Showing papers in "Biotechnology and Bioengineering in 1984"

Diffusion characteristics of substrates in Ca-alginate gel beads.

Abstract: The diffusion characteristics of several substrates of varying molecular sizes into and from Ca-alginate gel beads in well-stirred solutions were investigated. The values of the diffusion coefficient (D) of substrates such as glucose, L-tryptophan, and alpha-lactoalbumin [with molecular weight (MW) less than 2 x 10(4)] into and from the gel beads agreed with those in the water system. Their substrates could diffuse freely into and from the gel beads without disturbance by the pores in the gel beads. The diffusion of their substrates into and from the gel beads was also not disturbed by increasing the Ca-alginate concentration in the beads and the CaCl(2) concentration used in the gel preparation. In the case of higher molecular weight substances such as albumin (MW = 6.9 x 10(4)), gamma-globulin (MW = 1.54 x 10(5)) and fibrinogen (MW = 3.41 x 10(5)), the diffusion behaviors of the substrates into and from the gel beads were very different. No diffusion of their substrates into the gel beads from solutions was observed, and only albumin was partly absorbed on the surface of the gel beads. The values of D of their substrates from the gel beads into their solutions were smaller than their values in the water system, but all their substrates could diffuse from the gel beads. The diffusion of high molecular weight substrates was limited more strongly by the increase of Ca-alginate concentration in the gel beads than by the increase of the CaCl(2) concentration used in the gel preparation. Using these results, the capacity of Ca-alginate gel as a matrix of immobilization was discussed.

Alkaline peroxide delignification of agricultural residues to enhance enzymatic saccharification

Abstract: Approximately one-half of the lignin and most of the hemicellulose present in agricultural residues such as wheat straw and corn stover are solubilized when the residue is treated at 25 degrees C in an alkaline solution of hydrogen peroxide. The delignification reaction is most efficient when the ratio of hydrogen peroxide to substrate is at least 0.25 (w/w) and the pH is 11.5. The supernatant fraction from a given pretreatment, after addition of makeup peroxide and readjustment of the pH, can be recycled to treat at least six additional batches of substrate, resulting in a substantial concentration of hemicellulose and soluble lignin degradation products. Hydrolysis of the insoluble fraction with Trichoderma reesei cellulase after alkaline peroxide treatment yields glucose with almost 100% efficiency, based upon the cellulose content of the residue before treatment. These data indicate that alkaline peroxide pretreatment is a simple and efficient method for enhancing the enzymatic digestibility of lignocellulosic crop residues to levels approaching the theoretical maximum.

Effect of crystallinity and degree of polymerization of cellulose on enzymatic saccharification

Abstract: Lignocellulose materials were pretreated by methods known to improve enzymatic saccharification, and the percentage crystallinity (x-ray diffraction) and degree of polymerization were measured. It was observed that although the percentage crystallinity of cellulose was not altered by alkaline explosion (AE), carbon dioxide explosion (CE), ozone, and sodium chlorite treatment, very great increases were obtained in the extent of enzymatic saccharification. All the pretreatments studied except sodium chlorite caused significant reduction in degree of polymerization. It appears likely that the rate and extent of saccharification is governed by particle size, surface area, and degree of polymerization, since crystallinity effects alone do not explain the observed trends in the hydrolysis data.

Studies on on-line bioreactor identification. I. Theory.

Abstract: An integrated approach is presented for the on-line estimation of the state of a biochemical reactor from presently attainable real-time measurements. Elemental and macroscopic balances are used for the determination of the total rate of growth and state-of-the-art estimation techniques are subsequently employed for the elimination of process and measurement noises and the estimation of state variables and unknown culture parameters. The proposed approach is very flexible in that as new sensors become available they can be easily incorporated within the present framework to estimate new variables or improve the accuray of the old ones. The method does not require any model for the growth kinetics and is very successful in accurately estimating the above variables in the presence of intense noise and under both steady-state and transient conditions. State estimates obtained by the presented method can be used for the development of adaptive optimal control schemes as well as for basic studies of the characteristic properties of microbial cultures.

Algal autoflocculation—verification and proposed mechanism

Abstract: Biomass autoflocculation in outdoor algal cultures was found to be associated with increases of culture pH levels, due to CO(2) consumption by the algal photosynthetic activity. Under these alkaline conditions, some medium chemical ions precipitated together with the algal biomass. The chemical substances involved with the process and its dependence on pH value were studied by simulation of autoflocculation in laboratory experiments. Proper concentrations of calcium and orthophosphate ions in the medium are important for autoflocculation and, in order to attain it within the pH range 8.5-9.0, the culture should contain 0.1mM-0.2mM orthophosphate and 1.5mM-2.5mM calcium prior to raising the pH level. Calcium phosphate precipitates are considered as the flocculating agent which reacts with the negatively charged surface of the algae and promotes aggregation and flocculation.

Economic evaluation of alternative ethanol fermentation processes

Abstract: Eleven alternative fermentation schemes for ethanol production are compared. Conventional batch, continuous, cell recycle, and immobilized cell processes, as well as membrane, extraction, and vacuum processes which remove ethanol from the broth selectively as it is produced, are considered. The processes are compared on identical bases using a consistent model for the yeast metabolism. Both molasses and cellulose hydrolyzate are considered as feeds. Optimized ethanol plants, including feed preparation, fermentation, and product recovery sections are designed and total costs are projected.

Dephenolization of industrial wastewaters catalyzed by polyphenol oxidase.

Abstract: A new enzymatic method for the removal of phenols from industrial aqueous effluents has been developed. The method uses the enzyme polyphenol oxidase which oxidizes phenols to the corresponding o-quinones; the latter then undergo a nonenzymatic polymerization to form water-insoluble aggregates. Therefore, the enzyme in effect precipitates phenols from water. Polyphenol oxidase has been found to nearly completely dephenolize solutions of phenol in the concentration range from 0.01 to 1.0 g/L. The enzymatic treatment is effective over a wide range of pH and temperature; a crude preparation of polyphenol oxidase (mushroom extract) is as effective as a purified, commercially obtained version. In addition to phenol itself, polyphenol oxidase is capable of precipitating from water a number of substituted phenols (cresols, chlorophenols, naphthol, etc.). Also, even pollutants which are unreactive towards polyphenol oxidase can be enzymatically coprecipitated with phenol. The polyphenol oxidase treatment has been successfully used to dephenolize two different real industrial wastewater samples, from a plant producing triarylphosphates and from a coke plant. The advantage of the polyphenol oxidase dephenolization over the peroxidase-catalyzed one previously elaborated by the authors is that the former enzyme uses molecular oxygen instead of costly hydrogen peroxide (used by peroxidase) as an oxidant.

Computer model for glucose-limited growth of a single cell of Escherichia coli B/r-A.

Abstract: A computer model is described which is capable of predicting changes in cell composition, cell size, cell shape, and the timing of chromosome synthesis in response to changes in external glucose limitation. The model is constructed primarily from information on unrestricted growth in glucose minimal medium. The ability of the model to make reasonable quantitative predictions under glucose-limitation is a test of the plausibility of the basic biochemical mechanisms included in the model. Such a model should be of use in differentiating among competing hypotheses for biological mechanisms and in suggesting as yet unobserved phenomena. The last two points are illustrated with the testing of a mechanism for the control of the initiation of DNA synthesis and predictions on cell-width variations during the division cycle.

Cybernetic modeling of microbial growth on multiple substrates

Abstract: The internal regulatory processes, which underlie a variety of behavior in microbial growth on multiple substrates, are viewed as a manifestation of an invariant strategy to optimize some goal of the cells. A goal-seeking or cybernetic model is proposed here, with the optimization obased on a short-term perspective of response to the environment. The model parameters are determined from the growth data on single substrates. The model predicts the entire range of microbial growth behavior on multiple substrates from simultaneous utilization of all sugars to sequential utilization with pronounced diauxic lags. It is shown to predict the many variations of the diauxic phenomenon in different growth conditions. The transients in continuous culture growth on mixed substrates caused by varying the feed strategies are easily simulated by this model. The framework of this model can be applied to batch or continuous culture growth of many bacteria on different combinations of substrates.

Accumulation of heavy-metal ions by Zoogloea ramigera.

Abstract: Biomass has been produced from glucose using the organism Zoogloea ramigera 115. This biomass has been used to remove copper, cadmium, and uranyl ions from water solutions. The metal uptake was studied with two different methods: either by spectrophotometric measurements on the solutions after flocculation or by potentiometric measurements with amalgam electrodes in order to follow the entire complex formation. The metal-biopolymer interaction in 0.1M NaClO/sub 4/ is practically the same as when no neutral salt is added. The metal uptake is dependent on pH and is selective. A reversible process suitable for metal complexation is described.

Kinetics of the enzymatic hydrolysis of cellulose

Abstract: Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs.

Dissolved oxygen concentration profiles in a production-scale bioreactor.

Abstract: A five-compartment model for the liquid flow and the oxygen transfer into the liquid phase of a large-scale bioreactor is presented The aim of the model is to predict the following reactor operating variables: 1) the overall oxygen transfer capacity of the reactor; 2) the local liquid dissolved oxygen concentrations, for estimation of bad aerated zones which can introduce negative effects for the microorganisms and as a base for reliable scaledown experiments to estimate those effects; 3) the gas-phase exhaustion

Comparison of different strategies for the lipase-catalyzed preparative resolution of racemic acids and alcohols: Asymmetric hydrolysis, esterification, and transesterification.

Abstract: Lipase from Candida cylindracea has been found to be a highly stereospecific catalyst suitable for preparative resolution of racemic acids and alcohols. Using (R, S)-2-(p-chlorophenoxy) propionic acid (whose R isomer is a herbicide) and (R, S)-sec-butanol (a versatile synthon) as model compounds, three alternative approaches to lipase-catalyzed resolutions-asymmetric hydrolysis, esterification, and transesterification-have been compared. Enzymatic esterification in biphasic systems has been ruled out for preparative resolutions because addition of the acids lowers the pH of the aqueous phase thereby greatly reducing the efficiency of the procedure. Both enzymatic hydrolysis and biphasic transesterification afforded resolution of the racemates on a gram scale. From the standpoint of productivity, ease of product separation, and the amount of steps required, lipasecatalyzed asymmetric hydrolysis has been judged to be superior for the practical resolution of racemic acids, and lipase-catalyzed asymmetric transesterification to be the method of choice for the practical resolution of racemic alcohols.

Activity of Pseudomonas aeruginosa in biofilms: Steady state

Abstract: Aerobic glucose metabolism by Pseudomonas aeruginosa in steady-state biofilms at various substrate loading rates and reactor dilution rates was investigated. Variables monitored were substrate (glucose), biofilm cellular density, biofilm extracellular polymeric substance (EPS) density, and suspended cellular and EPS concentrations. A mathematical model developed to describe the system was compared to experimental data. Intrinsic yield and rate coefficients included in the model were obtained from suspended continuous culture studies of glucose metabolism by P. aeruginosa. Experimental data compared well with the mathematical model, suggesting that P. aeruginosa does not behave differently in steady-state biofilm cultures, where diffusional resistance is negligible, than in suspended cultures. This implies that kinetic and stoichiometric coefficients for P. aeruginosa derived in suspended continuous culture can be used to describe steady-state biofilm processes.

Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724.

Abstract: It is known that 2,3-butanediol is a potentially valuable chemical feedstock that can be produced from the sugars present in hemicellulose and celluose hydrolysates. Klebsiella oxytoca is able to ferment most pentoses, hexoses, and disaccharides. Butanediol appears to be a primary metabolite, excreted as a product of energy methabolism. The theoretical maximum yield of butanediol from monosaccharides is 0.50 g/g. This article describes the effects of pH, xylose concentration, and the oxygen transfer rate on the bioconversion of D-xylose to 2,3-butanediol. Product inhibition by butanediol is also examined. The most important variable affecting the kinetics of this system appears to be the oxygen transfer rate. A higher oxygen supply favors the formation of cell mass at the expense of butanediol. Decreasing the oxygen supply rate increases the butanediol yield, but decreases the overall conversion rate due to a lower cell concentration.

Factors affecting the pretreatment of biomass with gaseous ozone.

Abstract: Treatment of a wide variety of lignocellulosic biomass with gaseous ozone results in greatly enhanced susceptibility to cellulase enzyme hydrolysis and to digestion by rumen microorganisms so that it can be used as ruminant animal feed or for the production of glucose via enzymatic hydrolysis. By use of appropriate reaction conditions a useful degree of such pretreatment may be obtained in 1-2 h contact time with an ozone consumption of ca. 4-6% of the dry weight of the biomass.

Continuous ethanol fermentation using immobilized yeast cells

Abstract: Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.

Competitive adsorption of cellulase components and its significance in a synergistic mechanism

Abstract: Some studies on the adsorption of cellulase on cellulose revealed part of the mechanisms involved in the enzymatic hydrolysis of cellulose and provided some clues to the synergistic mechanism of cellulase complex. The adsorption of cellulase was significantly affected by the reaction conditions and physical chemical characteristics of cellulose. Endoglucanase consisted of adsorbable and nonadsorbable components. Cellobiohydrolase had the strongest adsorption affinity. Each cellulase component is postulated to have distinctly different adsorption sites on cellulose, corresponding to the active sites in the hydrolysis reaction. Competitive adsorption kinetics between cellulase components were also observed during the adsorption process. The degree of competitive adsorption was most remarkable when the composition of cellulase components was nearly the same as that in the crude cellulase complex. This seems to show the optimal relative composition of cellulase components. The synergism between cellobiohydrolase and endoglucananse could be elucidated more clearly by this competitive adsorption model of the reaction mechanism.

A comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood

Abstract: Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increases the glucose yield. Pore size measurements reveal that the increase in pore volume, in the size range of the cellulase molecule, following pretreatment for pine is only about one-half the value obtained with mixed hardwood. This suggests that pore volume is an important determinant of substrate-enzyme reactivity.

Increased digestibility of bagasses by pretreatment with alkalis and steam explosion

Abstract: Alkali treatment and steam explosion of bagasse were investigated in order to develop economical and effective methods of increasing the digestibility of bagasse. The treated bagasse was to be used as a substrate for the production of volatile fatty acids by anaerobic acidogenic bacteria. The alkalis examined were NaOH, NH(3) (aqueous), NaOH + NH(3), Ca(OH)(2), and Ca(OH)(2) + Na(2)CO(3), at ambient temperature and in combination with steam explosion at 200 degrees C, 6.9 MPa, and 5 min cooking times. Digestibilities of up to 733 g organic matter (OM)/kg bagasse dry matter (DM) were obtained for bagasse treated with NaOH and Ca(OH)(2) + Na(2)CO(3); less than 430 g OM was obtained for bagasse treated with aqueous NH(3); and up to 724 g OM was obtained for bagasse treated with Ca(OH)(2). This digestibility was only achieved by using high concentrations of Ca(OH)(2), i.e., 180-300 g/kg bagasse. Steam explosion increased the digestibility of bagasse up to 740 g OM in the presence of alkali but only to 610 g OM in the absence of alkali. The digestibility of bagasse without pretreatment was 190 g OM/kg bagasse DM. More than one-half the hemicellulose present was solubilized by pretreatment. The composition of the liquid fraction of steam-exploded material was examined and contained mainly xylose monomers and oligomers (112 g/kg original bagasse DM) and acetic acid (33 g/kg original DM). The relative costs of the alkalis used were obtained for the United States, Australia, and Europe. Lime [Ca(OH)(2)] was the least expensive alkali per unit of additional digestible OM obtained. Ammonia was the most expensive alkali to use, except in the United States where the difference in its cost relative to other alkalis was smaller. However, ammonia provides organic nitrogen for microbial growth, and could be recycled. With acidogenic fermentations, alkali is able to double as a neutralizing agent during fermentation. Thus, concentrations of alkali equal to that required for neutralization may be used in pretreatment. Concentrations of Ca(OH)(2) as high as 300 g/kg bagasse were needed for neutralization and should, therefore, be considered for pretreatment. Steam explosion of bagasse resulted in digestible, sterilized substrates of small particle size with readily separable liquid and pulp streams.

Genetically structured models forlac promoter-operator function in the Escherichia coli chromosome and in multicopy plasmids: Lac operator function.

Abstract: A mathematical model based on known molecular interactions has been formulated to describe quantitatively regulation of expression of the lactose (lac) operon in the Escherichia coli chromosome and in multicopy plasmids. This model is genetically structured such that a nucleotide sequence change affecting transcription initiation at the lac promoter-operator influences one or very few directly corresponding model parameters. The model simulates chromosomal lac operon function in good agreement with previous experimental measurements for many lacl and lacO mutant systems as well as for diploid cells which carry F'lac episomes. Simulation results clearly show the loss of cloned lac operator regulation as the plasmid copy number increases, in agreement with experimental trends. The importance of this class of models in designing DNAs, organisms, and reactors for precise regulation of cloned gene expression is discussed.

Recovery of uranium from biological adsorbents—desorption equilibrium

Abstract: Results are presented of the experimental investigations of uranium elution and reloading for the waste inactive biomass of Rhizopus arrhizus. The experimental data and the analysis of the present work suggest the following conclusions: recovery of uranium that has been taken up by R. arrhizus is possible by elution; of the six elution systems examined, sodium bicarbonate solutions appear to be the most promising because they can effect near complete uranium recovery and high uranium concentration factors; the bicarbonate solution causes the least damage to the biomass; solid-to-liquid ratios in bicarbonate elution systems can exceed 120:1 (mg:mL) for a 1N NaHCO/sub 3/ solution, with almost complete uranium recovery and eluate uranium concentrations of over 1.98 x 10/sup 4/ mg/L; mineral acids, although good elution agents, result in substantial damage to the biomass thus limiting the biomass reuse potential; sulfate ions in the elutions solution limit the elution potential of the biomass, possibly by conferring novel crystallinity to the cell wall chitin network and confining inside the chitin network more biosorbed uranium.

The effect of organosolv pretreatment on the enzymatic hydrolysis of poplar

Abstract: 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.

Kinetic studies of corn stover saccharification using sulphuric acid.

Abstract: The kinetics of crystalline cellulose and hemicellulose hydrolysis in corn stover were studied with a nonisothermal technique. Reactions were arrested at temperatures between 160 and 240 degrees C and product sugars were analyzed using a Bio-Rad HPX-85 liquid chromatographic column. A simple first-order series reaction model was used for both cellulose and hemicellulose hydrolysis reactions. Kinetic parameters were obtained for three different sulphuric acid concentrations (0.49, 0.92, and 1.47 wt %). Activation energies remained constant over this acid concentration range but the preexponential factors showed an increase with acid concentration. Relationships were obtained between the preexponential factors and acid concentrations. Cellulose hydrolysis and glucose degradation reactions were observed to be of higher order with respect to acid concentration in comparison with the previous studies with other raw materials.

Feed component inhibition in ethanolic fermentation by Saccharomyces cerevisiae.

Abstract: Inhibition by secondary feed components can limit productivity and restrict process options for the production of ethanol by fermentation. New fermentation processes (such as vacuum or extractive fermentation), while selectively removing ethanol, can concentrate nonmetabolized feed components in the remaining broth. Stillage recycle to reduce stillage waste treatment results in the buildup of nonmetabolized feed components. Continuous culture experiments are presented establishing an inhibition order: CaCl2, (NH4)2×SO4 > NaCl, NH4Cl > KH2PO4 > xylose, MgCl2 > MgSO4 > KCl. Reduction of the water activity alone is not an adequate predictor of the variation in inhibitory concentration among the different components tested. As a general trend, specific ethanol productivity increases and cell production decreases as inhibitors are added at higher concentration. We postulate that these results can be interpreted in terms of an increase in energy requirements for cell maintenance under hypertonic (stressed) conditions. Ion and carbohydrate transport and specific toxic effects are reviewed as they relate to the postulated inhibition mechanism. Glycerol production increases under hypertonic conditions and glycerol is postulated to function as a nontoxic osmoregulator. Calcium was the most inhibitory component tested, causing an 80%decline in cell mass production at 0.23 mol Ca2+/L and calcium is present at substantial concentration in many carbohydrate sources. For a typical final cane molasses feed, stillage recycle must be limited to less than onethird of the feed rate; otherwise inhibitory effects will be observed.

Cellular reporoduction and extracellular polymer formation by Pseudomonas aeruginosa in continuous culture

Abstract: The kinetics of cellular reproduction and the rate and extent of synthesis of extracellular polymeric substances (EPS) were investigated for P. aeruginosa growing in glucose-limited chemostats. mu(max) and K(s) estimates of 0.4 h(-1) and 2 mg glucose C/L, respectively, at 25 degrees C were obtained for this bacterium. The extent of EPS formation was inversely related to the growth rate of P. aeruginosa. The rate of EPS formation had both growth- and non-growth-associated components. The growth-associated polymer formation rate coefficient (k) was 0.3 mg polymer C/mg cellular C and the non-growth-associated polymer formation rate coefficient (k') was 0.04 mg polymer C/mg cellular C/h. The values for k and k' must be regarded as provisional since the product formation data were quite variable at low dilution rates. Estimates of the cellular (Y(x/s)) and polymer (Y(p/s)) yield coefficients were 0.3 mg cellular C/mg glucose C and 0.6 mg polymer C/mg glucose C, respectively. Most of the non-growth-associated consumption of glucose detected was due to exopolymer formation.

High-efficiency ethanol production from lignocellulosic residues pretreated with alkaline H2O2.

Abstract: Pretreatment should be economic and should not utilize toxic reagents. In this study locally obtained residues were used - wheat straw, cornstalks, corn husks and kenaf -as substrates. The high efficiency of glucose production from alkaline H/sub 2/O/sub 2/ pretreated lignocellulosic residues made these materials excellent substrates for ethanol production by Saccharomyces cerevisiae in combined saccharification/fermentation experiments. Results showed that overall efficiency of ethanol formation was 90% for pretreated corn cobs, stalks and husks compared to 50% for untreated materials. Yields from kenaf and oak were also enhanced although below the theoretical maximum. The lignin containing supernatant does not appear to be inhibitory to Saccharomyces cerevisiae growth or ethanol production. The improvement in conversion efficiency is apparently the result of the removal of about one half of the lignin along with an apparent reduction in the degree of crystallinity within the cellulose structure itself. 16 references.

Characterization of intracellular accumulation of poly-β-hydroxybutyrate (PHB) in individual cells of Alcaligenes eutrophus H16 by flow cytometry

Abstract: Poly-beta-hydroxybutyrate (PHB) accumulates in individual cells of Alcaligenes eutrophus in the form of refractile bodies which alter the light-scattering properties of individual cells. Flow cytometry has been applied to measure the distributions of single-cell light-scattering intensity in Alc. eutrophus populations during batch cultivation of the organism. These measurements clearly identify heterogeneities in the inoculum which influence the lag interval prior to beginning of exponential growth. Light-scattering distributions show greater homogeneity and are extremely similar during balanced, exponential growth. After exhaustion of the nitrogen source and with carbon source still available, significant PHB accumulations occur and the flow cytometry measurements reveal extreme heterogeneity in single-cell light-scattering properties. These measurements clearly demonstrate the potential advantages of single-cell light-scattering measurements by flow cytometry for analysis and control of certain fermentation processes. Single-cell light-scat light-scattering measurements in conjunction with flow sorting instrumentation have been applied to demonstrate enrichment of PHB-producing cells, initially present in a number concentration of 0.01%by a factor of 300 in a single pass. Flow cytometry-cell sorting technology should find significant application in strain improvement and mutant selection.

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose.

Abstract: alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.