bluuebunny/crossref_metadata_2025 huggingface dataset:
Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: I. Selective retainment of NADP(H) in a continuous reaction
Authors: Ikemi Masahisa , Koizumi Noriaki , Ishimatsu Yoshiaki
Published: December 2004

The concept of a charged membrane bioreactor (CMBR) has been proposed for continuous reactions of enzymatic reduction dependent upon the nicotinamide coenzyme NADP(H). It was found that a composite membrane with a negative charge, NTR 7410, could retain NADP(H) selectively without any chemical modification. Several permeation experiments have revealed that the retainment of a coenzyme is based on electrostatic repulsion of negative charges between the membrane and the phosphate moiety of NADP(H). The retainment ratio was reduced by the addition of inorganic salt, although it could be restored to 0.8 in the presence of albumin. A reactor equipped with a charged membrane as the coenzyme separator module was constructed and used in the continuous production of sorbitol. NADPH‐dependent aldose reductase isolated from Candida tropicalis IAM 12202 was used for the production of sorbitol from glucose. The coenzyme oxidized in this reaction was enzymatically regenerated by conjugation with glucose dehydrogenase, together with the coproduction of gluconic acid from glucose. With a substrate conversion of 85%, 100 g/L sorbitol was produced and equimolar gluconic acid was coproduced for more than 800 h, indicating that the reaction was efficiently coupled to the enzymatic regeneration. The initial high retainment ratio of the membrane was almost maintained throughout the entire reaction. Consequently, the turnover number of the coenzyme reached 106,000.
Production of dextransucrase and dextran by Leuconostoc mesenteroides immobilized in calcium‐alginate beads: II. Semicontinuous fed‐batch fermentations
Authors: El‐Sayed Abdel‐Halim M. M. , Mahmoud Wafaa M. , Coughlin Robert W.
Published: December 2004

Cells of Leuconostoc mesenteroides immobilized in calcium alginate beads were used to produce dextransucrase (DS) in three sequential cycles of semicontinuous fed‐batch fermentations. Each cycle consisted of a fed‐batch DS production period of 24 h followed by a batch dextran production period for another 24 h. Free, suspended cells were used in only one cycle of fed‐batch DS production followed by a dextran production period. It was impractically tedious to separate and reuse free cells. Increasing sucrose feed rate from 5 to 10 g/L h led to increases of the total enzymatic activity by about 88% with immobilized cells and by about 100% with free cells. In DS fed‐batch semicontinuous fermentation, total enzymatic activity produced by immobilized cells was 1.35 and 1.56 times greater than that produced by free cells with respective sucrose feeding rates of 10 and 5 g/L h. These increases in enzyme productivity with immobilized cells, however, required total overall operating times three times longer (three cycles) than with free cells (one cycle). Growing the microorganism at optimum conditions for DS production also increased the dextran yield and shortened the time of conversion of sucrose to dextran, regardless of whether the cells were free or immobilized. Moreover, during three cycles of semicontinuous operation (144 h) immobilized cells produced more than three times as much dextran as free cells during one cycle (24 h).
The separation of affinity flocculated yeast cell debris using a pilot‐plant scroll decanter centrifuge
Authors: Bentham A. C. , Bonnerjea J. , Orsborn C. B. , Ward P. N. , Hoare M.
Published: December 2004

The use of a scroll decanter centrifuge for the removal and dewatering of affinity‐flocculated yeast cell debris from a crude homogenate is described. Laboratory shear modulus measurements were used to compare the structure of flocculated and nonflocculated sediments and to indicate the dewatering conditions under which the sediment could be discharged from the centrifuge. The structure of the flocculated sediment was such that a dry beach could be used within the centrifuge while still being able to discharge the solids. The scroll decanter performance for recovery and dewatering of the flocculated homogenate was found to be independent of feed flow rate and differential scroll rate. Eighty‐five percent of the solid material was recovered from the flocculated homogenate while the extent of sediment dewatering resulted in the loss of only 7% of the soluble protein in the sediment. The supernatant clarity matched that achieved by low‐gravity laboratory centrifugation studies.
Stability and reaction characteristics of reverse‐phase evaporation vesicles (REVs) as enzyme containers
Authors: Sada Eizo , Katoh Shigeo , Terashima Masaaki , Shiraga Hiroshi , Miura Yasushi
Published: December 2004

The ion‐dependent enzymes, activities of which are affected by ions, were entrapped in the inside aqueous phase of reverse‐phase evaporation vesicles (REVs) to protect the enzyme activities from inhibitory cations. Their activities were still preserved in the presence of the inhibitory cations because the permeabilities of the inhibitory cations through the lipid membranes of REVs were much lower than those of substrates and products. The REVs were relatively stable in hypertonic condition, while they were unstable in hypotonic condition. Changes in osmotic pressure difference largely affected solute permeabilities of REVs. The shrinking and swelling of REVs resulting from osmotic pressure differences led to these changes in stabilities and solute permeabilities. Although the entrapment of the enzyme into REVs showed good protective effects against divalent cations, it was not effective against univalent cations. The measurements of cation permeabilities revealed that the enzyme trapped in the bilayer regions of REVs acts as a selective channel for the univalent cations. The REVs containing the enzyme could be used without any activity losses in a hollow fiber module.
Polarographic measurement of oxygen uptake using lipoxygenase in reverse micelles
Authors: Hochkoeppler Alejandro , Palmieri Sandro
Published: December 2004

Lipoxygenase‐catalyzed linoleic acid peroxidation was chosen as a model system to study the applicability of oxygraphy to monitor the oxygen uptake in organic solvents containing reverse micelles. Care was taken to control the oxygen back transfer from the atmosphere to the sample micellar solution, resulting in a significant improvement of electrode response. Under these conditions, lipoxygenase activity was linear up to 100 μg of enzyme. Given the quality of the calibration curve and the good correlation between lipoxygenase and ascorbate oxidase, the described technique is proposed as an alternative method for determining lipoxygenase activity in reverse micelles. The reliability of this technique was confirmed by the good agreement between polarography and classic spectrophotometry in kinetic studies. Preliminary experiments carried out on soybean cells solubilized in a Tween 85‐isopropylpalmitate system demonstrated that a light‐dependent oxygen uptake can be measured. The authors propose that the Clark‐type electrode be employed to study both the activity of oxidasic enzymes in reverse micelles and cell viability and physiology in organic solvents.
Depression of protein synthetic capacity due to cloned‐gene expression in E. coli
Authors: Wood Thomas K. , Peretti Steven W.
Published: December 2004

Recombinant bacterial systems exhibit limited capacities for heterologous protein production. As seen with this and other systems, cloned‐gene protein production reaches an upper limit despite further increases in gene dosage. A series of closely related plasmids which contain mutations affecting their copy number has been used to investigate the macromolecular impediments to enhanced recombinant protein production. Within a common host, HB101, the level of the ampicillin resistance gene, bla, was varied using five plasmids which differ solely in their replication machinery. Separate fermentations were conducted in which the plasmid copy number was varied from 0 to over 400 while the specific growth rate was fixed at 0.6 h−1 for each chemostat cultivation. The effects of constitutive expression of the bla gene as copy number was elevated were then determined using pulse‐labelling and RNA–DNA hybridizations. Specifically, the steady‐state level, synthesis rate, and stability of β‐lactamase messenger RNA and ribosomal RNA were determined as a function of copy number. The results indicate that as copy number rises, both β‐lactamase mRNA synthesis rates and steady‐state mRNA levels increase. Therefore, β‐lactamase production in these strains does not appear to be limited by the level of β‐lactamase mRNA. However, as the copy number was amplified, the stability of rRNA decreased to the point that steady‐state levels of rRNA decreased. These data indicate that a limitation develops within the translational capacity of the cell at high levels of cloned‐gene expression. The results suggest that strategies designed to enhance recombinant protein expression should include manipulation of translation as well as transcription.
Minimal nutritional requirements for immobilized yeast
Authors: Chen C. , Dale M. C. , Okos M. R.
Published: December 2004

The effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, “differential” plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 × 1011 cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35–65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75–95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth‐ and non‐growth‐associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).
Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. III: Mathematical model of cellular energetics and catabolism
Authors: Grosz Ron , Stephanopoulos Gregory
Published: December 2004

Mathematical models of the catabolic pathways, the utilization and waste of ATP, and the factors affecting yeast growth in a micro‐aerobic chemostat are presented. The models incorporate the intracellular metabolite and enzyme activity assays performed in Part II to explain the unusual macroscopic chemostat behaviors reported in Part I. The catabolic model successfully predicts a maximum in the specific ethanol productivity as a function of the intracellular ATP concentration. The ATP balance model enables the prediction of the intracellular ATP concentration and the ATP yield for given dissolved oxygen concentrations. Finally, in the context of a growth model, singularity theory provides a framework to explain the transition observed in Part I between hysteresis and the monotonic biomass versus oxygenation profiles in response to changes in the nutrient composition. The models serve to organize data and to concretely express proposed metabolic mechanisms and cause–effect hypotheses. The model is only applicable to the micro‐aerobic and excess glucose conditions encountered in this study.
Analysis of E. coli phoA‐lacZ fusion gene expression inserted into a multicopy plasmid and host cell's chromosome
Authors: Shin Pyong K. , Seo Jin‐Ho
Published: December 2004

The production of β‐galactosidase from the E. coli phoA‐lacZ fusion gene was studied to compare the gene expression behavior of two cloning methods: insertion to multicopy plasmids and integration into host cell's chromosome. The chromosome‐integrating strain showed more tight control of fusion gene expression levels than the plasmid‐containing strain. A 100‐fold enhancement of specific β‐galactosidase activity in the former strain was achieved in response to changes of initial inorganic phosphate concentration from 1 to 0.1 mM, whereas a 26‐fold increase was observed in the latter strain. The low degree of overexpression in the plasmid‐bearing cells was due to a combination of factors including leaky expression in repressed conditions and limitation of biosynthetic machinery in derepressed conditions. In a mixture of inorganic and organic phosphates, inorganic phosphate levels in the medium exhibited oscillatory behavior. The oscillation of inorganic phosphate is attributed to selective usage of inorganic phosphate followed by hydrolysis of organic phosphate to inorganic by alkaline phosphatase. The fluctuation of inorganic phosphate levels also caused the oscillation of β‐galactosidase activity.
Lewis cell studies to determine reactor design data for two‐liquid‐phase bacterial and enzymic reactions
Authors: Woodley J. M. , Brazier A. J. , Lilly M. D.
Published: December 2004

Substrate transfer rates from organic to aqueous phases were measured in the presence and absence of biocatalyst in the reaction medium, using modified Lewis cells. These measurements, in combination with intrinsic aqueous phase biocatalytic reaction kinetics, were used to confirm that benzyl acetate hydrolysis by pig liver esterase and toluene oxidation by a strain of Pseudomonas putida occur uniformly throughout the bulk of the aqueous phase. Such data may be used to provide a basis for two‐liquid‐phase biocatalytic reactor design.
NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes
Authors: Siano Steven A. , Mutharasan R.
Published: December 2004

A fiber‐optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic–anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose‐depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine‐depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate‐level phosphorylation, as shown by decreases of 35–52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6–38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.
A new method for direct selection of plasmid‐free segregants using mercury hypersensitivity as a phenotypic marker of bacterial plasmids
Authors: Mori Tsukasa , Kaga Takayuki , Toda Kiyoshi , Nakahara Hideomi , Ohtake Hisao
Published: December 2004

A new method was developed for direct selection of plasmid‐free segregants using mercury hypersensitivity (Hgss) as a phenotypic marker of bacterial plasmids. The Hgss marker originated from the 4.8‐kb EcoRI fragment H of the R‐factor R100. Since the EcoRI fragment H spans the majority of the mercury resistance operon (mer), but lacks the intact merA gene coding for the mercury reductase enzyme, this fragment conferred the Hgss phenotype. The Hgss marker was introduced into high‐copy‐number plasmids pUC18, pBR322, and pHSG298. Segregational loss of the Hgss plasmids caused a significant increase of resistance to Hg2+, and this allowed direct selection of plasmid‐free segregants on nutrient agars containing 1–2 μg HgCl2 ml−1. Plasmid‐loss segregants were estimated to appear at frequencies ranging from 10−3 to 10−7 for the tested high‐copy‐number plasmids. THe Hgss marker proved to be useful for direct selection of plasmid‐free segregants from a mixed population of plasmid‐harboring and plasmid‐free cells.
Mass transfer characterization of an airlift probe for oxygenating and mixing cell suspensions in an NMR spectrometer
Authors: Kramer Hans W. , Bailey James E.
Published: December 2004

The oxygen transfer characteristics of a 20‐mm O.D. airlift contactor fitted with an oxygen microelectrode were determined by steady‐state sulfite oxidation measurements. The volumetric mass transfer coefficient kLa was proportional to sparging power input per unit volume raised to a power which varied from 0.41 in water (coalescing bubbles) to 0.76 in NaCl solutions (noncoalescing bubbles). The highest observed kLa value was 0.012 s−1 which is sufficient to aerate Escherichia coli in an NMR spectrometer at moderate to high cell densities, depending on the physiological state of the cells.
Recombinant protein excretion in Escherichia coli JM103[pUC8]: Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability
Authors: Ryan Wen , Parulekar Satish J.
Published: December 2004

The presence of a high copy number plasmid (pUC8) was found to affect integrity of the cell envelope of Escherichia coli JM103, causing in turn significant release of the plasmid‐encoded protein (β‐lactamase). The alterations in cell membrane permeability were evident from the increased susceptibility of recombinant cells to deoxycholic acid and methylene blue, which did not have appreciable effect on plasmid‐free cells. The deteriorated cell membrane structure also resulted in a substantial reduction in specific growth rate and mass yield of plasmid‐bearing cells. Further enhancement in β‐lactamase excretion was achieved by permeabilizing cell membrane with ethylenediaminetetraacetate (EDTA) and phenethyl alcohol (PEA). Unlike other commonly used physical and chemical methods for releasing the enzymes accumulated in the cells, application of EDTA and PEA at appropriate concentrations neither led to cell death nor interrupted synthesis of the plasmid‐encoded protein. While in situ application of PEA was complicated due to interference with β‐lactamase activity, in situ application of EDTA was found to be an efficient way of releasing the recombinant protein without sacrificing its productivity. The experimental results demonstrate that the presence of EDTA and PEA can substantially reduce the growth rate differential between plasmid‐free and plasmid‐bearing cells, suggesting possible improvement of plasmid stability by application of these cell membrence‐permeabilizing agents on a periodic basis.
Lethal events during gas sparging in animal cell culture
Authors: Jöbses I. , Martens D. , Tramper J.
Published: December 2004

The lethal effects of gas sparging on hybridoma cells obtained from a chemostat culture were examined in a bubble column. Experiments were performed to identify and quantify the main hazardous event: bubble formation, bubble rising, or bubble breakup. The results indicate that bubble breakup is the main cause of cell death. The protective activity of the surfactant Pluronic F68 against sparging seems to result from a direct interaction with the cells rather than influencing bubble‐liquid interface properties.
Development of pure culture biofilms of P. putida on solid supports
Authors: Shrove Gina S. , Olsen Ronald H. , Vogel Timothy M.
Published: December 2004

Pseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined.

Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.

The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.
Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentation
Authors: Yabannavar V. M. , Wang D. I. C.
Published: December 2004

The use of immobilization in extractive lactic acid fermentation by Lactobacillus delbrueckii is preferred. In this article, the mathematical simulations to examine the influences of substrate and product transport were performed to assess the overall performance. The simulations showed that transport of the substrate in k‐carrageenan beads was not a rate limiting factor. However, the model observed significant buildup of inhibitory product in large beads. The model was validated through comparisons with the experimental results. Finally, the model was used to predict the performance of the extractive fermentation under different operating strategies.
Promotion of oxygen transfer in three‐phase fluidized‐bed bioreactors by floating bubble breakers
Authors: Kang Y. , Fan L. T. , Min B. T. , Kim S. D.
Published: December 2004

The objective of the present study was to investigate a method to enhance the volumetric rate of oxygen transfer in three‐phase fluidized‐bed bioreactors. The rates of oxygen transfer from air bubbles to viscous liquid media were promoted by floating bubble breakers in three‐phase fluidized beds operated in the bubble coalescing regime. The liquid‐phase volumetric oxygen transfer coefficient has been recovered by fitting the axial dispersion model to the resultant data, and its dependence on the experimental variables, such as the gas and liquid flow rates, particle size, concentration of bubble breakers, and liquid viscosity, has been examined. The results indicate that the liquid‐phase volumetric oxygen transfer coefficient can be enhanced up to 20‐25%. The coefficient exhibits a maximum with respect to the volume ratio of the floating bubble breakers to the fluidized solid particles; it increases with increases in the gas and liquid flow rates and size of fluidized particles, while it decreases with an increase in the liquid viscosity. An expression has been developed to correlate the liquid‐phase volumetric oxygen transfer coefficient with the experimental variables.
Photosynthetic activity of plant cells solubilized in water‐in‐oil microemulsions
Authors: Hochkoeppler A. , Luisi P. L.
Published: December 2004

With the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water‐in‐oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2‐ethyl‐hexyl‐sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N′, N′‐tetramethyl‐p‐phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.
Kinetics of loss of a recombinant plasmid in Bacillus subtilis
Authors: Shoham Yuval , Demain Arnold L.
Published: December 2004

Recombinant plasmid pCEDS is structurally unstable in Bacillus subtilis cultures. We have previously shown that stability can be independently increased by changing from a complex medium supporting high growth rates to a chemically‐defined medium supporting a lower growth rate and removal of a 4.77‐kb EcoRI fragment from pCED3 to give plasmid YS1. Further stabilization was achieved by combining the two approaches. In the present work, we show that the stabilization of the plasmid‐encoded LacZ+ phenotype can be explained solely by the effect on the growth rate ratio between cells containing modified and parental plasmids. By using modified stability experiments (where a single cell rather than a suspended colony was used to initiate growth), independent growth rate measurements, and a simple mathematical model, we can describe the kinetics of the loss of the LacZ+ phenotype in terms of two variables, α and p (where α is the ratio of growth rates between modified and parental cells, and p is the probability of obtaining modified cells from parental cells). Under the conditions tested, the average values of α were 1.52 for cultures growing in complex medium, 1.28 for cultures growing in defined medium, and 1.18 for cultures containing the modified plasmid pYS1 growing in complex medium. The calculated p values ranged between 10−8 and 10−10 under all conditions. Plasmid (pYS137) was used to directly estimate plasmid deletion rates in B. subtilis and it showed a rate between 5 × 10−8 and 1.1 × 10−9 deletions/cell/generation. In contrast to B. subtilis, there were no detectable differences in growth rates between Escherichia coli strains harboring plasmid pCEDS and plasmid‐free cells. These results explain the observed stability of pCEDS in E. coli cultures and indicate that readily detected instability in B. subtilis cultures can be the result of rare deletion events.
Oxygen transfer in slurry bioreactors
Authors: Kawase Yoshinori , Moo‐Young Murray
Published: December 2004

The oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40‐L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re‐examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.
Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: Stimulatory effect of carbon dioxide and ethylene on alkaloid production
Authors: Kim Dong‐II , Pedersen Henrik , Chin Chee‐Kok
Published: December 2004

Airlift bioreactor operations have been studied for the growth‐associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas‐stripping also played a significant role and it was discovered that CO2 and ethylene were important for product formation. By supplying a mixture of CO2 and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases.
Modeling linear and variable growth in phosphate limited suspension cultures of Opium poppy
Authors: Curtis Wayne R. , Hasegawa Paul M. , Emery Alden H.
Published: December 2004

In examining the growth kinetics of cell suspensions of opium poppy (Papaver somniferum), the increase in biomass with time was observed to be linear over the entire batch growth period of up to 20 days. Although batch growth profiles were reproducible utilizing the same inoculum, growth rates varied tremendously when experiments were inoculated with cells from different flasks. Both of these phenomena are difficult to explain with conventional batch growth models. In a series of a experiments, phosphate was determined to be the growth‐rate‐limiting substrate. By expressing growth rate in terms of the intracellular reserves of phosphorus, a growth model which expresses kinetics in terms of the intracellular phosphorus contents of the cells is shown to predict both linear growth character and inoculum dependent variability in growth. The stationary phase phosphate content of seven plant suspension cultures of different plant species was found to be comparable to phosphorus levels of phosphate‐starved poppy cells, which suggests that phosphate limitation may be common for plant tissue culture. The applicability of this model to other biological systems which display similar batch growth patterns when subjected to inorganic nutrient deprivation is discussed.
Continuous acetone‐butanol‐ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation
Authors: Friedl Anton , Qureshi N. , Maddox Ian S.
Published: December 2004

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m2 h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.
Propionic acid fermentation of lactose by Propionibacterium acidipropionici: Effects of pH
Authors: Hsu Sheng‐Tsiung , Yang Shang‐Tian
Published: December 2004

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was ∼0.23 h−1. The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from ∼33% (w/w) at pH 6.1–7.1 to ∼63% at pH 4.5–5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%–12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex‐tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.
Effect of light irradiation on anthocyanin production by suspended culture of Perilla frutescens
Authors: Zhong Jian‐jiang , Seki Tatsuji , Kinoshita Shin‐ichi , Yoshida Toshiomi
Published: December 2004

After a series of experiments on photoperiodicity and light intensity under daylight supplied by an ordinary fluorescent lamp in cultivations using a flask and a roux bottle, it was found that irradiation at 27.2 W/m2 for the whole period was effective for anthocyanin production by a suspended culture of Perilla frutescens (shiso). A high amount of anthocyanin pigments, 3.0 g/L, was obtained in a bubble column bioreactor after 10 days of cultivation at an aeration rate of 0.1 vvm with light irradiation at 27.2 W/m2, while 2 g/L was obtained at 13.6 W/m2 and very little at 54.4 W/m2. A high amount of anthocyanin pigments, 2.9 g/L, was also produced using an aerated and agitated bioreactor at an agitation speed of 130 rpm, an aeration rate of 0.1 vvm and light irradiation intensity of 27.2 W/m2. The amount of anthocyanin produced was more than twice that without light irradiation, Keeping the other cultivation conditions the same. The results obtained also showed that the amount of anthocyanin pigment accumulated in a shake flask could be rather well reproduced in bioreactors for both aerated culture, and aerated and agitated culture, by improving the conditions of light irradiation, which conspicuously affects metabolite formation.
Development of a kinetic model for the alcoholic fermentation of must
Authors: Caro I. , Pérez L. , Cantero D.
Published: December 2004

We Propose a kinetic expression which accounts for the temperature dependence of ethanol yield losses in batch alcoholic fermentation. Moreover, the characteristic parameters of the microbial growth equation have been calculated for Saccharomyces cerevisiae under typical wine industry conditions. A substrate consumption equation is established which minimizes possible model deviations in the latter process stages. Experimental data were obtained in the laboratory and the proposed equations were then applied at an industrial level (2.5 × 104 L) where they described the data well.
The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction
Authors: Laval Jean Marc , Moiroux Jacques , Bourdillon Christan
Published: December 2004

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi‐irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.
Implementation of an adaptive controller for the startup and steady‐state running of a biomethanation process operated in the CSTR mode
Authors: Renard P. , Van Breusegem V. , Nguyen M.‐T. , Naveau H. , Nyns E.‐J.
Published: December 2004

An adaptive control algorithm has been implemented on a biomethanation process to maintain propionate concentration, a stable variable, at a given low value, by steering the dilution rate. It was thereby expected to ensure the stability of the process during the startup and during steady‐state running with an acceptable performance. The methane pilot reactor was operated in the completely mixed, once‐through mode and computer‐controlled during 161 days. The results yielded the real‐life validation of the adaptive control algorithm, and documented the stability and acceptable performance expected.
Reaction kinetics in biofilms
Authors: Lewandowski Zbigniw , Walser Gabriele , Characklis William G.
Published: December 2004

A novel in situ microtechnique allows evaluating parameters of diffusion‐controlled reactions in biofilms. A microprobe, 15 μm in diameter, was used to simultaneously measure the dissolved oxygen concentration and the optical density at different depths in a submerged biofilm. Based on the results, the biofilm diffusion coefficient for dissolved oxygen, Df the dissolved oxygen flux through the biofilm surface, J02, and the half velocity coefficient, Ks, have been calculated.
Effect of photobioreactor inclination on the biomass productivity of an outdoor algal culture
Authors: Lee Yuan‐Kun , Low Chin‐Seng
Published: December 2004

The profiles of photon flux density incidented on a tubularloop photobioreactor in the day could be altered by inclining the bioreactor at an angle with the horizontal. The photon flux density at noon decreased with increasing angle of inclination, whereas the photon flux density in the early morning and late afternoon increased with increasing angle of inclination. The overall photosynthetic radiance received by the bioreactor inclined at 0, 25, 45, and 80° was 1:0.89:0.77:0.62. Regardless of the angle of bioreactor inclination, the overall biomass output rate of a fed‐batch culture over an 8‐h/day period was comparable (26–36 g‐biomass m−2 bioreactor surface area day−1). As a bioreactor inclined at an angle occupied smaller land area, and daily biomass output rate per land area of a bioreactor inclined at 80° (130 g‐biomass m−2 land) was about six times of that obtainable at horizontal position (21‐g biomass m−2 land). The bioenergetics growth yield from the absorbed photosynthetic radiance was not a constant but an inverse function of the photon flux density. The quasi‐steady state chlorophyll content of the Chlorella cells varied between 36 and 63 mg g−1 cells. Photoinhibition of the maximum photosynthetic capacity was not observed in this study.
Interaction of invertase with polyelectrolytes
Authors: Dautzenberg Herbert , Kötz Joachim , Philipp Burkart , Rother Gudrun , Schellenberger Alfred , Mansfeld Johanna
Published: December 2004

In connection with our work on polyelectrolyte complex formation with polyampholytes, the interaction between invertase and several linear polyelectorlytes has been investigated by means of turbidimetry, light scattering measurements, and determination of the enzyme activity. Polyelectrolyte complex formation of invertase was shown to occur with cationic polyelectrolytes only. The light‐scattering data yield information on aggregation and desegregation processes in complex formation. As indicated by our results, only a part of the protein molecules is engaged in this Coulombic interaction, and this part shows a rather small enzyme activity only. Thus, a direct interaction between invertase and a cationic polyelectrolyte is no effective approach to enzyme binding, but a complete immobilization of invertase can be achieved via an “inclusion flocculation” with a symplex formed by interaction between an anionic and a cationic linear polyelectrolyte or via immobilization in symplex microcapsules.
Measurement and simulation of the morphological development of filamentous microorganisms
Authors: Yang H. , Reichl U. , King R. , Gilles E. D.
Published: December 2004

Growth of Streptomyces tendae was investigated in submerged culture. Images of several mycelia were analyzed by means of an image‐processing system. The studies revealed that tip growth angles and branching outgrowth angles could be regarded as normally distributed. Based on these results, a random model for directional growth of hyphal tips as well as directional growth of branches is proposed. This model shows curved elongation of hyphal tips, so that the morphological development of a mycelium up to the formation of a pellet is predicted, similar to that observed in nature.
Agitation induced cell injury in microcarrier cultures. Protective effect of viscosity is agitation intensity dependent: Experiments and modeling
Authors: Lakhotia Sanjay , Papoutsakis Eleftherios T.
Published: December 2004

The effect of medium viscosity on the specific death rate of bovine embryonic kidney (BEK) cells cultured in spinner flask microcarrier cultures has been examined for various impeller speeds. Two types of media were used, a serum‐containing growth medium and a serum‐free maintenance medium. The latter does not support cell growth. We found that increasing medium viscosity suppresses cell death rates in both growth and maintenance medium cultures in an agitation‐intensity‐dependent fashion; the beneficial effect of medium viscosity in reducing the specific death rate is amplified as the agitation rate is increased. Furthermore, increasing medium viscosity has no effect on the specific death rate of the cells when the agitation rate is below a critical level. A model based on the turbulent energy content of eddies in the dissipation spectrum of turbulence of length scales on the order of magnitude of the microcarrier diameter and lower has been developed to account for cell death due to both bead‐to‐bead and bead‐to‐eddy interactions. The model constitutes a significant departure from previous efforts first because both types of interactions are accounted for simultaneously and second because the properties of a spectrum of eddies instead of the Kolmogorov‐scale eddy size alone are used in the model. The model explains the functional dependence of the specific death rates on the medium viscosity at varying agitation intensities.
Displacement of equilibrium in electroenzymatic reactor for acetaldehyde production using yeast alcohol dehydrogenase
Authors: Lortie Robert , Fassouane Aziz , Laval Jean‐Marc , Bourdillon Christian
Published: December 2004

Electrochemical regeneration of NAD was performed in a bench scale reactor in which yeast alcohol dehydrogenase catalyzed the oxidation of ethanol. By recycling one of the products of the reaction, it was possible to displace the equilibrium and favor the production of acetaldehyde. The flow‐through electrode was made of graphite felt and had a specific area of 275 cm−1. A mathematical model taking into account the enzymatic and electrochemical reaction rates as well as the mass transfer to the electrode was used to analyze the results. The limiting steps in the reactor are the electrochemical reaction for low potentials and the cofactor mass transfer for high potentials.
Effects of electroconductive heat treatment and electrical pretreatment on thermal death kinetics of selected microorganisms
Authors: Palaniappan Sevugan , Sastry Sudhir K. , Richter Edward R.
Published: December 2004

Suspensions of yeast cell (zygo Saccharomyces bailii) in a phosphate buffer solution were subjected to conventional (hot water) and ohmic (electric current) heating under identical temperature histories. Experiments were also conducted with cells of Escherichia coli to compare the lethal effect of combination of sublethal electrical preteatment and conventional heating with conventional heating. The kinetic parameters (D,Z,K and Ea) were determined for both organisms during different treatments. There was no significant difference in the death rate of yeast cells during conventional and ohmic heating at the voltage range used in this study. Results of electrical pretreatment and conventional heating on E. coli indicated differences under certain conditions when compared with pure conventional heating. Thus it is concluded that microbial death during ohmic heating was due primarily to thermal effects with no significant effect of electric current per se. Sublethal electrical pretreatment appears to offer potential for increased bacterial inactivation in certain cases.
Kinetics of lipase‐catalyzed esterification in supercritical CO2
Authors: Marty A. , Chulalaksananukul W. , Willemot R. M. , Condoret J. S.
Published: December 2004

This study compares two solvents for enzymatic reactions: supercritical carbon dioxide (SCCO2) and organic solvent (n‐hexane). The model reaction that was chosen was the esterification of oleic acid by ethanol catalyzed by an immobilized lipase fromMucor miehei(Lypozyme). The stability of the enzyme appeared to be quite good and similar in both media but was affected by the water content. Partition of water between solvents and immobilized enzyme has been calculated from experimental adsorption isotherms. The water content of the solid phase has a dramatic influence on the activity of the enzyme and its optimum value for activity was about 10% (w/w) in both media. A kinetic study enabled a Ping‐Pong Bi‐Bi reaction mechanism with inhibition by ethanol to be suggested. Despite some differences in kinetic constants, activity was in the same range in both media. Hypotheses for explaining the kinetic constant variations have been proposed and particular attention has been paid to the pH effects.