Showing papers in "Biotechnology and Bioengineering in 1993"

Biosorption of cadmium by biomass of marine algae

Abstract: Biomass of nonliving, dried brown marine algae Sargassum natans, Fucus vesiculosus, and Ascophyllum nodosum demonstrated high equilibrium uptake of cadmium from aqueous solutions. The metal uptake by these materials was quantitatively evaluated using sorption isotherms. Biomass of A. nodosum accumulated the highest amount of cadmium exceeding 100 mg Cd[sup 2+]/g (at the residual concentration of 100 mg Cd/L and pH 3.5), outperforming a commercial ion exchange resin DUOLITE GT-73. A new biosorbent material based on A. nodosum biomass was obtained by reinforcing the algal biomass by formaldehyde cross-linking. The prepared sorbent possessed good mechanical properties, chemical stability of the cell wall polysaccharides and low swelling volume. Desorption of deposited cadmium with 0.1-0.5 M HCl resulted in no changes of the biosorbent metal uptake capacity through five subsequent adsorption/desorption cycles. There was no damage to the biosorbent which retained its macroscopic appearance and performance in repeated metal uptake/elution cycles.

A mathematical model for dynamic simulation of anaerobic digestion of complex substrates: focusing on ammonia inhibition.

Abstract: A mathematical model for anaerobic degradation of complex organic material, such as manure, has been developed. The model includes an enzymatic hydrolytic step and four bacterial steps and involves 12 chemical compounds. The model focuses on ammonia inhibition and includes a detailed description of pH and temperature characteristics in order to accurately simulate free ammonia concentration. Free ammonia and acetate constitute the primary modulating factors in the model. The model has been applied for the simulation of digestion of cattle manure in continuously stirred tank reactors (CSTRs), and results compare favorably with experimental data.

Activity studies of eight purified cellulases: Specificity, synergism, and binding domain effects.

Abstract: The activities of six purified Thermomonospora fusca cellulases and Trichoderma reesei CBHI and CBHII were determined on filter paper, swollen cellulose, and CMC. A simple method to measure the soluble and insoluble reducing sugar products from the hydrolysis of filter paper was found to effectively distinguish between exocellulases and endocellulases. Endocellulases produced 34% to 50% insoluble reducing sugar and exocellulases produced less than 8% insoluble reducing sugar. The ability of a wide variety of mixtures of these cellulases to digest 5.2% of a filter paper disc in 16 h was measured quantitatively. The specific activities of the mixtures varied from 0.41 to 16.31 micromol cellobiose per minute per micromole enzyme. The degree of synergism ranged from 0.4 to 7.8. T. reesei CBHII and T. fusca E3 were found to be functionally equivalent in mixtures. The catalytic domains (cd) of T. fusca endocellulases E2 and E5 were purified and found to retain 93% and 100% of their CMC activity, respectively, but neither cd protein could digest filter paper to 5.2%. When E2cd and E5cd were substituted in synergistic mixtures for the native proteins, the mixtures containing E2cd retained 60%, and those containing E5cd retained 94% of the original activity. Addition of a beta-glucosidase was found to double the activity of the best synergistic mixture. Addition of CBHI to T. fusca crude cellulase increased its activity on filter paper 1.7-fold.

Prevascularization of porous biodegradable polymers.

Abstract: Highly porous biocompatible and biodegradable polymers in the form of cylindrical disks of 13.5 mm diameter were implanted in the mesentery of male syngeneic Fischer rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was smaller for amorphous polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology. Poly(L-lactic acid) (PLLA) devices of 5 mm thickness, 24.5% crystallinity, 83% porosity, and 166 μm median pore diameter were filled by tissue after 25 days. However, the void volume of prevascularized devices (4%) was minimal and not practical for cell transplantation. In contrast, for amporphous PLLA devices of the same dimensions, and the similar porosity of 87% and median pore diameter of 179 μm, the tissue did not fill completely prevascularized devices, and an appreciable percentage (21%) of device volume was still available for cell engraftment after 25 days of implantation. These studies demonstrate the feasibility of creating vascularized templates of amorphous biodegradable polymers for the transplantation of isolated or encapsulated cell populations to regenerate metabolic organs and tissues. © 1993 John Wiley & Sons, Inc.

Formation of microparticulate protein powder using a supercritical fluid antisolvent

Abstract: Gas antisolvent (GAS) expansion of dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMFA) solutions with supercritical carbon dioxide was used to produce biologically active powders of insulin. Powders with 90% of the particles smaller than 4 μm and 10% smaller than 1 μm were obtained under all conditions tested when the process was operated continuously, with small liquid droplets sprayed into a flowing supercritical continuum. Slow pressurization of the stagnant protein solution resulted in larger particles. In vivo tests on rats revealed no differences between the biological activity of processed and unprocessed insulin, GAS processing of organic solution appears to be a reliable and effective method for the production of dry, biologically active microparticulate powders of peptides and proteins. © 1993 John Wiley & Sons, Inc.

Study of the Enzymatic Hydrolysis of Cellulose for Production of Fuel Ethanol by the Simultaneous Saccharification and Fermentation Process

Abstract: The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and beta-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and beta-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and beta-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production.

Cadmium biosorption by Saccharomyces cerevisiae

Abstract: Cadmium uptake by nonliving and resting cells of Saccharomyces cerevisiae obtained from aerobic or anaerobic cultures from pure cadmium-bearing solutions was examined. The highest cadmium uptake exceeding 70 mg Cd/g was observed with aerobic baker's yeast biomass from the exponential growth phase. Nearly linear sorption isotherms featured by higher sorbing resting cells together with metal deposits localized exclusively in vacuoles indicate the possibility of a different metal-sequestering mechanism when compared to dry nonliving yeasts which did not usually accumulate more than 20 mg Cd/g. The uptake of cadmium was relatively fast, 75% of the sorption completed in less than 5 min.

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p‐xylene by two Pseudomonas isolates.

Abstract: Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene.

Effect of surfactants on cellulose hydrolysis

Abstract: The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low.

Removal of lead from aqueous solutions by Penicillium biomass.

Abstract: The removal of lead ions from aqueous solutions by adsorption on nonliving Penicillium chrysogenum biomass was studied. Biosorption of the Pb(+2) ion was strongly affected by pH. Within a pH range of 4 to 5, the saturated sorption uptake of Pb(+2) was 116 mg/g dry biomass, higher than that of activated charcoal and some other microorganisms. At pH 4.5, P. chrysogenum biomass exhibited selectivity for Pb(+2) over other metal ions such as Cd(+2), Cu(+2), Zn(+2), and As(+3) Sorption preference for metals decreased in the following order: Pb > Cd > Cu > Zn > As. The sorption uptake of Pb(+2) remained unchanged in the presence of Cu(+2) and As(+3), it decreased in the presence of Zn(+2), and increased in the presence of Cd(+2).

Biofiltration of methanol vapor.

Abstract: Biofiltration of solvent and fuel vapors may offer a cost-effective way to comply with increasingly strict air emission standards. An important step in the development of this technology is to derive and validate mathematical models of the biofiltration process for predictive and scaleup calculations. For the study of methanol vapor biofiltration, an 8-membered bacterial consortium was obtained from methanol-exposed soil. The bacteria were immobilized on solid support and packed into a 5-cm-diameter, 60-cm-high column provided with appropriate flowmeters and sampling ports. The solid support was prepared by mixing two volumes of peat with three volumes of perlite particles (i.e., peat-perlite volume ratio 2:3). Two series of experiments were performed. In the first, the inlet methanol concentration was kept constant while the superficial air velocity was varied from run to run. In the second series, the air flow rate (velocity) was kept constant while the inlet methanol concentration was varied. The unit proved effective in removing methanol at rates up to 112.8 g h(-1) m(-3) packing. A mathematical model has been derived and validated. The model described and predicted experimental results closely. Both experimental data and model predictions suggest that the methanol biofiltration process was limited by oxygen diffusion and methanol degradation kinetics.

A thermodynamically based correlation for maintenance gibbs energy requirements in aerobic and anaerobic chemotrophic growth.

Abstract: A thermodynamic framework has been provided for the description of maintenance requirements of microorganisms. The central parameter is the biomass specific Gibbs energy consumption for maintenance, m(E) (kJ/C-mol biomass . h). A large set of data has been used including (i) a large range of different organisms (bacteria, yeasts, plant cells), (ii) mixed cultures, (iii) heterotrophic and autotrophic growth, (iv) growth under aerobic and anaerobic conditions, and (v) a large temperature range (5-75 degrees C). It appears that only the temperature has a major influence, with an energy of activation of 69 kJ/mol. Different electron donors or electron acceptors only show a very minor influence on m(E). On the basis of the data set, temperature correlations of m(E) have been derived for aerobic and anaerobic growth. The generalized concept for maintenance Gibbs energy is used to establish a correlation which allows the estimation of the biomass yield on electron donor as a function of C-source, electron donor, electron acceptor, N source, growth rate, and temperature. The advantage of using the m(E) parameter over other maintenance-related parameters (like mu(e), m(O2), m(D), gamma(D)m(D)) is discussed.

Biochemical production capabilities of escherichia coli

Abstract: Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products.

Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies.

Abstract: Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies, In case of expression of eukaryotic proteins containing cysteine, which may form disulfide bonds in the native active protein, often nonnative inter- and intramolecular disulfide bonds exist in the inclusion bodies. Hence, several methods have been developed to isolate recombinant eukaryotic polypeptides from inclusion bodies, and to generate native disulfide bonds, to get active proteins. This article summarizes the different steps and methods of isolation and renaturation of eukaryotic proteins containing disulfide bonds, which have been expressed in E. coli as inclusion bodies, and shows which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins.

Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia Sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp.

Abstract: The toxicity of homologous series of organic solvents has been investigated for the gram-positive bacteria, Arthrobacter sp. and Nocardia sp., and the gram-negative bacteria, Acinetobacter sp. and Pseudomonas sp. The hydrophobicity of the solvent, expressed by its logP(octanol), proves to be a good measure for the toxicity of solvents in a two-phase system. The transition from toxic to nontoxic solvents occurs between logP(octanol) 3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logP(octanol), above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram-negative bacteria is found to be slightly higher than that of gram-positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two-phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one-phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram-positive Arthrobacter and the gram-negative Acinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al. Enzyme Microb. Technol. 1990, 12: 281-291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible.

Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study.

Abstract: The theoretical yield of poly-D(-)-3-hydroxybutyrate (PHB) has been estimated from the biochemical pathway leading to PHB when a carbohydrate (glucose), a C(1) compound (methanol), a C(2) compound (acetic acid), or a C(4) compound (butyric acid) is used as a carbon source. In estimating the yield, recycling (or regeneration) of NADP(+)/ (NADPH + H(+)) and NAD(+) /(NADH + H(+)) have been taken into account. A special emphasis is made on te regeneration of NADPH, which is the coenzyme of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADPH-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. An equation which predicts the overall yield of PHB when non-PHB residual biomass is actually formed has been derived as a function of both the theoretical yield and PHB content of the dry cell mass. The ratio of the overall yield to the theoretical yield is roughly proportional to the PHB content.

Evaluation of a hepatocyte‐entrapment hollow fiber bioreactor: A potential bioartificial liver

Abstract: We have developed a hepatocyte entrapment hollow fiber bioreactor for potential use as a bioartificial liver. Hepatocytes were entrapped in collagen gel inside the lumen of the hollow fibers. Medium was perfused through the intraluminal region after contraction of the hepatocyte-entrapment gel. Another medium stream, comparable to the patient's blood during clinical application, passed through the extracapillary space. Viability of hepatocytes remained high after 5 days as judged by the rate of oxygen uptake and viability staining. Urea and albumin synthetic activities were also sustained. Transmission electron microscopic examination demonstrated normal ultrastructural integrity of hepatocytes in such a bioreactor. With its sort-term, extracorporeal support of acute liver failure, the current bioreactor warrants further investigation. © 1993 John Wiley & Sons, Inc.

The Kinetics of Cometabolism

Abstract: Experimental observations indicate that the rates of cometabolic transformation are linked to the consumption of growth substrate during growth and to the consumption of cell mass and/or energy substrate in the absence of growth substrate. Three previously proposed models (models 1 through 3) describing the kinetics of cometabolism by resting cells are compared, and the interrelationships and underlying assumptions for these models are explored. Models 1 to 3 are shown to converge at high concentrations of the nongrowth substrate. An expression describing nongrowth substrate transformation in the presence of growth substrate is proposed, and this expression is integrated with an expression for cell growth to give a single unstructured model (model 4) that encompasses models 1 to 3 and describes cometabolism by both resting and growing cells. Model 4 couples transformation of nongrowth substrate to consumption of growth substrate and biomass, and predicts that cometabolism will result, and decreased specific growth rates for a cometabolizing population. Competitive inhibition can also be incorporated in the model. Experimental aspects of model calibration and verification are discussed. The need for models that distinguish between the exhaustion of cell activity and cell death is emphasized. © 1993 Wiley & Sons, Inc.

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

Abstract: When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH2, their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its “regular” counterpart. © 1993 John Wiley & Sons, Inc.

A statistical analysis of the effect of substrate utilization and shear stress on the kinetics of biofilm detachment

Abstract: One of the least understood processes affecting biofilm accumulation is detachment. Detachment is the removal of cells and cell products from an established biofilm and subsequent entrainment in the bulk liquid. The goal of this research was to determine the effects of shear stress and substrate loading rate on the rate of biofilm detachment. Monopopulation Pseudomonas aeruginosa and undefined mixed population biofilms were grown on glucose in a RotoTorque biofilm reactor. Three levels of shear stress and substrate loading rate were used to determine their effects on the rate of detachment. Suspended cell concentrations were monitored to determine detachment rates, while other variables were measured to determine their influence on the detachment rate. Results indicate that detachment rate is directly related to biofilm growth rate and that factors which limit growth rate will also limit detachment rate. No significant influence of shear on detachment rate was observed. A new kinetic expression that incorporates substrate utilization rate, yield, and biofilm thickness was compared to published detachment expressions and gives a better correlation of data obtained both in this research and from previous research projects, for both mono- and mixed-population biofilms. © John Wiley & Sons, Inc.

A model of biofilm detachment

Abstract: A general mathematical framework for modeling biofilm detachment is presented. The approach is founded on a material balance on biomass that equates the detachment rate to the product of a detachment frequency and a detaching particle mass. The model provides a theoretical basis for deriving many of the empirical detachment rate expressions in common use and can thus lend some insight into their physical and biological significance. By allowing for variation in the detachment frequency with depth in the biofilm, the model permits derivation of detachment expressions that reflect a dependence on chemical or physiological gradients in the biofilm. Analysis of literature data sets from two different biofilm systems suggests, in both cases, that detachment is a growth-associated phenomenon. © 1993 John Wiley & Sons, Inc.

Electric prompting and control of denitrification

Abstract: The effectiveness of a denitrification process which is driven and controlled by an electric current is demonstrated. Denitrifying microorganisms were immobilized on a carbon electrode and hydrogen was produced through the electrolysis of water. The hydrogen was utilized for the reduction of nitrate to N(2). The denitrification rate was a linear function of the electric current, and it was shown that about 1 mol of electron reduces 0.2 mol of nitrate to N(2) gas. These results exhibit that the proposed process is simple and feasible, especially for the treatment of low-strength nitrate solutions.

Dynamics of phenol degradation by Pseudomonas putida.

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

Substantial overproduction of antibodies by applying osmotic pressure and sodium butyrate.

Abstract: Much of the current cell technology has enabled increased antibody production levels due to judicious nutrient feeding to raise cell densities and design better bioreactors. This study demonstrates that hybridomas can be hyperstimulated to produce higher immunoglobulin (lg) levels by suppressing cell growth and increasing culture longevity through adaptation to higher osmolarity media and addition of sodium butyrate. Prior to adaptation, cells placed in higher osmotic pressures (350 and 400 mOsm) were severely suppressed in growth down to 25% of the control (300 mOsm), although total lg titers achieved were similar to the control, approximately 140 mg/L. After a week of adaptation to 350 and 400 mOsm media, cell growth was not as dramatically suppressed, but considerably higher lg levels were attained at these elevated osmolarities. The highest yield of 265 mg/L was obtained at 350 mOsm compared to 140 mg/L at 300 mOsm, while maximum viable cell numbers dropped from 35 x 10(5) cells/mL to 31 x 10(5) cells/mL and culture longevity was extended by 20 h more than the control. Sodium butyrate, known to enhance protein production in other cell types, was then supplemented at a range of concentrations between 0.01 and 0.4 mM to the 350 mOsm culture to further enhance the lg levels. Butyrate at a concentration of 0.1 mM, in combination with osmotic pressure at 350 mOsm, further elevated the lg levels to 350 mg/L. Concomitantly, maximum viable cell numbers were reduced to 22 x 10(5) cells/mL, but culture longevity was extended by 40 h in the 0.1 mM butyrate supplemented culture compared to the control condition. Specific antibody productivity, q(Mab), continued to stay high during the stationary phase and was further elevated during the decline phase: thus, overall lg levels can be increased by 2.3 times by combining osmotic pressure and butyrate treatment.

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon.

Abstract: The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain.

Production of indole alkaloids by selected hairy root lines of Catharanthus roseus.

Abstract: Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC-MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine. © 1993 John Wiley & Sons, Inc.

Industrial separation of carboxylic and amino acids by liquid membranes: applicability, process considerations, and potential advantage.

Abstract: Liquid–liquid extraction and membrane separation are well-known separation method of extensive industrial application. Their incorporation into liquid membranes has the potential of several advantages, some of which are of particular interest for the recovery of carboxylic and amino acids: selectivities higher than those attainable by current separation methods, saving on energy costs for final concentration of separated products, high fluxes, compact installation, and low capital and operation costs. Stability of the liquid advantages, can be secured by utilizing extractant blocking polymeric membranes, Applicability, process consideration, and economic implications for recovery for carboxylic and amino acids by various extractant/membrane combinations are discussed. © 1993 John Wiley & Sons, Inc.

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis.

Abstract: Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations.

Anaerobic waste-activated sludge digestion - A bioconversion mechanism and kinetic model

Abstract: The anaerobic bioconversion of raw and mechanically lysed waste-activated sludge was kinetically investigated. The hydrolysis of the biopolymers, such as protein, which leaked out from the biological sludge with ultrasonic lysis, was a first-order reaction in anaerobic digestion and the rate constant was much higher that the decay rate constant of the raw waste activated sludge. An anaerobic digestion model that is capable of evaluating the effect of the mechanical sludge lysis on digestive performance was developed. The present model includes four major biological processes-the release of intracellular matter with sludge lysis; hydrolysis of biopolymers to volatile acids; the degradation of various volatile acids to acetate; and the conversion of acetate and hydrogen to methane. Each process was assumed to follow first order kinetics. The model suggested that when the lysed waste-activated sludge was fed, the overall digestive performance remarkably increased in the two-phase system consisting of an acid forming process and a methanogenic process, which ensured the symbiotic growth of acetogenic and methanogenic bacteria.

A two-plane tubular photobioreactor for outdoor culture of Spirulina.

Abstract: A photobioreactor in the form of a 245-m-long loop made of plexiglass tubes having an inner diameter of 2.6 cm was designed and constructed for outdoor culture of Spirulina. The loop was arranged in two planes, with 15 8-m-long tubes in each plane. In the upper plane, the tubes were placed in the vacant space between the ones of the lower plane. The culture recycle was performed either with two airlifts, one per plane, or with two peristaltic pumps. The power required for water recycle in the tubular photobioreactor, with a Reynolds number of 4000, was 3.93 x 10(-2) W m(-2). The photobioreactor contained 145 L of culture and covered an overall area of 7.8 m(2). The photobioreactor operation was computer controlled. Viscosity measurements performed on Spirulina cultures having different biomass concentrations showed non-Newtonian behavior displaying decreasing viscosity with an increasing shear rate. The performance of the two-plane photobioreactor was tested under the climatic conditions of central Italy (latitude 43.8 degrees N, longitude 11.3 degrees E). A biomass concentration of 3.5 g L(-1) was found to be adequate for outdoor culture of Spirulina. With a biomass concentration of 6.3 g L(-1), the biomass output rate significantly decreased. The net biomass output rate reached a mean value of 27.8 g m(-2) d(-1) in July; this corresponded to a net photosynthetic efficiency of 6.6% (based on visible irradiance).