Tissue culture based screening for selection of high biomass and phenolic producing clonal lines of lavender using pseudomonas and azetidine-2-carboxylate.

Lavender is a good source of essential oils and phenolic metabolites for food, medicine, and cosmetic applications. Due to cross-pollination, lavender has substantial plant to plant variation and therefore a high degree of genetic inconsistency in the level of phytochemicals produced for diverse applications. Tissue culture methods, using benzyladenine-induced shoot organogenesis, were used to isolate clonal lines originating from individual heterozygous seeds among a heterogeneous seed population to exploit the genetic heterogeneity. Subsequently, in a two-step method, clonal shoots of each clonal line were evaluated for the ability to tolerate Pseudomonas inoculation and various levels (0-200 microM) of proline analogue, azetidine-2-carboxylate. On the basis of tolerance to Pseudomonas and proline analogue treatments, multiple shoot forming ability, biomass, rosmarinic acid, total phenolics, and total chlorophyll, 20 separate clonal lines were screened and isolated for further vegetative propagation and evaluation. From the clonal lines isolated, lines LH-14, LH-15, LH-17, and LH-11 showed the best potential for overexpression of phenolic metabolites in response to Pseudomonas and proline analogue.

Synthesis and properties of lignin peroxidase from Streptomyces viridosporus T7A.

The production of lignin peroxidase by Streptomyces viridosporus T7A was studied in shake flasks and under aerobic conditions in a 7.5-L batch fermentor. Lignin peroxidase synthesis was found to be strongly affected by catabolite repression. Lignin peroxidase was a non-growth-associated, secondary metabolite. The maximum lignin peroxidase activity was 0.064 U/mL at 36 h. In order to maximize lignin peroxidase activity, optimal conditions were determined. The optimal incubation temperature, pH, and substrate (2,4-dichlorophenol) concentration for the enzyme assays were 45 degrees C, 6, and 3 mM, respectively. Stability of lignin peroxidase was determined at 37, 45, and 60 degrees C, and over the pH range 4-9.

Omega- and Omega+ production in central Pb + Pb collisions at 40 and 158A GeV.

Results are presented on Omega production in central Pb+Pb collisions at 40 and 158A GeV beam energy. For the first time in heavy ion reactions, rapidity distributions and total yields were measured for the sum Omega(-) + Omega(+) at 40A GeV and for Omega(-) and Omega(+) separately at 158A GeV. The yields are strongly underpredicted by the string-hadronic UrQMD model but agree better with predictions from hadron gas models.

System-size dependence of strangeness production in nucleus-nucleus collisions at squareroot[sNN]=17.3 GeV.

Emission of pi+/-, K+/-, phi, and Lambda was measured in near-central C+C and Si+Si collisions at 158 AGeV beam energy. Together with earlier data for p+p, S+S, and Pb+Pb, the system-size dependence of relative strangeness production in nucleus-nucleus collisions is obtained. Its fast rise and the saturation observed at about 60 participating nucleons can be understood as the onset of the formation of coherent systems of increasing size.

Effects of p-cresol and chlorophenols on pentachlorophenol biodegradation.

The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. (c) 1995 John Wiley & Sons Inc.

The use of alpha-galactosidase and invertase in hollow fiber reactors.

Invertase and alpha-galactosidase have been immobilized in hollow fiber cartridges with no detectable enzyme leakage and used for the hydrolysis of sucrose and raffinose, respectively. For both hollow fiber immobilized enzymes nearly complete substrate conversion is possible. Enzyme stabilities in polysulfonate hollow fibers which have been preconditioned with bovine albumin approach the stabilities of the free enzymes.

Two-stage continuous fermentation of Saccharomycopsis fibuligeria and Candida utilis.

Biomass production and carbohydrate reduction were determined for a two-stage continuous fermentation process with a simulated potato processing waste feed. The amylolytic yeast Saccharomycopsis fibuligera was grown in the first stage and a mixed culture of S. fibuligera and Candida utilis was maintained in the second stage. All conditions for the first and second stages were fixed except the flow of medium to the second stage was varied. Maximum biomass production occurred at a second stage dilution rate, D(2), of 0.27 h (-1). Carbohydrate reduction was inversely proportional to D(2), between 0.10 and 0.35 h (-1).

Production of Major Extracellular Enzymes during Lignocellulose Degradation by Two Streptomycetes in Agitated Submerged Culture.

Streptomyces viridosporus T7A and S. badius 252 were grown in 1 to 2% (wt/vol) slurry cultures with mineral salts solution containing 0.6% yeast extract and 100/200 mesh ground and extracted corn lignocellulose at 37 degrees C. Enzyme activities rapidly increased in the first 3 to 4 days and then declined and remained at a relatively constant level. Concentrations of endoglucanase and xylanase produced by S. badius were lower than those produced by S. viridosporus. However, the lignin-peroxidase peak concentration was threefold higher than with S. viridosporus and was obtained at 9 to 10 days of incubation. By polyacrylamide gel analysis, it was determined that peroxidases from both species consisted of four enzymes, with only one, the lignin peroxidase, having high activity. A culture pH of 8.5 was preferable for lignocellulose degradation by S. badius.

A model for continuous fermentations with amylolytic yeasts.

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.

Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor.

beta-D-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of beta-D-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.

Kinetics of Growth and Amylase Production of Saccharomycopsis fibuligera on Potato Processing Wastewater.

The kinetics of growth and amylase production of Saccharomycopsis fibuligera were studied in a chemostat on a synthetic potato processing blancher water. Dilution rates (D) from 0.101 to 0.480 h were examined. A mathematical model based on the Monod equation was developed. The yield of cell mass from carbohydrates was constant and equal to 0.84. The maximum specific growth rate and the Monod constant were determined to be 0.596 h and 0.226 mg/ml, respectively. An equation for the steady-state starch concentrations was empirically derived. The steady-state noncarbohydrate carbon levels rose linearly with D. Reducing sugars were the growth-limiting substrate, and their steady-state levels conformed to Monod kinetics. The yield of amylase from the cell mass (Y(z)) declined as D rose and was described by the equation Y(z) = (-8.005D + 4.076). The model predicted that the maximum production of cell mass should occur at D = 0.35 h and the maximum production of amylase should occur at D = 0.22 h. The mathematical model presented agreed with the experimental results in its prediction of the steady-state level of reducing sugar, starch, cell mass, and amylase concentrations as well as the productivity of amylase.

Catabolite repression of amylase synthesis in yeast.

Amylase synthesis by the yeasts Saccharomycopsis fibuligera and Schwanniomyces castellii and alluvius is repressed by glucose. Steady state continuous culture data for amylase activity, E, biomass concentration, X, and reducing sugar concentration, S, were fitted to the three-parameter catabolite repression model E/X = [1 + a(S/X)]/[1 + b(S/X)], and biomass productivity, DX, and amylase productivity, DE, were determined for S. castellii and S. alluvius.

Metal binding by pyridine-2,6-bis(monothiocarboxylic acid), a biochelator produced by Pseudomonas stutzeri and Pseudomonas putida.

Pyridine-2,6-bis(monothiocarboxylic acid) (pdtc), a natural metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida that promotes the degradation of carbon tetrachloride, was synthesized and studied by potentiometric and spectrophotometric techniques. The first two stepwise protonation constants (pK) for successive proton addition to pdtc were found to be 5.48 and 2.58. The third stepwise protonation constant was estimated to be 1.3. The stability (affinity) constants for iron(III), nickel(II), and cobalt(III) were determined by potentiometric or spectrophotometric titration. The results show that pdtc has strong affinity for Fe(III) and comparable affinities for various other metals. The stability constants (log K) are 33.93 for Co(pdtc)2(1-); 33.36 for Fe(pdtc)2(1-); and 33.28 for Ni(pdtc)2(2-). These protonation constants and high affinity constants show that over a physiological pH range the ferric pdtc complex has one of the highest effective stability constants for iron binding among known bacterial chelators.

Gel entrapped L-asparaginase: kinetic behavior and antitumor activity.

L-Asparaginase from Escherichia coli was immobilized by entrapment in a gel based on poly(2-hydroxyethyl methacrylate) with an activity as high as 730 I.U./g of dry gel. The apparent Michaelis constant for these gels was similar to that of the free enzyme. At 37 degrees C the immobilized enzyme had a half-life of more than 40 days, in vitro. The gel was freeze-dried, crushed and sieved to pass a 38 mum screen, giving a median particle size of 12 mum. C3H mice were injected intraperitoneally with 40 I.U. of L-asparaginase; the peak plasma activity after 4 hours was only 0.9 I.U. for the gel entrapped enzyme compared to a peak activity of 5.0 I.U. after 2 hours for the native L-asparaginase. Ninety percent of the plasma enzyme activity for the gel entrapped case was sedimentable at 21,000 X g, indicating a small leakage of the enzyme from the gel; the clearance for the enzyme activity in plasma had an initial half-life of 13 hours in contrast to a half-life of 2 hours for the native preparation. After intraperitineal injection of 5.0 I.U. into C3H mice, plasma L-asparagine fell to undetectable levels for 4 days and reappeared by day 8 for both the native and immobilized enzymes. Subcutaneously transplanted 6C3HED murine lymphoma was inhibited by 35, 78 and 100% after single intraperitoneal injections of immobilized L-asparaginase of 2, 4 and 8 I.U., respectively, as compared to 36, 53 and 86% for the native enzyme by the 14th day. Body weight changes after receiving immobilized L-asparaginase were essentially similar to those of animals receiving a comparable dose of native enzyme. These results indicate that while most of the immobilized L-asparaginase remains at the injection site, it produces a significant plasma L-asparagine depression and antitumor acitivity comparable to that of the native preparation without major toxicity.

Ethanol fermentation in a continuous tower fermentor.

A mutant of Saccharomyces cerevisiae, which forms large, multicellular flocs in liquid culture, rapidly fermented media containing high concentrations of glucose (100-180 g/L) in a continuous nonaerated tower fermentor at 30 degrees C. The fermentor operated continuously for seven months. Batch and tower fermentor data were fitted to a kinetic model incorporating linear ethanol inhibition and Monod dependence on glucose. Conversion, ethanol yield, and ethanol productivity were related to the apparent fermentation time for initial glucose concentrations of 130 and 180 g/L. Productivities of 8-12 g ethanol/L h were achieved through the yeast bed giving conversions exceeding 90% of the theoretical yield.

Products of Anaerobic 2,4,6-Trinitrotoluene (TNT) Transformation by Clostridium bifermentans.

Experiments to elucidate the 2,4,6-trinitrotoluene (TNT)-transforming activity of Clostridium bifermentans LJP-1 identified reductive TNT transformations that ultimately produced as end products triaminotoluene (TAT) and phenolic products of TAT hydrolysis. An adduct of TAT, apparently formed by condensation of TAT and pyruvic aldehyde (methyl glyoxal), was also detected.

Lambda and lambda production in central Pb-Pb collisions at 40, 80, and 158A GeV.

Production of Lambda and Antilambda hyperons was measured in central Pb-Pb collisions at 40, 80, and 158A GeV beam energy on a fixed target. Transverse mass spectra and rapidity distributions are given for all three energies. The Lambda/pi ratio at midrapidity and in full phase space shows a pronounced maximum between the highest BNL Alternating Gradient Synchrotron and 40A GeV CERN Super Proton Synchrotron energies, whereas the Lambda/pi ratio exhibits a monotonic increase.

Evidence for an exotic S= -2, Q= -2 baryon resonance in proton-proton collisions at the CERN SPS.

Results of resonance searches in the Xi(-)pi(-), Xi(-)pi(+), Xi;(+)pi(-), and Xi;(+)pi(+) invariant mass spectra in proton-proton collisions at sqrt[s]=17.2 GeV are presented. Evidence is shown for the existence of a narrow Xi(-)pi(-) baryon resonance with mass of 1.862+/-0.002 GeV/c(2) and width below the detector resolution of about 0.018 GeV/c(2). The significance is estimated to be above 4.2sigma. This state is a candidate for the hypothetical exotic Xi(--)(3/2) baryon with S=-2, I=3 / 2, and a quark content of (dsdsu). At the same mass, a peak is observed in the Xi(-)pi(+) spectrum which is a candidate for the Xi(0)(3/2) member of this isospin quartet with a quark content of (dsus[-]d). The corresponding antibaryon spectra also show enhancements at the same invariant mass.