bluuebunny/crossref_metadata_2025 huggingface dataset:
A simplified calculation method applied to enzyme inactivation during drying
Authors: Liou J. K. , Luyben K. Ch. A. M. , Bruin S.
Published: December 2004

An approximate method for solving the nonlinear diffusion problem in the case of a power‐function variation of the diffusion coefficient with concentration has been applied to a drying process with simultaneous enzyme inactivation. Experimental results obtained by air drying of soybean lipoxygenase entrapped in a glucose calcium‐alginate gel are in good agreement with the predicted behavior, whereas hardly any differences occur between the results obtained with the approximate method and those obtained by a numerical solution of the original model.
Sand administration as an instrument for biofilm control of Pseudomonas putida ATCC 11172 in chemostat cultures
Authors: Molin Göran , Nilsson Inge
Published: December 2004

Pseudomonas putida ATCC 11172 was grown in chemostat on L‐asparagine or phenol as the sole, limiting carbon and energy source. The growth characteristics of a culture where a biofilm was present, were compared with one where the biofilm was strongly reduced by the grinding and shearing effect of sand suspended in the culture. In the presence of the intact biofilm, the curve of steady‐state biomass versus dilution rate diverged greatly from the theoretical pattern predicted by conventional chemostat models. The sand strongly retarded the biofilm formation and to a high degree restored the shape of the biomass versus dilution rate curve to a more conventional pattern. The maximum specific growth rate (μmax) could not be calculated from the biofilm cultures. However using the sand cultures, μmax was determined to 0.64 h−1 with L‐asparagine as the carbon source and 0.49 h−1 with phenol which compare favorably with the respective μmax values calculated from batch cultures.

Incorporation of sand into strongly agitated cultures is recommended as an efficient and simple means of controlling biofilm formation in continuous cultures. The method may enable the gathering of basic kinetic data difficult to obtain in the presence of biofilm.
Utilization of short chain monocarboxylic acids in an effluent of a petrochemical industry by Acinetobacter calcoaceticus
Authors: Du Preez J. C. , Lategan P. M. , Toerien D. F.
Published: December 2004

The aqueous effluent generated by the Fischer‐‐Tropsch process, containing a total of 13 g/L C2–C5 monocarboxylic acids, was investigated as a potential substrate for the production of single‐cell protein (SCP). A bacterial isolate, Acinetobacter calcoaceticus, could utilize all the acids in the effluent simultaneously in chemostat cultures, and no residual acids were detected in the culture below a dilution rate of 0.78 h−1. The critical dilution rate was 1.04 h−1. The maintenance energy requirement of the cells growing on the monocarboxylic acid mixture was considerably lower than that of cells growing on acetate as the sole carbon source. Enrichment of the effluent with ethanol to increase the biomass concentration was successful and still allowed the simultaneous and efficient utilization of all the carbon sources, but resulted in a decrease of the critical dilution rate by ca. 20%.
Direct measurement of carbon substrate oxidation and incorporation patterns in RuMP‐type methylotrophs: Chemostatic cultures of Methylomonas L3
Authors: Chu I‐Ming , Bussineau Christopher M. , Papoutsakis Eleftherios Terry
Published: December 2004

A technique using C‐14 isotope tracers to probe the branching of carbon flow in methylotrophic bacteria has been devised and applied to continuous steady‐state cultures. Methylomonas L3, a strain which utilizes the KDPG/TA variant of the ribulose monophosphate cycle for carbon fixation, was employed in the experimental studies. The actual in vivo rates of substrate‐carbon incorporation into biomass, both direct and via CO2, and of the two carbon oxidation schemes were determined in three different steady‐state cultures. The results show that the carbon substrate is oxidized predominantly via formate (the linear oxidation scheme), and that the cyclic scheme of oxidation is minimally, if at all, utilized. The carbon incorporation and oxidation patterns appear to vary considerably with the dilution rate and the inoculum history. The experimental accuracy of the new technique is discussed in detail.
Potentiometric system for selective formate measurement and improvement of response characteristics by permeation of cells
Authors: Ho May Y. K. , Rechnitz G. A.
Published: December 2004

A formate‐selective biocatalytic potentiometric electrode system based on whole cells Pseudomonas oxalaticus has been developed. Permeation of the Gram‐negative Pseudomonas oxalaticus microbial cells by EDTA is shown to improve the response slope from 38 mV/decade to 49 mV/decade, resulting in increased sensitivity. Out of 13 possibly interfering compounds tested, only pyruvate and lactate showed moderate response.
Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli
Authors: Seo Jin‐Ho , Bailey James E.
Published: December 2004

Plasmid–host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and β‐lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of α‐methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to α‐methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of β‐lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.
Immobilization of enzyme onto poly(ethylene–vinyl alcohol) membrane
Authors: Imai Kiyokazu , Shiomi Tomoo , Uchida Kozo , Miya Masamitsu
Published: December 2004

Invertase was ionically bound to the poly(ethylene–vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2‐dimethyl‐aminoacetoaldehyde dimethylacetal (AAA) and 3‐(N, N‐dimethylamino‐n‐propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, Km, was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemar origin in the same way. The amount and activity of immobilized glucoamylase were much less than of invertase.
Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid
Authors: Mizutani Satoru , Mori Hironori , Shimizu Shoichi , Sakaguchi Kenji , Kobayashi Takeshi
Published: December 2004

Escherichia coli harboring a recombinant plasmid was cultivated in fed‐batch culture to enhance production of a gene product. Expression of the leucine gene from Thermus thermophilus in the recombinant plasmid was examined by the assay of β‐isopropylmalate dehydrogenase activity at 75°C. When E. coli was cultivated in medium without leucine, biomass concentration reached 15 g/L and the specific activity became 0.082 U/mg protein. When leucine was fed in the medium throughout cultivation, although biomass concentration reached 63 g/L, the specific activity decreased to 0.016 U/mg protein. When E. coli was cultivated in medium containing 1 g leucine/L, the specific activity remained virtually constant (about 0.13 U/mg protein) and biomass concentration reached 32 g dry cells/L. In these cultivations, growth yields of several amino acids and glucose were examined. When leucine was not added to the medium, growth yields except for histidine were lowest. When leucine was fed throughout the cultivation, growth yields of glucose and tryptophan were highest. The pH‐stat was useful for feeding amino acids.
Adsorption equilibrium in immuno‐affinity chromatography with polyclonal and monoclonal antibodies
Authors: Sada Eizo , Katoh Shigeo , Sukai Kiyoshi , Tohma Masaaki , Kondo Akihiko
Published: December 2004

The effects of pH, ionic strength, anion species, and antibody concentration on the adsorption equilibrium between immobilized antibodies and antigens were studied by use of anti‐BSA, anti‐HSA, anti‐BlgG, and monoclonal anti‐HSA coupled to Sepharose 4B. The polyclonal antibodies possessed average binding affinities of the order of 108M−1, and the heterogeneity was accounted for by assuming a normal distribution of the free energy of antibody–antigen combination. The monoclonal antibody, on the other hand, showed a homogeneous affinity of the Langmuir type. Bound antigens could be eluted by lowering pH or adding a chaotropic anion, and their purity was very high. The antibody ligand was sufficiently stable for repeated use.
Enzymatic saccharification of pretreated rice straw and biomass production
Authors: Araujo A. , D'Souza J.
Published: December 2004

A comparative study on the saccharification of pretreated rice straw was brought about by using cellulase enzyme produced by Aspergillus terreus ATCC 52430 and its mutant strain UNGI‐40. The effect of enzyme and substrate concentrations on the saccharification rate at 24 and 48 were studied. A syrup with 7% sugar concentration was obtained with a 10% substrate concentration for the mutant case, whereas a syrup with 6.8% sugar concentration was obtained with 3.5 times concentrated enzyme from the wild strain. A high saccharification value was obtained with low substrate concentration; the higher the substrate concentration used, the lower the percent saccharification. The glucose content in the hydrolysate comprised 80–82% of total reducing sugars; the remainder was cellobiose and xylose together. The hydrolysate supported the growth of yeasts Candida utilis and Saccharomyces cerevisiae ATCC 52431. A biomass with a 48% protein content was obtained. The essential amino acid composition of yeast biomass was determined.
An immobilized enzyme reactor for the detoxification of bilirubin
Authors: Sung Cynthia , Lavin Arthur , Klibanov Alexander M. , Langer Robert
Published: December 2004

An immobilized enzyme reactor has been developed for the degradation of bilirubin as a potential treatment for neonatal jaundice. It utilizes the enzyme bilirubin oxidase from Myrothecium verrucaria, which in the presence of molecular oxygen converts bilirubin to biliverdin and other products that are much less toxic than bilirubin. Bilirubin oxidase was covalently attached to agarose beads using cyano transfer activation. Forty percent of the specific activity of bilirubin oxidase was retained after immmobilization, and preparations with 20 units of enzymatic activity per gram of drained wet weight of gel were obtained. The stability of bilirubin oxidase at pH 7.4 and 37°C was improved fivefold by immobilization. A 15‐mL column containing immobilized bilirubin oxidase, through which a 37°C solution of 332μM bilirubin and 450μM human serum albumin in 0.05M phosphate buffer (pH 7.4) was passed at 1 mL/min, converted more than 60 percent of the bilirubin per pass. The substrate specificity of the enzyme and the small volume of the reactor are important characteristics for this clinical application where it is desirable to remove only one compound from the blood and to minimize the volume of blood in the extracorporeal circuit. This reactor, by detoxifying the jaundiced infant's blood of bilirubin, would eliminate the risks associated with the use of donor blood as is done currently in treating severe neonatal jaundice.
Simultaneous utilization of methanol–glucose mixtures by Hansenula polymorpha in chemostat: Influence of dilution rate and mixture composition on utilization pattern
Authors: Egli Thomas , Bosshard Christoph , Hamer Geoffrey
Published: December 2004

The utilization of mixtures of methanol (C1) and glucose (C6) of different composition by the methylotrophic yeast Hansenula polymorpha was studied in carbon‐limited chemostat culture. For all mixtures tested a similar utilization pattern was observed: At low dilution rates both carbon sources were utilized simultaneously, but at high dilution rates the cells used glucose only and the unutilized methanol accumulated in the culture medium. When grown with C1 only, the cells exhibited a critical dilution rate Dc(C1) of 0.19 h−1, but when C1–C6 mixtures were used as the carbon and energy substrate, the yeast was able to completely utilize C1 at dilution rates considerably higher than Dc(C1). The dilution rate at which the transition from C1–C6 growth to C6 growth occurred (Dt) was strictly dependent on the composition of the C1–C6 mixture in the feed, and Dt increased with decreasing proportions of C1 in the mixture. During mixed substrate growth the formation of biomass from the two substrates was additive. The results reported indicate that the utilization of C1–C6 mixtures and hence Dt in H. polymorpha are subject to two different regulatory regimes. When the cells were growing with C1–C6 mixtures containing more than 60% C1, the transition form C1–C6 to C6 growth was most probably influenced by the maximum C1 oxidizing capacity of the cells, whereas for growth with mixtures containing less than 40% C1, a growth rate of 0.28–0.30 h−1 seemed to be the limiting barrier for the simultaneous utilization of the components of the binary carbon and energy substrate mixture.
Enhancement of growth and ferrous iron oxidation rates of T. Ferrooxidans by electrochemical reduction of ferric iron
Authors: Yunker S. B. , Radovich J. M.
Published: December 2004

Thiobacillus ferrooxidans, the bacterium most widely used; in bioleaching or microbial desulfurization studies, was grown in an electrolytic bioreactor containing a synthetic, ferrous sulfate medium. Passage of current through the medium reduced the bacterially generated ferric iron to the ferrous iron substrate. When used in conjunction with an inoculum that had been adapted to the electrolytic growth conditions, this technique increased the protein (cell) concentration by 3.7 times, increased the protein (cell) production rate by 6.5 times, increased the yield coefficient (cellular efficiency) by 8.0 times, and increased the ferrous iron oxidation rate by 1.5 times at 29°C, compared with conventional cultivation techniques. A Monod‐type equation with accepted values for the maximum specific growth rate could not account for the increased growth rate under electrolytic conditions.
Invertase immobilized on macroporous polystyrene: Properties and kinetic characterization
Authors: Mansfeld J. , Schellenberger A.
Published: December 2004

Invertase from baker's yeast (Saccharomyces cerevisiae) was covalently bound via benzoquinone and glutaraldehyde to a macroporous polystyrene anion exchanger. The behavior of the invertase–polystyrene complexes in batch and packed‐bed reactors was characterized kinetically. In addition to kinetic studies on sucrose hydrolysis at low initial substrate concentrations, the dependence of conversion degree on flow rate at high, industrially used substrate concentrations was determined. The described invertase–polystyrene complexes are suitable for technical application in the production of glucose–fructose mixtures because of their high specific and relative activities, as well as the good hydrodynamical and mechanical properties of the polystyrene matrix.
Effects of dissolved oxygen shock on the stability of recombinant Escherichia coli containing plasmid pKN401
Authors: Hopkins David J. , Betenbaugh Michael J. , Dhurjati Prasad
Published: December 2004

The effect of dissolved oxygen shock on the stability of recombinant Escherichia coli cells containing plasmid pKN401 was investigated. The recombinant cells were stable in control batch experiments in media with and without ampicillin. However, these recombinant cells were highly unstable under conditions where a dissolved oxygen shock was induced. The results have implications for design of aerated reactors for recombinant cells.
Nature of yeast cells immobilized by radiation polymerization. Activity dependence on the water content of polymer carriers
Authors: Fujimura T. , Kaetsu I.
Published: December 2004

Higher activity of ethanol production due to higher density of yeast cells was observed in yeast cells immobilized with artificial polymer carriers of higher water content based on methoxypolyethyleneglycol methacrylate and 2‐hydroxyethylacrylate. These polymer carriers were prepared by radiation‐induced polymerization below 0°C. Yeast cells were immobilized with these carriers by adsorption method during multiplication. Two possible reasons for higher activity were discussed.
Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis
Authors: Brownell Harold H. , Saddler John N.
Published: December 2004

Pretreatment methods were compared with steam explosion, and differing views on the relative importance of mechanical and chemical effects were outlined. Hydrolysis was desirable; pyrolysis was undesirable. The effects of initial moisture content on steam consumption, mechanism and rate of heat transfer, pentosan solubilization, and subsequent glucose yield were summarized. The insignificant effect, after treatment at 240°C, of 90% pressure bleed‐down before explosion on subsequent simultaneous saccharification and fermentation (SSF) yields was described. Treatment at 190°C with complete bleed‐down (no explosion), when compared with that at 240°C with explosion from full pressure, showed at least as good solubilizatoin of pentosan, enzymatic hydrolysis, and SSF but showed greater pentosan destruction for the same degree of pentosan removal. Water washing of unexploded steamed aspenwood chips was at least as efficient as that of similarly treated but exploded chips. Scanning electron micrographs of unexploded chips showed extensive rupturing of vessel pit membranes and other morphological features associated with steam‐exploded wood. Neither the explosion nor the high temperatures (above 190°C) are necessary.
Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports
Authors: Shimizu K. , Ishihara M.
Published: December 2004

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl4) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10–50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl‐β‐glucoside, and aryl‐β‐xyloside were 3–53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.
Single‐cell protein from methanol with Enterobacter aerogenes
Authors: Gnan Said Omar , Abodreheba Ali Omar
Published: December 2004

An identifiedEnterobacter aerogenesutilizing methanol as a sole carbon source was studied for the optimization of biomass production and the reduction of its nucleic acid content. Results indicated that the highest yield and conversion were obtained at 0.5% methanol. The addition of seawater as a source of trace elements has an adverse effect. However, the addition of urea as source of nitrogen enhanced the growth of E. aerogenes. Heat shock at 60°C for 1 min followed by incubation at 50°C for 2 h caused 72.6% reduction in the nucleic acid.
Inhibition or initiation of a radical polymerization reaction by an ultraviolet‐induced enzymatic process
Authors: Van Deynse D. , Desie G. , De Schryver F. C. , Leenders L.
Published: December 2004

α‐Chymotrypsin was modified to a light‐controllable enzyme derivative by acylating active serine 195 residue with a cinnamoyl group or analogue. Upon UV irradiation the acylgroup could be isomerized, leading to release of the inhibiting group. Enzymatic activity could thus be regulated by means of UV light. A full 100% inhibition of the enzymatic activity could not be reached by the cinnamoyl derivative. Only posttreatment with diisopropylfluorophosphate yields a fully inactive enzyme derivative. The shelf‐life of the inhibited enzyme was rather poor. Only freeze‐dried samples could be used for several months without significant recovery of activity. Adapting the sensitivity of the system to visible light seems limited to the size of an enzyme's active site. Combination of the enzymatic system producing an inhibitor or an initiator with a polymerization reaction can result in a photographic process with a higher amplification factor.
Growth and enzyme synthesis during continuous culture of Trichosporon cutaneum on phenol
Authors: Spånning Åke , Neujahr Halina Y.
Published: December 2004

The soil yeast Trichosporon cutaneum was grown in continuous culture with phenol as the sole carbon source. The cultures were operated as carbon‐limited chemostats or as steady‐state continuous cultures without carbon limitation. Selected comparative runs were also conducted on glucose or acetate as carbon source. In addition to growth parameters, the activities of several intracellular enzymes were determined, comprising those directly involved in the degradation of phenol as well as auxiliary enzymes required for the generation of reducing power. All enzymes were assayed in detergent‐permea‐bilized cells. Phenol was found to serve as an excellent carbon source, comparable to glucose or acetate. The utilization of phenol in T. cutaneum is very efficient as indicated by a low maintenance requirement (0.01 g phenol/g cells·h). The cell yields obtained were on the order of 0.8 g cells/g phenol. Although the phenol‐limited chemostats were run with fully phenol‐induced cells, a further increase in the activities of isocitrate DH(NADP+), maleate DH and the phenol‐degrading enzymes occurred after transition to nonlimiting condition. Enzyme activities increased in parallel with increasing phenol levels in the effluent, as well as with increasing toxicity. The significance of this phenomenon is discussed. The significance of this phenomenon is discussed. This elevation in enzyme activities in not related to an increase in specific growth rate.
Analysis of the productivity of a continuous algal culture system
Authors: Radmer R. , Behrens P. , Arnett K.
Published: December 2004

We describe a first‐principles analysis of a system for the continuous culture of the green alga Scenedesmus obliquus under light‐limiting conditions. According to this analysis, the productivity of the algal culture is given by the relation Y = EmI0AK(1 – e−αcl) – GRcV, where Y = yield (g cells/h), Em = 0.20 (the maximum attainable photosynthetic conversion on an energy basis), A = illuminated area (m2), K = 0.156[(g cells/h/W), the energy equivalent of the algae], I0 = light intensity (W/m2), α = extinction coefficient (L/cm/g),c = cell concentration (g/L), I = light path (cm), R = respiration rate (g carbon/g cells/h), V = culture volume (L), and G = ratio of g cells to g carbon (2.04). This formula is completely determined and has no free adjustable parameters. Using parameter values determined independently, the model accurately predicted the relationship of productivity to cell density in the culture system.
Improvement of product yields by temperature‐shifting of Escherichia coli cultures containing plasmid pOU140
Authors: diPasquantonio Vincent M. , Betenbaugh Michael J. , Dhurjati Prasad
Published: December 2004

Temperature shifting was investigated as a means of improving cloned‐gene product yields form a recombinant Escherichia coli containing the temperature‐sensitive plasmid, pOU 140. In a series of shaker flask fermentations recombinant cells were thermally induced for different time periods. The growth, stability, and plasmid product levels were followed, and the results indicate the existence of an induction time period that maximizes product yield. A sustained thermal induction results in recombinant cell death and instability, while exposure to a runaway temperature for minimal time periods does not give sufficiently high product yields. At intermediate cycling times, however, the recombinant cells remain stable, and the plasmid replication region is activated, resulting in higher product yields.
Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator
Authors: Chang Ho Nam , Kyung Yun‐Seung , Chung Bong Hyun
Published: December 2004

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate‐limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.
Methane recovery from water hyacinth through whole‐cell immobilization technology
Authors: Annachhatre A. P. , Khanna P.
Published: December 2004

The concepts of feed pretreatment, phase separation, and whole‐cell immobilization technology have been incorporated in this investigation for the development of rational and cost‐effective two‐ and three‐stage methane recovery systems from water hyacinth (WH)

Analyses of laboratory data reveal that a three‐stage system could be designed with an alkali pretreatment stage [3.6% Na2CO3 + 2.5% Ca(OH)2 W/W, 24 h HRT] followed by an open acid reactor (2.1 days HRT) and closed immobilized methane reactor (12 h HRT), providing steady‐state COD conversion of 62–65%, TVA conversion of 91–95%, and gas productivity of 4.08–5.36 L/L reactor volume/day with 82% methane. A gas yield of 50 L/kg WH/day (dry wt basis) at 35–37°C is possible with this system. Insulation bricks, with particle size distribution of 500–3000 μm, were used as support material in the reactors at organic loading rate of 20 kg COD/m3 day. The reactors matured in 15–18 weeks

Substantial reduction in retention time for the conversion of volatile acids in immobilized methane reactors prompted further research on the combined immobilized reactor to make possible an additional reduction in the cost of a WH‐based biogas system. Evaluation of laboratory data reveals that a two‐stage system could be designed with an open alkali pretreatment stage and a combined immobilized reactor (12 h HRT), providing steady‐state COD conversion of 53% and gas productivity of 3.1 L/L reactor volume/day with 86% methane. A gas yield of 44 L/kg WH/day (dry wt basis) at 35–37°C could be obtained from this system. Insulation bricks, with 500–1000 μm particle size distribution, was used as support material at an organic loading rate of 15 kg COD/m3 day. Notwithstanding the fact that the technology in this study has been developed with water hyacinth as substrate, the implicit principles could be extended to any other organic substrate.
Polymerization of phenols catalyzed by peroxidase in nonaqueous media
Authors: Dordick Jonathan S. , Marletta Michael A. , Klibanov Alexander M.
Published: December 2004

Polymers produced by horseradish‐peroxidase‐catalyzed coupling of phenols have been explored as potential substitutes for phenol‐formaldehyde resins. To overcome low substrate solubilities and product molecular weights in water, enzymatic polymerizations in aqueous‐organic mixtures have been examined. Peroxidase vigorously polymerizes a number of phenols in mixtures of water with water‐miscible solvents such as dioxane, acetone, di‐methylformamide, and methyl formate with the solvent content up to 95%. As a result, various phenolic polymers with average molecular weights from 400 to 2.6 × 104 D were obtained depending on the reaction medium composition and the nature of the phenol. Peroxidase‐catalyzed copolymerization of different phenols in 85% dioxane was demonstrated. Poly(p‐phenylphenol) and poly(p‐cresol) were enzymatically prepared on a gram scale. They had much higher melting points, and in addition, poly(p‐phenylphenol) was found to have a much higher electrical conductivity than phenol‐formaldehyde resins.
Mixing and power characteristics of drag board device in shallow pool
Authors: Cárdenas A. , Markovits A.
Published: December 2004

Mixing and stirring of a 20‐m2 shallow pool by means of a drag board device has been investigated. The board closes the pool cross section except for a slit of a few centimeters above the bottom, and it is slowly moved back and forth, forcing the water to run through the slit and thereby creating a turbulent backwhirl. Power drawn and the drag on the board has been measured together with the velocities of the water at different locations in the wake of the board. Power number Np has been correlated with the Reynolds NRe and the bottom clearance numbers Nc by the expressions

Power and Reynolds numbers are defined as usual with the square root of the board‐immersed‐area as the characteristic length. The bottom clearance number is defined as the ratio of the water depth in the pool to the difference between water depth and the board width immersed in water. Flow pattern behind the board consists of large vortex loops causing the fluid to circulate from bottom to top and producing a thorough mixing effect. The drag board seems to have several advantages over conventional paddle wheels for the mixing and stirring of algal cultures in shallow ponds.
Reaction properties of hydrophobic enzymes and their immobilization on phospholipid composite membranes
Authors: Sada Eizo , Katoh Shigeo , Terashima Masaaki , Kikuchi Yasufumi
Published: December 2004

Reaction rates of hydrophobic enzymes, aminopeptidase, and alkaline phosphatase, in microsomes prepared from the porcine brush border membrane and in vesicles pre pared from microsomes and phospholipids were measured at various temperatures. Interactions between the hydrophobic enzymes and the phospholipid layers are discussed as well as the effects of fluidity change of phospholipid layers on enzyme activity. Further, reaction properties and stabilities of the immobilized vesicles containing microsomal enzymes were studied.
An investigation of a fertilizer–tap water medium for mass algal production in outdoor plastic‐enclosed systems
Authors: Geldenhuys D. J. , Walmsley R. D. , Toerien D. F.
Published: December 2004

The feasibility of using two fertilizers (urea plus superphosphate) in tap water as a medium for the mass culture of green algae (Scenedesmus and Ankistrodesmus) in outdoor plastic‐enclosed minipond systems was investigated. Experiments in which the basic fertilizer‐tap water medium was enriched with micro‐ and/or macronutrients revealed no nutrient deficiency symptoms in the algal biomass produced. Biomass production was found to be quantitatively related to the concentration of fertilizer added and maximal production (> 15 g/m2 day) was achieved following the addition of 30 mg N/L (1.89 g N/m2 day) and 4.5 mg P/L (0.28 g P/m2/day).
Amylolysis of raw corn by Aspergillus niger for simultaneous ethanol fermentation
Authors: Han Inyee Y. , Steinberg M. P.
Published: December 2004

The novelty of this approach was hydrolysis of the raw starch in ground corn to fermentable sugars that are simultaneously fermented to ethanol by yeast in a non‐sterile environment. Thus, the conventional cooking step can be eliminated for energy conservation. A koji of Aspergillus niger grown on whole corn for 3 days was the crude enzyme source. A ratio of 0.2 g dry koji/g total solids was found sufficient. Optimum pH was 4.2. Ethanol concentration was 7.7% (w/w) in the aqueous phase with 92% raw starch conversion. Agitation increased rate. Sacharification was the rate‐limiting step. The initial ethanol concentration preventing fermentation was estimated to be 8.3% by weight.
Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma viride
Authors: Beldman G. , Voragen A. G. J. , Rombouts F. M. , Searle‐van Leeuwen M. F. , Pilnik W.
Published: December 2004

Adsorption on crystalline cellulose of six endoglucanases (Endo I, II, III, IV, V and VI; 1, 4‐β‐D‐glucan glucanohydrolase, EC 3.2.1.4) and two exoglucanases (Exo II and III; 1,4‐β‐D‐glucan cellobiohydrolase, EC 3.2.1.92), purified from a commercial cellulase preparation of Trichoderma viride origin, was studied. Endo I, III, and V adsorbed strongly on Avicel cellulose, while adsorption of Endo II, IV, and VI was much lower. Also, the two exoglucanases could be divided into one enzyme (Exo III) that had a high adsorption affinity and another enzyme (Exo II) that adsorbed only moderately. Adsorption data fitted the Langmuir‐type adsorption isotherm. However, adsorption was only partially reversible with respect to dilution. No relation could be found between adsorption affinity and degree of randomness in cellulose hydrolysis, measured as the diversity of released hydrolytic products. Kinetic measurements indicated that only part of the adsorbed enzyme molecules are hydrolytically active.
Influence of very small bubbles on kLa measurement in viscous microbiological cultures
Authors: Kawase Y. , Moo‐Young M.
Published: December 2004

In gas–liquid dispersions based on viscous non‐Newtonian fluids, numerous very small bubbles are formed due to their high residence times in contacting devices such as bubble columns. The influence of these small bubbles on the measurement of the volumetric mass transfer coefficient in the contactor by the dynamic CO2 gas analysis method is discussed. It is found that their effect on this measuring technique is insignificant compared to that when using the conventional dissolved‐oxygen technique.
The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae
Authors: Iversen J. J. Lønsmann
Published: December 2004

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth‐limiting glucose concentration in media without pH control was increased from 1.0 g carbon L−1 to 2.2 g carbon L−1, the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30–200 μM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH‐dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (YATP) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y′ATP) was 9.4 and compared well with the true molar growth yield coefficient (Y), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy‐limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy‐limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y′ATP of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.
Dynamics of chemostat in which one microbial population grows on multiple complementary nutrients
Authors: Pavlou Stavros
Published: December 2004

The equations of a chemostat in which one microbial population grows on multiple rate‐limiting nutrients are formulated. The dynamics of a chemostat involving growth on complementary nutrients is studied through stability analysis of the system of equations. Some conditions are derived that relate the dynamic behavior of the chemostat to its operating conditions and can be applied to any model for the specific growth rate of the population. It is shown that, if maintenance of the population is neglected, the system exhibits no sustained or damped oscillations. If maintenance of the population is considered, damped oscillations are observed for some operating conditions.
Performance evaluation of ethanol fermentor systems using a vector‐valued objective function
Authors: Tsuji Satoshi , Shimizu Kazuyuki
Published: December 2004

It is shown that the performance evaluation using a vector‐valued objection function whose components are the product productivity, the product concentration, and the substrate conversion is quite useful in getting deeper insight into the development of new processes and in determining the operating point. Particular attention is focused on the ethanol fermentation using variety of systems such as the conventional chemostat system, multiple fermentor system, cell recycle system, extractive fermentor system, cell recycle system, extractive fermentor system, and immobilized cell system. The contour map and the projection of the noninferior set are used in investigating the performance improvement and the trade‐offs among performance indexes.
Modeling the kinetics of immobilized glucose oxidase
Authors: Parker J. W. , Schwartz C. S.
Published: December 2004

Determination of glucose concentrations in fluids frequently requires the application of immobilized glucose oxidase. An accurate description of the immobilized enzyme kinetics is critical for such applications. In this study, the overall rate of reaction of immobilized glucose oxidase is investigated theoretically. A novel steady‐state model based on a ping‐pong kinetic mechanism for glucose oxidase is developed. Numerical studies are used to examine the parameter sensitivity of this model. The enzyme loading, matrix thickness and geometric con figuration are found to have a significant influence on substrate uptake by insolubilized glucose oxidase.

Aditionally, this new model is compared with a previously developed model based on an alternative ping‐pong kinetic mechanism. Under steady‐state conditions, no significant difference between the two models is apparent when appropriate kinetic parameters are applied to each of the models. The model developed herein is also compared with models utilizing the simplifying assumption of Michaelis–Mented kinetics for substrate reaction. Numerical studies indicate that under most realizable biological conditions, a model based on ping‐pong kinetics should be applied to accurately describe substrate uptake by immobilized glucose oxidase.
Inner filter effects and their interferences in the interpretation of culture fluorescence
Authors: Srinivas S. P. , Mutharasan R.
Published: December 2004

Inner filter effects and their interferences in the measurement and interpretation of culture fluorescence are discussed. An approximate light intensity model for a typical open‐ended culture fluorescence measuring device is developed for calculating the fluorescence response of a component of interest in a general three component solution. The model is tested using well de fined synthetic fluorescent systems. The model is then extended for correlating culture fluorescence with cell density and metabolic state of microbial cultures based on a lumping approximation. The extended model has been utilized to derive culture fluorescence parameters of yeast culture at three distinct metabolic states.
Production and application of methylotrophic yeast Pichia pastoris
Authors: Duff Sheldon J. B. , Murray William D.
Published: December 2004

Pichia pastoris is a methylotrophic yeast that makes use bf the enzyme alcohol oxidase to catalyze the first step of the dissimilatory pathway that enables it to grow on methanol. Because of its stability and low substrate specificity, alcohol oxidase is of considerable interest for a range of biotechnological processes. Various feeding regimes were evaluated in an effort to increase the biomass concentration and productivity that could be achieved from fermentations using this organism. Through continuous or semicontinuous feeding, biomass concentrations were increased 10‐fold over those achieved in batch fermentations. In subsequent trials, nongrowing whole cells were applied successfully to convert ethanol to acetaldehyde. Quantitative conversions of 20‐g/L solutions of ethanol have been achieved in 2 h, and acetaldehyde concentrations of up to 35 g/L have been achieved using extended reaction times of 5 h. The conversion reaction was limited by end product inhibition and by acetaldehyde holdup within the yeast cells.
Kinetics of microbial growth on mixtures of pentachlorophenol and chlorinated aromatic compounds
Authors: Klec̆ka Gary M. , Maier Walter J.
Published: December 2004

Batch experiments were conducted to examine the effects of several substrate analogs on the degradation of pentachlorophenol by an enrichment culture of pentachlorophenol‐utilizing bacteria. The presence of substrate analogs which were unable to serve as a carbon source for growth of the culture (e.g., 3,5,6,‐trichloro‐2‐pyridinol, 2,4‐dichlorophenoxyacetic acid) decreased the rate of pentachlorophenol degradation. The presence of a utilizable substrate analog (e.g., phenol, 2,4,5‐trichlorophenol) also inhibited the initial rate of pentachlorophenol degradation; however, the overall removal rate was accelerated due to an increase in cell mass concentration as a result of simultaneous growth on both substrates. These effects were shown to be predicted by a mathematical model based on a modified Monod equation. Kinetic parameters obtained from the results of laboratory studies can be used for further process analysis to define the optimal conditions for the biological treatment of complex mixtures of phenolic compounds.
Kinetics of bilirubin oxidase and modeling of an immobilized bilirubin oxidase reactor for bilirubin detoxification
Authors: Million Claudy J. P. , Klibanov Alexander M. , Langer Robert
Published: December 2004

The unbound bilirubin concentration and the enzymatic rate of bilirubin degradation by bilirubin oxidase in bilirubin‐serum albumin solutions have been investigated experimentally and theoretically. A stoichiometric bilirubin‐serum albumin binding analysis shows that the unbound bilirubin concentration depends only on the molar ratio of the total bilirubin concentration to the total serum albumin concentration. From the theoretical analysis and the measured unbound bilirubin concentrations, serum albumin may be modelled as a molecule having two binding sites, primary and secondary, with stoichiometric equilibrium constants of K1 = 6 × 107M−1 and K2 = 4.5 × 106M−1, respectively. The rate of total bilirubin degradation in bilirubin‐serum albumin mixtures is zero order. An immobilized bilirubin oxidase reactor model, which shows good agreement with experimental bilirubin conversions, is presented. At a flow rate of 1 mL/min with a 8‐mL reactor volume, a 50% bilirubin conversion per pass was observed with an inlet bilirubin concentration of 350μM and a serum albumin concentration of 500μM.
Experimental simulation of oxygen profiles and their influence on baker's yeast production: II. Two‐fermentor system
Authors: Sweere A. P. J. , Janse L. , Luyben K. Ch. A. M. , Kossen N. W. F.
Published: December 2004

A reactor configuration consisting of two reactors with an exchange flow was used for the experimental simulation of large‐scale conditions. The influence of fluctuations in oxygen concentration on the growth and metabolite production of baker's yeast was investigated by sparging one fermentor with air and one with nitrogen gas. It was found that the biomass yield decreased and the metabolite formation increased with rising circulation time (longer oxygen‐unlimited and oxygen‐limited periods). Not only was the performance of the oxygen‐limited fermentor characterised by (partly) reductive metabolism, but that of the oxygen‐unlimited fermentor as well. The results of the experiments in this reactor system were compared with those from the experiments carried out in a one‐fermentor system with periodically changing oxygen concentrations. The formation of acetic acid, which is characteristic for transient states, showed a distinct difference between the two reactor systems.
Semicontinuous diffusion fermentation of fodder beets for fuel ethanol and cubed protein feed production
Authors: Gibbons William R. , Westby Carl A. , Arnold Eugene
Published: December 2004

A novel, semicontinuous diffusion fermentation system was used to produce fuel ethanol and a cubed protein feed (CPF) from fodder beets at an intermediate scale. In the process, fodder beet cubes were augered diagonally upward against a flow of 0.26N H2SO4 and yeast in a tubular fermentor. Exiting one end of the fermentor was CPF, while fermented beer [6–9% (v/v) ethanol] exited the other end. Retention times for beer and CPF were 264 and 72 h, respectively. Contamination was controlled by maintaining the fermentation pH between 2.1 and 2.6 using H2SO4. Production costs for a greatly scaled‐up (times 1400) conceptual version of this system (using a continuous rather than a semicontinuous processing mode) were projected by calculation to be $0.529/L for 95% ethanol (net of a $0.112/L credit for CPF). The calculated energy balance (energy output‐energy input ratio) was estimated to be 3.04. In calculating the energy balance, the output energy of the CPF and input energy for growing the fodder beets were not included. A design for the scaled‐up plant is provided.