Kinetics and morphology of glucose-limited cultures of moulds grown in a chemostat and on solid media.

The affinity (KS value) of Geotrichum candidum for glucose determined from chemostate cultures was ca. 1 mg/1. KS values for glucose was also estimated from the radial growth rates of colonies of G. candidum and Neurospora crassa grown on media solidified with agar or silica gel. An assessment is made of the use of colony radial growth rate to determine substrate affinities. The length of apical and intercalary hyphal comparte ments, internode length and the diameter of leading hyphaat the margin of colonies grown on solid media were all reduced at low glucose concentrations.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K_s, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K_s, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K_s, about 30 mM).     

Potential of pure and mixed cultures of Cladosporium cladosporioides and Geotrichum candidum for application in bioremediation and detergent industry

The effect of ethoxylated oleyl–cetyl alcohol (Henkel, “Merima”, Serbia) on the growth and metabolic activity of Cladosporium cladosporioides, Geotrichum candidum and their mixed culture was in the focus of this paper. The cultures were grown in Czapek-Dox liquid nutrient medium with the addition of 0.5% pollutant and without it. The physico-chemical and biochemical changes of pH, the total biomass dry weight, the quantity of free and total organic acids, proteolytic activity and the quality of carbohydrates were evaluated from 4th to 19th day of fungal growth. The pollutant caused an inhibitory effect on biomass dry weight of C. cladosporioides and G. candidum for 10.36% and 4.65% respectively, and stimulatory effect on biomass of mixed culture for 3.80%. The pollutant had influence on the decrease in pH value of the media in the phase of culture growth, and pH changes were correlated with the amount of excreted total organic acids. The highest quantity of free and total organic acids was noted in media with pollutant of mixed culture and C. cladosporioides, respectively. The alkaline protease activities of C. cladosporioides, G. candidum and mixed culture were enhanced by addition of pollutant for 56.88%, 55.84% and 30.94% respectively. The obtained results indicate the potential of both pure and mixed cultures in mycoremediation environment contaminated by alcohol ethoxylated and detergent industry.     

Kinetics of Denitrifying Growth by Fast-Growing Cowpea Rhizobia

Two fast-growing strains of cowpea rhizobia (A26 and A28) were found to grow anaerobically at the expense of NO₃⁻, NO₂⁻, and N₂O as terminal electron acceptors. The two major differences between aerobic and denitrifying growth were lower yield coefficients (Y) and higher saturation constants (K_s) with nitrogenous oxides as electron acceptors. When grown aerobically, A26 and A28 adhered to Monod kinetics, respectively, as follows: K_s, 3.4 and 3.8 μM; Y, 16.0 and 14.0 g · cells eq⁻¹; μ_max, 0.41 and 0.33 h⁻¹. Yield coefficients for denitrifying growth ranged from 40 to 70% of those for aerobic growth. Only A26 adhered to Monod kinetics with respect to growth on all three nitrogenous oxides. The apparent K_s values were 41, 270, and 460 μM for nitrous oxide, nitrate, and nitrite, respectively; the K_s for A28 grown on nitrate was 250 μM. The results are kinetically and thermodynamically consistent in explaining why O₂ is the preferred electron acceptor. Although no definitive conclusions could be drawn regarding preferential utilization of nitrogenous oxides, nitrite was inhibitory to both strains and effected slower growth. However, growth rates were identical (μ_max, 0.41 h⁻¹) when A26 was grown with either O₂ or NO₃⁻ as an electron acceptor and were only slightly reduced when A28 was grown with NO₃⁻ (0.25 h⁻¹) as opposed to O₂ (0.33 h⁻¹).     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.     

Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K₂HPO₄ reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.     

Determination of Tissue Thermal Conductivity by Measuring and Modeling Temperature Rise Induced in Tissue by Pulsed Focused Ultrasound

A tissue thermal conductivity (K_s) is an important parameter which knowledge is essential whenever thermal fields induced in selected organs are predicted. The main objective of this study was to develop an alternative ultrasonic method for determining K_s of tissues in vitro suitable for living tissues. First, the method involves measuring of temperature-time T(t) rises induced in a tested tissue sample by a pulsed focused ultrasound with measured acoustic properties using thermocouples located on the acoustic beam axis. Measurements were performed for 20-cycle tone bursts with a 2 MHz frequency, 0.2 duty-cycle and 3 different initial pressures corresponding to average acoustic powers equal to 0.7 W, 1.4 W and 2.1 W generated from a circular focused transducer with a diameter of 15 mm and f-number of 1.7 in a two-layer system of media: water/beef liver. Measurement results allowed to determine position of maximum heating located inside the beef liver. It was found that this position is at the same axial distance from the source as the maximum peak-peak pressure calculated for each nonlinear beam produced in the two-layer system of media. Then, the method involves modeling of T(t) at the point of maximum heating and fitting it to the experimental data by adjusting K_s. The averaged value of K_s determined by the proposed method was found to be 0.5±0.02 W/(m·°C) being in good agreement with values determined by other methods. The proposed method is suitable for determining K_s of some animal tissues in vivo (for example a rat liver).     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

Studies on the relationship between specific growth rate and concentration of nitrogen source for heterogeneous microbial populations of sewage origin

Batch experiments were run using heterogeneous populations to determine whether a hyperbolic equation of the type suggested by Monod could be used to depict the relation between specific growth rate, μ, and NH₃-N concentration when ammonia N was the growth-limiting nutrient. The heterogeneous populations employed were developed from sewage seed grown on glucose at various levels of nitrogen and various dilution rates in completely mixed continuous flow reactors. It was found that the hyperbolic function could be used. Values of μ_m in the range of 0.4–0.7 hr^?1 were observed, and values of K_s, in general, ranged from 1.5 to 4.0 mg/l. Variation in the values of these growth “constants” did not follow any discernible pattern related to past growth history (i.e., COD:N ratio or dilution rate at which the cells were previously grown).     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Evolution of Hypervariable Region 1 of Hepatitis C Virus in Primary Infection

The hypervariable region 1 (HVR-1) of the putative envelope encoding E2 region of hepatitis C virus (HCV) RNA was analyzed in sequential samples from three patients with acute type C hepatitis infected from different sources to address (i) the dynamics of intrahost HCV variability during the primary infection and (ii) the role of host selective pressure in driving viral genetic evolution. HVR-1 sequences from 20 clones per each point in time were analyzed after amplification, cloning, and purification of plasmid DNA from single colonies of transformed cells. The intrasample evolutionary analysis (nonsynonymous mutations per nonsynonymous site [K_a], synonymous mutations per synonymous site [K_s], K_a/K_s ratio, and genetic distances [gd]) documented low gd in early samples (ranging from 2.11 to 7.79%) and a further decrease after seroconversion (from 0 to 4.80%), suggesting that primary HCV infection is an oligoclonal event, and found different levels and dynamics of host pressure in the three cases. The intersample analysis (pairwise comparisons of intrapatient sequences; rK_a, rK_s, rK_a/rK_s ratio, and gd) confirmed the individual features of HCV genetic evolution in the three subjects and pointed to the relative contribution of either neutral evolution or selective forces in driving viral variability, documenting that adaptation of HCV for persistence in vivo follows different routes, probably representing the molecular counterpart of the viral fitness for individual environments.     

GLUCOSE TRANSPORT SYSTEMS AND GROWTH CHARACTERISTICS OF BRACTEACOCCUS MINOR1,2

Bracteacoccus minor grows on glucose both in the dark and in the light. However, the growth rates at 50 ft-c or higher in the presence of glucose are considerably lower than those in inorganic media at the same intensities. The lowered growth rates in the presence of glucose appear to be related to changes in metabolism toward increased production of storage carbohydrates. Glucose shortens the length of the lag phase at high light intensities and increases the length of the exponential phase at all light intensities, resulting in very high cell yields compared to cultures grown in inorganic media. B. minor has 2 transport systems for glucose: (1) a high affinity system, K_S=1 × 10^?5 M, which is formed in the dark in the absence of external glucose; and (2) a low affinity system, K_S=5 × 10^?4 M, which appears to be constitutive. At high concentrations of glucose there is also significant free diffusion of glucose into the cells. The glucose analog, 3-O-methyl glucose, is also taken up by the inducible system, but at a lower rate than glucose. It is Acumulated, about 2000 times in a 20-min incubation period, indicating active transport. Cycloheximide inactivates the high affinity system to the same extent as high light intensities, and also prevents induction of this system in the dark. Rates of photosynthesis are inversely correlated to glucose uptake rates over a range of light intensities of pre-incubation. The possession of a light-regulated high-affinity transport system as well as a constitutive low-affinity system for glucose is probably of competitive advantage to this alga in the soil environment.     

Regulation of the intracellular potassium concentration in Escherichia coli B 525

The mutant Escherichia coli B 525 requires histidine, leucine and methionine and an elevated extracellular K⁺ concentration for growth, and is unable to retain K⁺ tightly inside the cells when incubated in media supplemented with glucose, arabinose, galactose or lactose as the sole energy and carbon source. The loss of K⁺ from the cells of B 525 can be prevented by adding histidine and leucine, which react specifically and only in combination. In media supplemented with glycerol as the substrate, with glucose and NH₄⁺, or with glucose under anaerobic conditions, a stationary level of K⁺ inside the cells can be obtained without the addition of histidine-leucine.On the addition of ribose to glycerol-adapted cells of B 525 preincubated in glycerol media, the intracellular K⁺ decreased immediately and markedly. This decrease can be overcome by the addition of histidine-leucine.     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Batch Tests To Determine Activity Distribution and Kinetic Parameters for Acetate Utilization in Expanded-Bed Anaerobic Reactors

Batch tests to measure maximum acetate utilization rates were used to determine the distribution of acetate utilizers in expanded-bed sand and expanded-bed granular activated carbon (GAC) reactors. The reactors were fed a mixture of acetate and 3-ethylphenol, and they contained the same predominant aceticlastic methanogen, Methanothrix sp. Batch tests were performed both on the entire reactor contents and with media removed from the reactors. Results indicated that activity was evenly distributed within the GAC reactors, whereas in the sand reactor a sludge blanket on top of the sand bed contained approximately 50% of the activity. The Monod half-velocity constant (K_s) for the acetate-utilizing methanogens in two expanded-bed GAC reactors was searched for by combining steady-state results with batch test data. All parameters necessary to develop a model with Monod kinetics were experimentally determined except for K_s. However, K_s was a function of the effluent 3-ethylphenol concentration, and batch test results demonstrated that maximum acetate utilization rates were not a function of the effluent 3-ethylphenol concentration. Addition of a competitive inhibition term into the Monod expression predicted the dependence of K_s on the effluent 3-ethylphenol concentration. A two-parameter search determined a K_s of 8.99 mg of acetate per liter and a K_i of 2.41 mg of 3-ethylphenol per liter. Model predictions were in agreement with experimental observations for all effluent 3-ethylphenol concentrations. Batch tests measured the activity for a specific substrate and determined the distribution of activity in the reactor. The use of steady-state data in conjunction with batch test results reduced the number of unknown kinetic parameters and thereby reduced the uncertainty in the results and the assumptions made.     

Growth of Geotrichum candidum and Penicillium camemberti Cultivated on Liquid Media Correlated with Ammonia and Methanethiol Emission

The growth of Geotrichum candidum and Penicillium camemberti plays an important role in the ripening of Camembert‐type cheeses, but the monitoring of the corresponding kinetics for fungal cocultures on solid media appears difficult. Continuous and non‐intrusive methods to characterize the growth of both species (like the monitoring of the emissions of ammonia and volatile sulphur compounds) may be highly relevant, under the condition that such emissions could be correlated with growth. This would be easier to investigate in submerged culture, since total biomass concentration is known to vary in proportion to broth turbidity. For this reason, growth kinetics, ammonia and flavour gas emission of both Geotrichum candidum and Penicillium camemberti grown separately in submerged cultures under the conditions of low aeration rate and uncontrolled pH were continuously recorded.In the basal medium (peptone+lactate supplemented with both glutamic acid and methionine [1 g/l]each), no significant gas emission was observed during the growth of both fungi. Ammonia and sulphur gas emissions by G. candidum were a little stimulated by supplementing the basal medium with trace elements, and, at a larger extent, by the addition of inorganic phosphate: Such a gaseous emission took place at the end of the growth phase of G. candidum. Irrespective of the basal medium supplementation, no significant emission ofammonia and sulphur gas was observed during the growth of P. camemberti. For the media and strains used, ammonia and volatile sulphur compounds emissions unequivocally showed the growth of Geotrichum candidum.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

Kinetics of Methyl t-Butyl Ether Cometabolism at Low Concentrations by Pure Cultures of Butane-Degrading Bacteria

Butane-oxidizing Arthrobacter (ATCC 27778) bacteria were shown to degrade low concentrations of methyl t-butyl ether (MTBE; range, 100 to 800 μg/liter) with an apparent half-saturation concentration (K_s) of 2.14 mg/liter and a maximum substrate utilization rate (k_c) of 0.43 mg/mg of total suspended solids per day. Arthrobacter bacteria demonstrated MTBE degradation activity when grown on butane but not when grown on glucose, butanol, or tryptose phosphate broth. The presence of butane, tert-butyl alcohol, or acetylene had a negative impact on the MTBE degradation rate. Neither Methylosinus trichosporium OB3b nor Streptomyces griseus was able to cometabolize MTBE.