Prediction of gas-liquid mass transfer coefficient in sparged stirred tank bioreactors

Oxygen mass transfer in sparged stirred tank bioreactors has been studied. The rate of oxygen mass transfer into a culture in a bioreactor is affected by operational conditions and geometrical parameters as well as the physicochemical properties of the medium (nutrients, substances excreted by the micro-organism, and surface active agents that are often added to the medium) and the presence of the micro-organism. Thus, oxygen mass transfer coefficient values in fermentation broths often differ substantially from values estimated for simple aqueous solutions. The influence of liquid phase physicochemical properties on kLa must be divided into the influence on k(L) and a, because they are affected in different ways. The presence of micro-organisms (cells, bacteria, or yeasts) can affect the mass transfer rate, and thus kLa values, due to the consumption of oxygen for both cell growth and metabolite production. In this work, theoretical equations for kLa prediction, developed for sparged and stirred tanks, taking into account the possible oxygen mass transfer enhancement due to the consumption by biochemical reactions, are proposed. The estimation of kLa is carried out taking into account a strong increase of viscosity broth, changes in surface tension and different oxygen uptake rates (OURs), and the biological enhancement factor, E, is also estimated. These different operational conditions and changes in several variables are performed using different systems and cultures (xanthan aqueous solutions, xanthan production cultures by Xanthomonas campestris, sophorolipids production by Candida bombicola, etc.). Experimental and theoretical results are presented and compared, with very good results.     

Bactericidal activity of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines, on phytopathogenic Xanthomonas campestris pv. vesicatoria cells

The ability of glycinecin A, a bacteriocin derived from Xanthomonas campestris pv. glycines 8ra, to kill closely related bacteria has been demonstrated previously by our group. In the present study, we aimed at determining the glycinecin A-induced cause of death. Treatment with glycinecin A caused slow dissipation of membrane potential and rapid depletion of the pH gradient. Glycinecin A treatment also induced leakage of potassium ions from X. campestris pv. vesicatoria YK93-4 cells and killed sensitive bacterial cells in a dose-dependent manner. Sensitive cells were killed within 2 h of incubation, most likely due to the potassium ion efflux caused by glycinecin A. These results suggest that the bactericidal mechanism of action of glycinecin A is correlated with the permeability of membranes to hydroxyl and potassium ions, leading to the lethal activity of the bacteriocin on the target bacteria.     

Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides.

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharide-negative mutants of X. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.     

Inhibition of bioluminescence in Photobacterium phosphoreum by sulfamethizole and its stimulation by thymine

In bioluminescent bacteria very few agents have been reported that can selectively inhibit the luminescence. In sensitivity tests with Photobacterium phosphoreum, using 55 different antibiotics, it was found that sulfamethizole, an inhibitor of dihydropteroate synthetase and the formation of folic acid, inhibited bioluminescence more than growth. Likewise, in mutants requiring thymine for growth, the luminescence per cell was much less in a medium low in thymine. In neither case could the decreased specific luminescence be attributed to a decrease in the cellular level of luciferase or aldehyde factor; the involvement of additional but unidentified factors in the regulation of in vivo bioluminescence is postulated.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris

Previously, genomic banks of Xanthomonas campestris were constructed in Escherichia coli, using mobilizable broad-host-range cosmids as the vectors. Following conjugal transfer, genes involved in the biosynthesis of xanthan polysaccharide (XPS) were cloned by the ability to restore the mucoid phenotype to the non-mucoid mutants. In this study, all clones were transferred into the wild-type strain Xc17 to evaluate the effects of the cloned genes on XPS production. Most clones showed no significant effect; however, two plasmids, pP2401 and pP2201, caused 10 and 15% yield increases, respectively, compared with that of controls. While it was not clear how pP2201 caused the yield increase, the effect of pP2401 seemed to result from elevated phosphomannose isomerase activity. Since XPS synthesis in X. campestris is a very efficient process, only relatively small increases are to be expected; an enhancement of productivity by 10-15% is important to the commercial production of xanthan.     

Xanthan production in bubble column and air-lift reactors

A bubble column (0.05 m(3)) and an air-lift fermentor (1.2 m(3)) were used for the production of the exocellular microbial polysaccharide xanthan with Xanthomonas campestris in a synthetic medium. Upon oxygen depletion in the liquid, the xanthan production rate dropped sharply and then became a linear function of the oxygen transfer rate. The volumetric mass transfer coefficients for oxygen conformed to the correlation of Suh et al. Using this correlation in combination with the model for xanthan batch fermentation suggested by Peters et al., the xanthan fermentations in the bubble column were well described. The model also correctly predicted the time course of the molecular weight of the polysaccharide even when a complex medium was used. In the air-lift fermentor, however, the xanthan production rate and the xanthan yields with respect to oxygen and glucose were lower than expected at the overall oxygen transfer rate. The poor performance of the air lift was traced back to the lack of any oxygen supply in the downcomer.     

Use of bioluminescence for detection of genetically engineered microorganisms released into the environment.

The persistence and movement of strain JS414 of Xanthomonas campestris pv. campestris, which was genetically engineered to bioluminesce, were monitored during a limited field introduction. Bioluminescence and traditional dilution plate counts were determined. Strain JS414 was applied to cabbage plants and surrounding soil by mist inoculation, by wound inoculation, by scattering infested debris among plants, and by incorporating bacteria into the soil. Bioluminescent X. campestris pv. campestris was detected in plant samples and in the rhizosphere up to 6 weeks after inoculation. Movement to uninoculated plants was detected on one occasion, but movement from the immediate release area was not detected. Strain JS414 was detected in soil samples beneath mist- and wound-inoculated plants only at intentionally infested locations and in aerial samples only on the day of inoculation. Our bioluminescence methods proved to be as sensitive as plating methods for detecting the genetically engineered microorganisms in environmental samples. Our results demonstrate that transgenic incorporation of the luxCDABE operon provides a non-labor-intensive, sensitive detection method for monitoring genetically engineered microorganisms in nature.     

Survival and persistence of bioluminescent Xanthomonas campestris pv. campestris on host and non-host plants in the field environment

Dispersal and persistence of a pathogenic strain of Xanthomonas campestris pv. campestris , genetically engineered to bioluminesce, was followed in and on host and non-host plants in the field environment. Black rot susceptible cabbage plants were mist inoculated with the bioluminescent strain only, or were mist inoculated with X. campestris pv. vesicatoria or a weakly pathogenic strain of X. c. campestris 1 week before challenge inoculation with the bioluminescent strain. Growth of the bioluminescent strain was detected with a low-light, charge-coupled device camera or through bioluminescence measurements of broth-enrichment cultures of leaf disk samples. Bioluminescent X. c. campestris could often be observed as populations on symptomless leaves or in lesions, and persisted as a vascular endophyte for more than 6 months throughout the winter growing season. Dispersal to cruciferous and non-cruciferous weeds was frequently detected. Pre-inoculation with X. c. vesicatoria or the weakly pathogenic X. c. campestris did not significantly affect the movement and persistence of the bioluminescent strain nor reduce the incidence of black rot disease.     

Characterization of bioluminescent derivatives of assimilable organic carbon test bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

The A protein of the filamentous bacteriophage Cf of Xanthomonas campestris pv. citri.

Filamentous bacteriophages have very strict host specificities. Experiments were performed to investigate whether the A protein of the filamentous phage Cf, which infects Xanthomonas campestris pv. citri but not X. campestris pv. oryzae, is involved in determining Cf's host specificity. The gene encoding the A protein of Cf was cloned and expressed in X. campestris pv. citri. The genomic DNA of another filamentous bacteriophage, Xf, which infects X. campestris pv. oryzae but not X. campestris pv. citri, was then introduced by electroporation into X. campestris pv. citri that had expressed the A protein of Cf. The progeny phages thus produced were able to infect both X. campestris pv. oryzae and X. campestris pv. citri, indicating that the A protein of Cf was incorporated into the viral particles of Xf and conferred upon Xf the ability to infect the host of Cf. Inactivation of the A protein gene abolished the infectivity of Cf. The results of this study indicate that the A protein of Cf is responsible for controlling the host specificity of Cf.     

Studies of the adenylate and pyridine nucleotide pools during nitrogenase ‘switch-off’ inRhodospirillum rubrum

Summary When ammonium ions are added to a nitrogen fixing culture ofRhodospirillum rubrum, nitrogenase activity decreases due to inactivation of the Fe-protein. We have studied the adenylate and pyridine nucleotide pools during switch-off using the sensitive bioluminescence method. Immediately after the addition of ammonium ions there is a decrease in the ATP pool which is quickly reversed and no change is seen during the switch-off period. The pyridine nucleotide pools also do not change significantly during the switch-off. Consequently we conclude that changes in the pools studied were not the signal promoting inactivation of the Fe-protein.     

Effect of concentrating and exposing the bioluminescent bacteria to the non‐luminescent allo‐bacterial extracellular products on their luminescence

Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal process to recognize the strength of its own population to start luminescence in harmony. There is a mechanism in these bacteria to also recognize inter‐species strength. When there is a higher number of these bacteria, the possibility and frequency of cell–cell physical contact will be high. In this study, the physical proximity was artificially enhanced between cells and the effect on luminescence in the concentrated cells in the normal culture medium and in the presence of other non‐bacterial cell‐free supernatants was investigated. The role of such physical contact in the quorum sensing in the bioluminescence is not known. Increase in the luminescence of V. fischeri when concentrated shows that the presence of physical proximity facilitates the quorum sensing for their bioluminescence. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of stress-responsive genes, hrcA-grpE-dnaK-dnaJ, from phytopathogenic Xanthomonas campestris

Sequencing of a 6.4-kb DNA fragment, cloned from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17 revealed five ORFs whose deduced amino acid sequences show strong similarities to the bacterial HrcA, GrpE, DnaK, DnaJ, and PdxK. The four heat shock genes are organized in the order hrcA-grpE-dnaK-dnaJ, a genome organization found in many gram-positive bacteria, but only in one gram-negative species (Xylella fastidiosa). These observations suggest that the HrcA-CIRCE system, comprising at least four genes arranged in this order, already existed for the regulation of stress responses before bacteria diverged into gram-negative and gram-positive groups. Primer-extension results suggested the presence of promoters at the regions upstream of grpE and dnaK. In the presence of stress, heat or ethanol (4%), the X. campestris pv. campestris 17 grpE and dnaK promoters were induced two- to three-fold over controls. Since the grpE and dnaK promoters possess E. coli sigma(32) promoter-like sequences, they are functional in E. coli, although at levels much lower than in X. campestris pv. campestris 17. Furthermore, expression of the X. campestris pv. campestris 17 dnaK promoter in E. coli was elevated by the cloned X. campestris sigma(32) gene, indicating that the cognate sigma(32) works more efficiently for the X. campestris promoters.     

Structures of the O-polysaccharide chains of the lipopolysaccharides of Xanthomonas campestris pv phaseoli var fuscans GSPB 271 and X campestris pv malvacearum GSPB 1386 and GSPB 2388

O-polysaccharides of phytopathogenic bacteria Xanthomonas campestris were isolated by mild acid degradation of the lipopolysaccharides and studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structures of the repeating units of the polysaccharides of X. campestris pv. phaseoli var. fuscans GSPB 271 (1). and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388 (2). were established:The O-polysaccharides of X. campestris are structurally similar to those of some Pseudomonas syringae strains.     

Exogenous compounds in studying the mechanism of electron-excited state formation in bioluminescence

Using a series of exogenous fluorescent molecules as potential energy acceptors, the hypothesis on the activity of the upper electron-excited states in bioluminescence was tested. The results in bacterial and firefly bioluminescent enzyme systems were compared. Similar activity to the energetic precursor in bacterial bioluminescence was not proven in the case of the firefly system, the result of a very efficient intramolecular energy transfer in the emitter of the firefly bioluminescence. The influence of a number of metallic salts on a bacterial bioluminescent enzyme system was studied. Bioluminescence inhibition coefficients were compared to the free energies of electron withdrawing of cations. The correlation shows that inhibition and activation of luminescence intensity result from the effects of cations on electron transfer in the bioluminescent system.     

四种黄单胞菌的基因芯片检测方法的建立

结合双重PCR和基因芯片技术同时检测和鉴定我国检疫性细菌,包括水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)、水稻细菌性条斑病菌(X.oryzae pv.oryzicola,Xooc)、柑桔溃疡病菌(X.axonopodis pv.citri,Xac)以及严重危害十字花科作物的甘蓝黑腐病菌(Xanthomonas campestris pv.campestris,Xcc)。以铁载体受体(Putative siderophore receptor)基因序列和RNA多聚酶西格玛因子(RNA polymerase sigma factor,rpoD)基因序列为靶标,设计引物和特异性探针能够同时检测这4种重要的病原菌。对17个细菌菌株进行芯片检测,仅4种靶标菌得到阳性结果,证明此方法具有很高的特异性。4种致病菌基因组DNA的检测灵敏度约为3 pg。检测结果表明,建立的基因芯片检测方法特异性强,能实现上述4种黄单胞菌的准确检测和鉴定,具有良好的应用前景。     

Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample. Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX bacteria were mutagenized with luxCDABE operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with p-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.     

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.