magnetite with glutaraldehyde in low yield due to the inactivation of the enzyme. However in the presence of 1% sucrose, the total activity recovered was 79% of the initial activity and the preparation contained 4.4 mg/g of active invertase. Immobilized invertase was less active than invertase when acting on oligosaccharides of the raffinose family. The immobilized enzymes could be easily recovered, from solutions or suspensions, magnetically.
TL;DR: A model has been developed which predicts the rate of decomposition of peroxide and inactivation of catalase in a flow reactor and first order dependence on peroxide concentration is assumed.
Abstract: Immobilized beef liver catalase has been used in a flow reactor to decompose hydrogen peroxide; at the same time the catalase is inactivated by its substrate. A model has been developed which predicts this rate of decomposition of peroxide and inactivation of catalase. First order dependence on peroxide concentration is assumed. The model was verified by experiment for a range of operating conditions and then used to predict the effects of a change in operating variables.
TL;DR: The death kinetics of a strain of Saccharomyces cerevisiae were studied in an industrial scale spray drier and it was found that during drying, although the rate of death is high, the activation energy is greatly decreased over that of death in aqueous solution.
Abstract: The death kinetics of a strain of Saccharomyces cerevisiae were studied in an industrial scale spray drier. In solution studies, the death kinetics of yeast was found to be comparable to pathogen destruction. From the studies in drying of yeast a prediction of a 4 log cycle decrease in viable cells of pathogens could be made for normal processing conditions. This should insure the safety of spray-dried foods unless after contamination occurs. It was found that during drying, although the rate of death is high, the activation energy is greatly decreased over that of death in aqueous solution (reduction from 130 kcal/mole to 5 kcal/mole). The reduction in Ea may be attributed to the thermodynamic compensation phenomenon in which the resulting negative entropy of reaction acts to protect the cells through a water–protein interaction. However, the possibility of a change in death mechanism cannot be precluded. Overall, these results suggest the danger in extrapolating death kinetics to high temperature.
TL;DR: A theoretical treatment has been worked out for the kinetics of solid‐supported enzyme systems, with diffusive and electrostatic effects taken into account.
Abstract: A theoretical treatment has been worked out for the kinetics of solid-supported enzyme systems, with diffusive and electrostatic effects taken into account. A utilization factor, defined as the ratio of the actual reaction rate to the rate of substrate consumption in the outer solution, is defined, and equations to evaluate the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product inhibition (noncompetitive), and (e) product inhibition (anticompetitive). When the solid-supported enzymes obey a Michaelis-Menten relationship, an equation for the apparent Michaelis constant is given and a criterion for insignificant diffusion effects is shown. A substrate-inhibited enzyme reaction may display multiple steady-state behavior, and a criterion for uniqueness is presented. In the case of product-inhibited enzyme reactions, the utilization factor is always less than that which corresponds to a Michaelis-Menten relationship. Equations to evaluate the apparent Michaelis and inhibition constants are given.
TL;DR: Disintegration of microorganisms in a continuously working industrial homogenizer has been studied and this type of disintegrator can be used for disruption of cells in order to produce single‐cell protein, active enzymes and other valuable cell components.
Abstract: Disintegration of microorganisms in a continuously working industrial homogenizer has been studied. The homogenizer consists of rotating discs in a cylinder filled with glass beads. Different parameters for disintegration of baker's yeast were investigated. The disintegration process is a first‐order reaction and it is influenced by the flow rate of the suspension and by the agitator speed. At a flow rate of 200 liters/hr about 85% of the yeast cells can be disrupted in a single pass through the disintegrator. This type of disintegrator can be used for disruption of cells in order to produce single‐cell protein, active enzymes and other valuable cell components.
TL;DR: One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor, and immobilized enzyme stability is an important factor in enzyme reactor design.
Abstract: One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor. Consequently immobilized enzyme stability is an important factor in enzyme reactor design. The performance of the reactor will decrease if during operation the support material disintegrates into smaller particles that pass out of the reactor system.
When β-galactosidase is immobilized by covalent attachment to AE-cellulose, the smaller particles have a higher activity. After subjection of the immobilized enxyme to a shear stress the average particle size decreases and the total enzymic activity increases. A loss of small particles from the reactor, although constituting a small weight percent loss of support, will result in a disproportionately large loss in activity. The relevance of these observations to reactor performance is discussed.
TL;DR: The temperature‐dependent endogenous metabolism model of single species continuous culture dynamics is utilized in the computer simulation of the Kalman filter state estimation technique, and the state estimator is illustrated in the context of conventional control strategies.
Abstract: The temperature-dependent endogenous metabolism model of single species continuous culture dynamics is utilized in the computer simulation of the Kalman filter state estimation technique. Parameters of the nonlinear equations can be “tracked” while variance in measured states can be damped. The state estimator is illustrated in, the context of conventional control strategies.
TL;DR: At low substrate concentrations and flow rates the reactions are largely diffusion‐controlled, the enhanced Km(app) values being largely if not entirely due to the diffusion control; ionic strength studies showed electrostatic repulsion effects to be unimportant.
Abstract: L-Asparaginase has been attached by chemical means to the inner surface of nylon tubing. An experimental study has been carried out of the flow kinetics for such a system, asparagine solutions at various concentrations being passed through two lengths of tubing at various flow rates. Measurements were made of the concentration of the product ammonia at the tube exit, and of the rate of formation of ammonia, under the various conditions. Apparent Michaelis constants, Km(app), were some three orders of magnitude higher than the Km for the enzyme in free solution (∼13 × 10−6JM). The results were analyzed with respect to the theoretical treatment described in the preceding paper (Kobayashi and Laidler), three different methods being employed. It is concluded that at lower substrate concentrations and flow rates the reactions are largely diffusion-controlled, the enhanced Km(app) values being largely if not entirely due to the diffusion control; ionic strength studies showed electrostatic repulsion effects to be unimportant. At high concentrations and high flow rates (when the diffusion layer is of negligible thickness) the diffusional effects are minimized, and Km(app) approaches the true Km value for the immobilized enzyme.
TL;DR: Glass‐immobilized A. niger laclases (lactase‐BG) retained much hydrolytic activity after storage and periodic use for 165 days at 55°C and pH 4.5.
Abstract: Partially purified lactoses (β-D-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus niger, Ladobacillus helveticus, and Saccharomyces lactis were immobilized on diazotized porous glass particles (mean pore diameter, 86.5 nm: particle size diameters, 75–125 μm). In acid whey containing 4–4.5% lactose, A. niger lactase gave the highest activity (89 μmoles lactose hydrolyzed/g glass, min) at 55°C and pH 4.5. Glass-immobilized A. niger laclases (lactase-BG) retained much hydrolytic activity after storage and periodic use for 165 days at 55°C. For values of X greater than 30%, hydrolysis of 0.12M lactose in acid whey by a continuous flow column packed with 2 ml of lactase-BG particles could be correlated by X = 17.2(V/F) + 12.5 where X = lactose hydrolysis, percent of lactose originally present; V = volume of packed bed of lactase-BG, ml; F = flow rate of acid whey, ml/min.