Aperfeiçoamento do processo de fermentação láctica em diferentes hortaliças e avaliação de aspectos econômicos e energéticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção de L-ácido lático a partir de células bacterianas imobilizadas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of Oat Hull Hemicellulosic Hydrolysate Fermentability Employing Pichia stipitis

Oat hull hemicellulosic hydrolysate obtained by diluted acid hydrolysis was employed as fermentation medium for Pichia stipitis cultivation. A comparison between the use of treated hydrolysate with 1% activated charcoal to reduce the toxic compounds generated during the hydrolysis process and untreated hydrolysate as a control was conducted. In the cultures using treated hydrolysate the total consumption of glucose, low xylose consumption and ethanol and glycerol formation were observed. The medium formulated with untreated hydrolysate showed morphological cell modifications with consequently cell death, no ethanol formation and formation of glycerol as byproduct of fermentative process, probably as a response to stressful conditions to yeast due to presence of high concentration of toxic compounds. Thus, further studies are suggested in order to determine the best conditions for hydrolysis and detoxification of the hydrolysate to improve the fermentative performance of P. stipitis.

Proceedings of the Nineteenth Annual Biochemical Engineering Symposium

The nineteenth symposium was held at the University of Missouri–Columbia on April 22, 1989. A total of eighteen papers were scheduled for presentation, of which nine were in poster session. Finally, fifteen papers were presented and sixteen were submitted for this proceedings. It was attended by 53 participants from five institutions. A sixth group (from Colorado State University) was kept from attending the symposium due to mechanical problems on the road and we missed them. Since they worked hard at their presentations, I requested CSU-group to submit their papers for the proceedings and I am happy that they did. ContentsMathematical modelling of a flour milling system. K. Takahashi, Y. Chen, J. Hosokoschi, and L. T. Fan. Kansas State University A novel solution to the problem of plasmid segregation in continuous bacterial fermentations. K.L. Henry, R. H. Davis, and A. L. Taylor. University of Colorado Modelling of embryonic growth in avian and reptile Eggs. C.L. Krause, R. C. Seagrave, and R. A. Ackerman. Iowa State University Mathematical modeling of in situ biodegradation processes. J.C. Wu, L. T. Fan, and L. E. Erickson. Kansas State University Effect of molecular changes on starch viscosity. C.H. Rosane and V. G. Murphy. Colorado State University Analysis of two stage recombinant bacterial fermentations using a structured kinetic model. F. Miao and D. S. Kampala. University of Colorado Lactic acid fermentation from enzyme-thinned starch by Lactobacillus amylovorus. P.S. Cheng, E. L. Iannotti, R. K. Bajpai, R. Mueller, and s. Yaeger. University of Missouri–Columbia Solubilization of preoxidized Texas lignite by cell-free broths of Penicillium strains. R. Moolick, M. N. Karim, J. C. Linden, and B. L. Burback. Colorado State University Separation of proteins from polyelectrolytes by ultrafiltration. A.G. Bazzano and C. E. Glatz. Iowa State University Growth estimation and modelling of Rhizopus oligosporus in solid state fermentations. D.-H. Ryoo, V. G. Murphy, M. N. Karim, and R. P. Tengerdy. Colorado State University Simulation of ethanol fermentations from sugars in cheese whey. C.J. Wang and R. K. Bajpai. University of Missouri–Columbia Studies on protoplast fusion of B. licheniformis. B. Shi, Kansas State University Cell separations of non-dividing and dividing yeasts using an inclined settler. C.-Y. Lee, R. H. Davis, and R. A. Sclafani. University of Colorado Effect of·serum upon local hydrodynamics within an airlift column. G.T. Jones, L. E. Erickson, and L. A. Glasgow. Kansas State University Optimization of heterologous protein secretion in continuous culture. A. Chatterjee, W. F. Remirez, and R. H. Davis. University of Colorado An improved model for lactic acid fermentation. P. Yeh, R. K. Bajpai, and E. L. Iannotti. University of Missouri–Columbia

Proceedings of the 22nd Annual Biochemical Engineering Symposium

This is the twenty-second of a series of symposia devoted to talks and posters by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, sixteenth, and twenti~th were hosted by Kansas State University, the second and fourth by the University of Nebraska- Lincoln, the sixth, seventh, tenth, thirteenth, seventeenth, and twenty-second by Iowa State University, the eighth, fourteenth, and nineteenth by the University of Missouri-Columbia, the eleventh, fifteenth, and twenty-first by Colorado State University, and the eighteenth by the University of Colorado. Next year's symposium will be at the University of Oklahoma. Symposium proceedings are edited and issued by faculty of the host institution. Because final publication usually takes place in refereed journals, articles included here are brief and often cover work in progress. ContentsC. A. Baldwin, J.P. McDonald, and L. E. Erickson, Kansas State University. Effect of Hydrocarbon Phase on Kinetic and Transport Limitations for Bioremediation of Microporous Soil J. C. Wang, S. K. Banerji, and Rakesh Bajpai, University of Missouri-Columbia. Migration of PCP in Soil-Columns in Presence of a Second Organic Phase Cheng-Hsien Hsu and Roger G. Harrison, University of Oklahoma. Bacterial Leaching of Zinc and Copper from Mining Wastes James A. Searles, Paul Todd, and Dhinakar S. Kompala, University of Colorado. Suspension Culture of Chinese Hamster Ovary Cells Utilizing Inclined Sedimentation Ron Beyerinck and Eric H. Dunlop, Colorado State University. The Effect of Feed Zone Turbulence as Measured by Laser Doppler Velocimetry on Baker's Yeast Metabolism in a Chemostat Paul Li-Hong Yeh, GraceY. Sun, Gary A. Weisman, and Rakesh Bajpai, University of Missouri-Columbia. Effect of Medium Constituents upon Membrane Composition of Insect Cells R. Shane Gold, M. M. Meagher, R. Hutkins, and T. Conway, University of Nebraska-Lincoin. Ethanol Tolerance and Carbohydrate Metabolism in Lactobacilli John Sargantanis and M. N. Karim, Colorado State University. Application of Kalman Filter and Adaptive Control in Solid Substrate Fermentation D. Vrana, M. Meagher, and R. Hutkins, University of Nebraska-Lincoln. Product Recovery Optimization in the ABE Fermentation Kalyan R. Tadikonda and Robert H. Davis, University of Colorado. Cell Separations Using Targeted Monoclonal Antibodies Against Surface Proteins Meng H. Heng and Charles E. Glatz, Iowa State University. Charged Fusion for Selective Recovery of B-Galactosidase from Cell Extract Using Hollow Fiber Ion-Exchange Membrane Adsorption Hsiu-Mei Chen, Peter J. Reilly, and Clark Ford, Iowa State University. Site-Directed Mutagenesis to Enhance Thermostability of Glucoamylase from Aspergillus: A Rational Approach P. Tuitemwong, L. E. Erickson, and D. Y. C. Fung, Kansas State University. Applications of Enzymatic Hydrolysis and Fermentation on the Reduction of Flatulent Sugars in the Rapid Hydration Hydrothermal Cooked Soy Milk Sanjeev Redkar and Robert H. Davis, University of Colorado. Crossflow Microfiltration of Yeast Suspensions Linda Henk and James C. Linden, Colorado State University, and Irving C. Anderson, Iowa State University. Evaluation of Sorghum Ensilage as an Ethanol Feedstock Marc Lipovitch and James C. Linden, Colorado State University. Stability and Biomass Feedstock Pretreatability for Simultaneous Saccharification and Fermentation Ali Demirci, Anthony L. Pometto Ill, and Kenneth E. Johnson, Iowa State University. Application of Biofilm Reactors in Lactic Acid Fermentation Michael K. Dowd, Peter I. Reilly, and WalterS. Trahanovsky, Iowa State University. Low Molecular-Weight Organic Composition of Ethanol Stillage from Corn Craig E. Forney, Meng H. Heng, John R. Luther, Mark Q. Niederauer, and Charles E. Glatz, Iowa State University. Enhancement of Protein Separation Using Genetic Engineering J. F. Shimp, J. C. Tracy, E. Lee, L. C. Davis, and L. E. Erickson, Kansas State University. Modeling Contaminant Transport, Biodegradation and Uptake by Plants in the Rhizosphere Xiaoqing Yang, L. E. Erickson, and L. T. Fan, Kansas State University. Modeling of Dispersive-Convective Characteristics in Bioremediation of Contaminated Soil Jan Johansson and Rakesh Bajpai, University of Missouri-Columbia. Fouling of Membranes J. M. Wang, S. K. Banerji, and R. K. Bajpai, University of Missouri-Columbia. Migration of Sodium-Pentachorophenol (Na-PCP) in Unsaturated and Saturated Soil-Columns J. Sweeney and M. Meagher, University of Nebraska-Lincoln. The Purification of Alpha-D-Glucuronidase from Trichoderma reesei

Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium

This volume contains the Proceedings of the Twenty-Sixth Annual Biochemical Engineering Symposium held at Kansas State University on September 21, 1996. The program included 10 oral presentations and 14 posters. Some of the papers describe the progress of ongoing projects, and others contain the results of completed projects. Only brief summaries are given of some of the papers; many of the papers will be published in full elsewhere. A listing of those who attended is given below. ContentsForeign Protein Production from SV40 Early Promoter in Continuous Cultures of Recombinant CHO Cells - Gautam Banik, Paul Todd, and Dhinakar Kampala Enhanced Cell Recruitment Due to Cell-Cell Interactions - Brad Farlow and Matthias Nollert The Recirculation of Hybridoma Suspension Cultures: Effects on Cell Death, Metabolism and Mab Productivity - Peng Jin and Carole A. Heath The Importance of Enzyme Inactivation and Self-Recovery in Cometabolic Biodegradation of Chlorinated Solvents - Xi-Hui Zhang, Shanka Banerji, and Rakesh Bajpai Phytoremediation of VOC contaminated Groundwater using Poplar Trees - Melissa Miller, Jason Dana, L.C. Davis, Murlidharan Narayanan, and L.E. Erickson Biological Treatment of Off-Gases from Aluminum Can Production: Experimental Results and Mathematical Modeling - Adeyma Y. Arroyo, Julio Zimbron, and Kenneth F. Reardon Inertial Migration Based Separation of Chlorella Microalgae in Branched Tubes - N.M. Poflee, A.L. Rakow, D.S. Dandy, M.L. Chappell, and M.N. Pons Contribution of Electrochemical Charge to Protein Partitioning in Aqueous Two-Phase Systems - Weiyu Fan and Charles C. Glatz Biodegradation of Some Commercial Surfactants Used in Bioremediation - Jun Gu, G.W. Preckshot, S.K. Banerji, and Rakesh Bajpai Modeling the Role of Biomass in Heavy Metal Transport Ln Vadose Zone - K.V. Nedunuri, L.E. Erickson, and R.S. Govindaraju Multivariable Statistical Methods for Monitoring Process Quality: Application to Bioinsecticide Production by 73 89 Bacillus Thuringiensis - c. Puente and M.N. Karim The Use of Polymeric Flocculants in Bacterial Lysate Streams - H. Graham, A.S. Cibulskas and E.H. Dunlop Effect of Water Content on transport of Trichloroethylene in a Chamber with Alfalfa Plants - Muralidharan Narayanan, Jiang Hu, Lawrence C. Davis, and Larry E. Erickson Detection of Specific Microorganisms using the Arbitrary Primed PCR in the Bacterial Community of Vegetated Soil - X. Wu and L.C. Davis Flux Enhancement Using Backpulsing - V.T. Kuberkar and R.H. Davis Chromatographic Purification of Oligonucleotides: Comparison with Electrophoresis - Stephen P. Cape, Ching-Yuan Lee, Kevin Petrini, Sean Foree, Micheal G. Sportiello and Paul Todd Determining Singular Arc Control Policies for Bioreactor Systems Using a Modified Iterative Dynamic Programming Algorithm - Arun Tholudur and W. Fred Ramirez Pressure Effect on Subtilisins Measured via FTIR, EPR and Activity Assays, and Its Impact on Crystallizations - J.N. Webb, R.Y. Waghmare, M.G. Bindewald, T.W. Randolph, J.F. Carpenter, C.E. Glatz Intercellular Calcium Changes in Endothelial Cells Exposed to Flow - Laura Worthen and Matthias Nollert Application of Liquid-Liquid Extraction in Propionic Acid Fermentation - Zhong Gu, Bonita A. Glatz, and Charles E. Glatz Purification of Recombinant T4 Lysozyme from E. Coli: Ion-Exchange Chromatography - Weiyu Fan, Matt L. Thatcher, and Charles E. Glatz Recovery and Purification of Recombinant Beta-Glucuronidase from Transgenic Corn - Ann R. Kusnadi, Roque Evangelista, Zivko L. Nikolov, and John Howard Effects of Auxins and cytokinins on Formation of Catharanthus Roseus G. Don Multiple Shoots - Ying-Jin Yuan, Yu-Min Yang, Tsung-Ting Hu, and Jiang Hu Fate and Effect of Trichloroethylene as Nonaqueous Phase Liquid in Chambers with Alfalfa - Qizhi Zhang, Brent Goplen, Sara Vanderhoof, Lawrence c. Davis, and Larry E. Erickson Oxygen Transport and Mixing Considerations for Microcarrier Culture of Mammalian Cells in an Airlift Reactor - Sridhar Sunderam, Frederick R. Souder, and Marylee Southard Effects of Cyclic Shear Stress on Mammalian Cells under Laminar Flow Conditions: Apparatus and Methods - M.L. Rigney, M.H. Liew, and M.Z. Southard

Iron and aluminium and hydrogen peroxide measured on water bottle samples during Marion Dufresne cruise ANTARES-II

Total dissolvable iron (TDFe), particulate iron (PFe) and hydrogen peroxide (H2O2 measurements were performed along a N-S transect in the upper 250 m in the Southern Ocean (62°00E/66°42S - 49°00S, ANTARES II cruise, February 1994). TDFe was organically extracted (APDC/DDDC-chloroform) and analysed by Graphite Furnace Atomic Absorption Spectrometry (GFAAS), PFe was analysed by GFAAS following a strong mixed-acid leach, and H2O2 was analysed on board by fluorometry. The respective detection limits are equal to 0.13 nmol/kg, 0.02 nmol/kg, and 3.0 nmol/kg. TDFe concentrations vary from 0.4 to 6.2 nmol/kg and profiles are not completely depleted in the surface. PFe concentrations vary from 0.02 to 0.2 nmol/kg. Iron/carbon (Fe/C) uptake ratios for phytoplankton were calculated either from seawater or particle measurements. They are variable along the transect but are consistent when they could be compared. All the observed ratios are within the range of values proposed for the Fe/C uptake ratios by phytoplankton. Using our uptake ratio calculated in the Permanent Open Ocean Zone (4 x 10**?6 mol/mol), we estimate that the primary production which can be supported by the iron input flux into the surface waters is two times higher than the measured primary production in the same area. In the surface waters, H2O2 concentrations vary from 5.0 to 19.7 nmol/kg. Such low concentrations are due to strong vertical mixing, low dissolved organic matter concentrations and the latitude of the site.

Mathematical Modeling for Studying Microbial Processes – Some Examples

Mathematical modeling may have different purposes in chemical and biochemical engineering sciences. One of them is to confirm or to reject kinetic models for certain processes, or to evaluate the importance of some transport phenomena on the net chemical or biochemical reaction rate. In the present paper different microbial processes are considered and modeled for evaluation of kinetic constants for batch and continuous processes accomplished by free and immobilized microbial cells. The practical examples are from the field of wastewater treatment and biosynthesis of products, like enzymes, lactic acid, gluconic acid, etc. By the aid of mathematical modeling the kinetics and the type of inhibition are specified for microbial wastewater denitrification and biodegradation of halogenated hydrocarbons. The importance of free and immobilized cells and their separate contribution to the overall microbial process is also evaluated for some fermentation processes: gluconic acid production, dichloroethane biodegradation, lactic acid fermentation and monochloroacetic acid biodegradation.

Caracterização físico-química e química do fruto do juazeiro (Ziziphus joazeiro Mart) e avaliação da sua conservação por fermentação láctica

Considering the plant biodiversity in the Brazilian Northeast, whose components can be inserted into sustainable production systems, the jujube (Ziziphus joazeiro Mart.) emerges as to recovery of its fruit. The present study has as objective to characterize the fruit of the jujube under the physical, physicochemical and chemical approach and assess its conservation by spontaneous lactic fermentation under the influence of chloride, sodium, calcium and potassium. According to the legislation, vegetable acidified by fermentation that is subjected to lactic acid fermentation in order to achieve a final product pH less than or equal to 4.5. The results of the physical, chemical and physico-chemistry of ripe fruit jujube showed the potential of this species for agro-processing. The yield of edible portion (91.83%), soluble solids content (18,98º Brix), titratable acidity (0.14% citric acid), pH (5.30) and its composition, divided in moisture (79.01%), protein (2.01%), lipids (0.52%), carbohydrate (17.59%), fiber, ash (0.76%) and its minerals were consistent with the characteristic profile fruits, thus favoring the development of spontaneous lactic fermentation. The minimum pH and titratable acidity observed maximum in the fermentation process under the influence of mixtures of salts (NaCl and KCl NaCl2) values ranged from 3.4 to 3.7 and from 0.54 to 0.95 (% lactic acid), respectively. The profile of the lactic fermentation of fruit of jujube in brine, fermented microbiological quality and the result of analysis of primary sensory prepared preserved, the application of endorsed by the consumer sensory evaluation, more particularly, derived from fermented fruit preserved in the presence of chloride sodium, in accordance with the traditional techniques of lactic fermentation of vegetables. The results of sensory evaluation conducted with 100 consumers (tasters) revealed an acceptance rate equal to 78% of the preserve. Despite restrictions on the sensory acceptability of fermented under the influence of salts (KCl and CaCl2) substitutes sodium chloride, preserved these perspectives presented to balance the optimization of mixtures, health product safety and consumer awareness towards prefer a more healthy product with reduced sodium content.

In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from glucooligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.

In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota

Fermentation properties of oligosaccharides derived from lactulose (OsLu) and lactose (GOS) have been assessed in pH-controlled anaerobic batch cultures using lactulose and Vivinal-GOS as reference carbohydrates. Changes in gut bacterial populations and their metabolic activities were monitored over 24 h by fluorescent in situ hybridization (FISH) and by measurement of short-chain fatty acid (SCFA) production. Lactulose-derived oligosaccharides were selectively fermented by Bifidobacterium and lactic acid bacterial populations producing higher SCFA concentrations compared to GOS. The highest total SCFA production was from Vivinal-GOS > lactulose > OsLu > GOS. Longer incubation periods produced a selective fermentation of OsLu when they were used as a carbon source reaching the highest selective index scores. The new oligosaccharides may constitute a good alternative to lactulose, and they could belong to a new generation of prebiotics to be used as a functional ingredient for improving the composition of gut microflora.

Effects of nutritional factors on growth of Lactobacillus fermentum mixed with Saccharomyces cerevisiae in alcoholic fermentation

The growth of Lactobacillus fermentum was studied in mixed culture with Saccharomyces cerevisiae during alcoholic fermentation of high test molasses (HTM). Yeast extract or a group of 17 amino acids caused a strong and fast decrease in yeast viability due to the strong increase of acidity produced by bacteria. Pure culture of Lactobacillus fermentum in dry sugar cane broth confirmed amino acids as the main nutrients needed to stimulate the growth of bacterial contaminant during alcoholic fermentation. The absence of L. fermentum growth was obtained when leucine: isoleucine or valine were not added to the medium. Phenylalanine, alanine, glutamic acid, cystine, proline, histidine, arginine, threonine, tryptophane, serine and methionine inhibited the bacterial growth at least in one of the cultures of L. fermentum tested.

Effect of 3,4,4 '-trichlorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation

3,4,4'-trichlorocarbanilide (TCC) was rested as a new method of bacterial growth control for S. cerevisiae alcoholic fermentations of diluted high test molasses (HTM). Minimal inhibitory concentration (MIC) was tested to determine the necessary concentration of TCC to control bacterial growth. The fed-batch alcoholic fermentation process was used with cell recycle similar to industrial conditions and Lactobacillus fermentum CCT 1407 was mixed in the first inoculum to grow with the yeast. Yeast extract was added into the must to stimulate bacterial growth. The best results of TCC's MIC to bacterial growth of Lactobacillus fermentum and Leuconostoc mesenteroides (< 0.125-1.0 mu g/ml) and Saccharomyces cerevisiae (16 mu g/ml) occurred when it was combined with sodium dodecylsulphate (SDS) in a 1: 4 TCC/SDS ratio (wt/wt) in distilled water solution. 1.8 g/l TCC entrapped in calcium alginate added to the must with yeast extract inhibited the growth of Lactobacillus fermentum CCT 1407 maintaining a controlled acidity, higher yeast viability and up to 20.8% of improvement in the average of alcoholic efficiency. Addition of 0.075 g/l TCC entrapped in calcium alginate and 1.67 mg/l SDS in the wort with yeast extract (0-5.0 g/l), inhibited and controlled the extensive bacterial contamination for 19 cycles of fermentation. (C) 1998 Published by Elsevier B.V. Ltd.

Coordination reactions in the extraction of cerium(IV) and fluorine(I) by DEHEHP from mixed nitric acid and hydrofluoric acid solutions

The coordination reactions during the solvent extraction of cerium(IV) and fluorine(l) from mixed nitric acid and hydrofluoric acid solutions by di-(2-ethylhexyl)-2-ethylhexylphosphonate, L (DEHEHP) in heptane have been investigated. The extraction data have been analyzed by graphical methods taking into account all plausible species extracted into the organic phase. Different variables influencing the extraction of Ce(IV), such as the concentrations of nitrate ions, hydrofluroric acid, nitric acid, and extractant have been studied. The results demonstrate that DEHEHP can extract not only Ce(NO3)(4) as Ce(NO3)4.2L and HF as HF (.) H2O (.) L, but both together as Ce(HF)(NO3)(4) (.) L. The extraction equilibrium equations are determined according to slope analysis and IR spectra. The equilibrium constants of the extracted complexes have been calculated, taking into account complexation between the metal ion and inorganic ligands in the aqueous phase and all plausible complexes extracted into the organic phase. It is also shown that boric acid, which was added into the mixed solutions to complex with F(I) is not extracted by DEHEHP, and neither does it affect the extraction of cerium(IV) and HF, nor change the extraction mechanism.

THERMODYNAMICS OF AMINO-ACID DISSOCIATION IN MIXED-SOLVENTS .3. GLYCINE IN AQUEOUS GLUCOSE SOLUTIONS FROM 5-DEGREES-C TO 45-DEGREES-C

The first thermodynamic dissociation constants of glycine in 5, 15 mass % glucose + water mixed solvents at five temperatures from 5 to 45-degrees-C have been determined from precise emf measurements of a cell without liquid junction using hydrogen and Ag-AgCl electrodes and a new method of polynomial approximation proposed on the basis of Pitzer's electrolytic solution theory in our previous paper. The results obtained from both methods agree within experimental error. The standard free energy of transfer for HCl from water to aqueous mixed solvent have been calculated and the results are discussed.