Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

Xylanolytic anaerobic thermophiles from Icelandic hot-springs

Anaerobic enrichment cultures inoculated with neutral and alkaline (pH 7.0–9.0) sediment and biomat samples from hot-springs in Hveragerdi and Fluir, Iceland, were screened for growth on beech xylan from pH 8.0 to 10.0 at 68° C: no growth occured in cultures above pH 8.4. Five anaerobic xylanolytic bacteria were isolated from enrichment cultures at pH 8.4; all five microbes were Gram-positive rods with terminal spores, and produced CO₂, H₂, acetate, lactate and ethanol from xylan and xylose. One of the isolates, strain A2, grew from 50 to 75° C, with optimum growth near 68° C, and from pH 5.2 to 9.0 with an optimum between 6.8 and 7.4. Taxonomically, strain A2 was most similar to Clostridium thermohydrosulfuricum. At pH 7.0, the supernatant xylanases of strain A2 had a temperature range from 50 to 78° C with an optimum between 68 and 78° C. At 68° C, xylanase activity occurred from pH 4.9 to 9.1, with an optimum from pH 5.0 to 6.6. At pH 7.0 and 68° C, the K _m of the supernatant xylanases was 2.75 g xylan/l and the V _max was 2.65 × 10^–6 kat/l culture supernatant. When grown on xylose, xylanase production was as high as when grown on xylan. Correspondence to: B. K. Ahring     

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.     

Rapid in vitro adventitious shoot propagation of <Emphasis Type="Italic">Scopolia parviflora</Emphasis> through rhizome cultures for enhanced production of tropane alkaloids

A rapid micropropagation system for Scopolia parviflora Nakai (Solanaceae), a rare medicinal plant native to Korea, was established using rhizome cultures. Shoots that originated from adventitious shoots of the rhizome were multiplied when the rhizomes were cultured on half-strength B5 liquid medium supplemented with various growth regulators. Optimum shoot multiplication was observed in half-strength B5 medium containing 3% (w/v) sucrose and 5.77 M gibberellic acid (GA₃). Each rhizome gave rise to an average of 12 shoots. Shoot elongation and root induction from multiple shoots occurred on growth regulator-free half-strength B5 solid medium. Healthy plantlets were transferred to a peat moss:vermiculite mixture for acclimatization, which was successful. The concentrations of tropane alkaloids, hyoscyamine and scopolamine were determined in different tissues of native growing plants, in vitro-propagated plants and acclimatized plants by high-performance liquid chromatography. The analysis revealed that the levels of hyoscyamine and scopolamine were higher in in vitro-propagated plants than in the native growing plants. When the rhizome was cut into segments and transferred to optimal culture conditions for multiple shoot propagation, only 12 weeks were required to produce a mature plant. We conclude that in vitro propagation techniques through rhizome cultures provide an efficient and rapid method for shoot propagation of S. parviflora.Abbreviations BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - HPLC High-performance liquid chromatography - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid     

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, -glucosidase and acid phosphatase, secreted by the cells during growth, and also -amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H₂O₂ from the cells (oxidative burst). H₂O₂ released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H₂O₂ is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H₂O₂ is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H₂O₂. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H₂O₂ and, when present together with elicitor, induction was synergistically increased.     

Competition for ethanol between sulfate-reducing and fermenting bacteria

Competition for ethanol between the sulfate-reducing bacteria Desulfobulbus propionicus, Desulfotomaculum orientis, Desulfovibrio vulgaris Marburg, Desulfovibrio gigas, Desulfovibrio desulfuricans Essex and the fermenting bacteria Pelobacter propionicus and Acetobacterium carbinolicum were studied in batch culture. A number of these bacteria was also chosen for competition experiments under ethanol limitation in chemostat culture. The maximum growth rates determined by washout experiments were higher for the fermenting bacteria (_max=0.096 resp. 0.335h^–1) than for the sulfate-reducing bacteria (_max0.03h^–1). In contrast, the saturation concentrations for half maximum growth rates (K_s values) for ethanol were lower for the sulfate-reducing bacteria (K_s5 M) than for the fermenting bacteria (K_s50 M). In batch culture competition experiments the fermenting bacteria turned out to be the better competitors, degrading 51–80% of the ethanol added. In competition experiments with ethanollimited chemostat cultures the sulfate-reducing bacteria Desulfobulbus propionicus and Desulfovibrio vulgaris outcompeted Pelobacter propionicus at dilution rates below their maximum specific growth rates. At a high dilution rate, a fast growing population of Desulfobulbus propionicus originated and was enriched in the chemostat during the competition experiment.     

In vitro propagation of cassava (Manihot esculenta Crantz)

A method is presented for the rapid in vitro propagation of cassava (Manihot esculenta Crantz). Nodal explants were induced to grow as multiple-shoot cultures on a medium containing 1.0 M 6-benzylamino purine (BAP), supplemented with 0.25 M -naphthaleneacetic acid (NAA). Nodes were removed from the shoots after three weeks of growth and subcultured on fresh culture medium. An average of 7.0 nodes were produced from each explanted node after three weeks in culture. Nodal explants were transferred to a medium containing 2.5 M indole-3-butyric acid (IBA) to improve root initiation on the developing plantlets. Plant establishment was possible upon transfer to soil. In vitro propagation offers enhanced rates of multiplication over more conventional methods of propagation. In addition, in vitro propagation facilitates the storage and international exchange of cassava germplasm.     

Tight control of growth and cell differentiation in photoautotrophically growing moss (<Emphasis Type="Italic">Physcomitrella</Emphasis><Emphasis Type="Italic">patens</Emphasis>) bioreactor cultures

The use of the moss Physcomitrella patens as a production system for heterologous proteins requires highly standardised culture conditions. For this purpose a semi-continuous photoautotrophic bioreactor culture of Physcomitrella was established. This culture grew stably for 7 weeks in a 5-l bioreactor with a dilution rate of 0.22/day. Enrichment of the air for aeration in a batch bioreactor culture with 2% (v/v) CO₂ resulted in an increase in the specific growth rate to 0.57/day. Changes in the pH of the semi-continuous bioreactor culture medium between pH 4.5 and pH 7.0 influenced protonema differentiation; however it did not negatively affect the growth rate compared to uncontrolled pH. The advantages of Physcomitrella as a system for the production of heterologous proteins in plants are discussed.     

The effect of oxygen partial pressure in bioreactors on cell proliferation and subsequent differentiation of somatic embryos of Cyclamen persicum

The effect of the relative oxygen partial pressure (pO₂) in bioreactors on cell proliferation and subsequent differentiation of somatic embryos from suspension cultures of Cyclamen persicum Mill. was investigated. The growth rate of cell line 3738-VIII in growth-regulator containing medium in bioreactors at 5% pO₂ was slightly reduced in comparison to 10% and 20% pO₂. Cultures growing at 40% pO₂ had a lower growth rate, a markedly reduced cell viability and showed a decrease of the medium pH to 3.5. Because a pH-control with a setpoint of 3.3 caused cell death within 4 days, it was assumed, that the reason for the poor cell proliferation and viability in the cultures at 40% pO₂ was an effect of medium acidification rather than of the high O₂ partial pressure. A significantly higher number of germinating embryos was obtained from the cultures grown at 40% pO₂ than from those grown in flasks or in bioreactors at 5%, 10% and 20% pO₂. These results were specific for cell line 3738-VIII. Another cell line, 3736-12, did not show marked differences in cell proliferation, viability, pH or subsequent regeneration of somatic embryos when grown at different O₂ partial pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and interferon-γ production in batch culture of CHO cells

The relationship between growth and interferon- (IFN-) production in the recombinant cell line CHO 320 was studied by varying the foetal calf serum (FCS) concentration. The specific growth rate varied with the initial FCS concentration in a manner which could be well fitted by the Monod model. TheK _s and _max values were found to be 0.771% (v/v) serum and 0.031 h^–1 respectively. The average specific IFN- production rates during the exponential phase increased with increasing FCS concentration. A good correlation between specific production rate and specific growth rate was found in all phases of the culture except the lag phase and it was clearly demonstrated that IFN- production was growth associated. Specific glucose and glutamine utilisation rates were inversely related to specific growth rates.     

Kinetics of substrate inhibition of exponential yeast growth

This work concerns mathematical modeling of the rate of microbial growth on inhibitory levels of nutrients as affected by pH, concentration of the nutrients, temperature, cultivation method, and method of data analysis. Candida utilis (ATCC 9226) was grown with sodium acetate as growth-limiting carbon and energy source in mineral salts medium in shake flask and continuous cultures to study inhibition by excess acetate. Differential shake flask cultures were grown at low yeast concentrations at temperatures (T) of 25 and 30°C, pH's between 5.5 and 7.0, and acetate concentrations (S) between 0.25 and 3.0% (w/v). Growth data were exponential with correlation coefficients greater than 0.995 in 49 of 56 experiments; the lowest correlation coefficient was 0.986. Specific growth rates (μ) determined by graphical methods showed only fair correlation with those determined by regression analysis. Both sets of specific growth rate data were grouped at constant T and pH and fitted to the three parameter equation, The improvement in use of the fitted equation instead of the mean value was significant with greater than 70% confidence in all (14 groups) and 90% confidence in only half of the data groups; the correlation does not improve with the increasing acetate inhibition at lower pH. Both defects in the model and insufficient data at each pH are responsible. A modified six parameters with hydrogen ion concentration(H⁺) as follows: Specific growth rates calculated with the six parameter equation matched observed values in all groups of isothermal data better than the means with greater than 99% confidence. The six parameter model adequately represents effects of acetate and hydrogen ion concentrations under constant or slowly changing environmental conditions and balanced growth; although better models probably exist. Thus steady-stste and transient continuous culture experiments agreed with many published growth yields, but specific growth rates could only be predicted qualitatively from the model fit to the shake flask data. The data and present models could be incorporated into published models for transient growth at low nutrient concentrations to correlate and perhaps predict microbial growth kinetics over a much wider range of growth conditions than now possible.     

Regulation of β-exoglucanase activity production by Saccharomyces cerevisiae in batch and continuous culture

The rate of synthesis and secretion of exo-1–3--glucanase activity closely paralleled the specific rate of growth in exponentially growing Saccharomyces cerevisiae cells in batch culture. When the stationary phase was reached both synthesis and secretion stopped. No activity was synthesized when the cells were maintained in carbon sources that did not allow them to grow. Studies in continuous culture indicate a strong relationship between the synthesis of exoglucanase activity and the specific growth rate. These results are taken as evidence of an essential role of this activity during the yeast budding cycle.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - S_r glucose concentration in the sterile reservoir; , glucose concentration at the steady state - biomass density at the steady state - G glucanase activity - Q _g specific exoglucanase synthesis rate     

Evaluation of different bio-kinetic parameters of Curvularia lunata at different environmental conditions

Growth kinetic parameters for Curvularia lunata were determined in yeast extract, peptone and dextrose medium under different environmental conditions. The values of specific growth rate () were found to be different at different cultivation (pH and temperature) conditions. At the optimum growth conditions (pH 7.0 and temperature 28 °C) the values of specific growth rate and maintenance coefficient for C. lunata were maximum (0.19 h^-1) and minimum (0.04 h^-1) respectively, whereas the growth yield coefficient (Y_EG) decreased with the increase of cultivation temperature. The values of saturation constant (K_S) did not change appreciably with the change of cultivation conditions.     

Effects of Culture Method and Growth Phase on Free Lipid Composition of Yersinia pseudotuberculosis

The influence of culture method (free-floating cells in liquid nutrient broth or bacteria attached to agar surface on solid agarized medium of the same formulation) and bacterial age on the composition of free lipids in Yersinia pseudotuber-culosis (O:Ib serovar, strain KS 3058) grown in the cold (5°C) has been investigated. The specific growth rate of the bacteria on solid medium was about threefold less than that in liquid medium. The qualitative composition of phospholipids and fatty acids only slightly depended on the bacterial culture method. At the same time, the colonially growing cultures contained somewhat more total lipids, they synthesized more phospholipids, in the linear growth phase they contained more lysophosphatides, and they had higher fatty acid unsaturation index and higher pathogenic potential than their planktonic counterparts grown in otherwise identical conditions. The bacterial growth phase influenced the amount of 3-hydroxytetrade-canoic acid and, indirectly, that of lipopolysaccharide. The dynamics of changes in the amount of this acid with bacterial age was opposite in the surface and broth cultures.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

Differential expression of a cell wall-localized peroxidase isoenzyme capable of oxidizing 4-hydroxystilbenes during the cell culture of grapevine (Vitis vinifera cv. Airen and Monastrell)

Differential expression and localization of peroxidase isoenzymes capable of oxidizing 4-hydroxystilbenes was studied during establishment of callus cultures from Vitis vinifera Airen (anthocyanin-non accumulating) and Monastrell (anthocyanin-accumulating) berries. Callus formation from mesocarp tissues was accompanied by differential expression of several peroxidase isoenzyemes located in cell walls, among which only peroxidase isoenzyme A₁ was capable of oxidizing 4-hydroxystilbene to any great extent. Likewise, grape cell cultures were capable of accumulating the grape stilbene phytoalexin, resveratrol. However, -viniferin, the most powerful phytoalexin in grapevines and previously considered as the product of peroxidase-mediated oxidative coupling of two resveratrol moieties, was only detectable in trace amounts. Since grapevine suspension cell cultures were unable to produce H₂O₂ as revealed by the luminol test, H₂O₂ production by the cultured cells appears to be one of the main factors which limits resveratrol oxidation in the cell walls of grapevine cells cultured in suspension.     

Metabolism of Acetivibrio cellulolyticus during optimized growth on glucose,cellobiose and cellulose

Summary The growth of Acetivibrio cellulolyticus in 2.5 l batch cultures was optimized by controlling the growth pH at 6.7, the dissolved inorganic sulphide concentration at 0.4–0.6 mM, and by constant removal of hydrogen from the cultures by sparging with N₂/CO₂ or N₂ gas. An initial ethanol concentration of 0.15% (w/v) in cellobiose media resulted in specific growth rates which were reduced by about 75% compared to growth rates of 0.17 h^–1 in control cultures. Acetivibrio cellulolyticus had to be adapted for growth on glucose and ¹⁴C-radiotracer studies indicated that glucose was metabolized by the Embden-Meyerhof pathway. The specific growth rate (=0.03h^–1) and molar growth yield (Y_glucose=21.5) were considerably lower than those obtained (=0.17 h^–1, Y_cellobiose=68.9) in cellobiose media. A Y_ATP of 12.8 was obtained during growth on cellobiose. The mol product formed per mol Avicel cellulose fermented (on anhydroglucose equivalent basis) were 3.70 H₂, 2.64 CO₂, 0.73 acetate, 0.39 ethanol and 0.03 total soluble sugars on glucose basis. Maximum cellulase activity was observed in cellulose-grown cultures.National Research council of Canada No. 20826     

Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

The effect of long term CO2 enrichment on the growth,biomass partitioning and mineral nutrition of Sitka spruce (Picea sitchensis (Bong.) Carr.)

Sitka spruce [Picea sitchensis (Bong.) Carr.] seedlings were grown for 3 years in an outside control plot or in ambient (355 mol mol^-1) or elevated (ambient + 350 mol mol^-1) atmospheric CO₂ environments, within open top chambers (OTCs) at the Institute of Terrestrial Ecology, Edinburgh. Sequential harvests were carried out at the end of each growing season and throughout the 1991 growing season, five in all. Plants grown in elevated CO₂ had, (i) 35 and 10% larger root/shoot ratios at the end of the first and third season, respectively, (ii) significantly higher summer leader extension relative growth rates, which declined more rapidly in early autumn than ambient grown plants, (iii) after three growing seasons a significantly increased mean annual relative growth rate, (iv) consistently lower foliar nutrient concentrations, and (v) after two growing seasons smaller total projected needle areas. Plants grown inside OTCs were taller, heavier and had a smaller root/shoot ratio than those grown outside the chambers. There was no effect of CO₂ concentration on Sitka spruce leaf characteristics, although leaf area ratio, specific leaf area and leaf weight ratio all fell throughout the course of the 3 year experiment.