Bacterial chemotaxis towards polysaccharide pectin by pectin-binding protein.

As opposed to typical bacteria exhibiting chemotaxis towards low-molecular-weight substances, such as amino acids and mono/oligosaccharides, gram-negative Sphingomonas sp. strain A1 shows chemotaxis towards alginate and pectin polysaccharides. To identify the mechanism of chemotaxis towards macromolecules, a genomic fragment was isolated from the wild-type strain A1 through complementation with the mutant strain A1-M5 lacking chemotaxis towards pectin. This fragment contained several genes including sph1118. Through whole-genome sequencing of strain A1-M5, sph1118 was found to harbour a mutation. In fact, sph1118 disruptant lost chemotaxis towards pectin, and this deficiency was recovered by complementation with wild-type sph1118. Interestingly, the gene disruptant also exhibited decreased pectin assimilation. Furthermore, the gene product SPH1118 was expressed in recombinant E. coli cells, purified and characterised. Differential scanning fluorimetry and UV absorption spectroscopy revealed that SPH1118 specifically binds to pectin with a dissociation constant of 8.5 µM. Using binding assay and primary structure analysis, SPH1118 was predicted to be a periplasmic pectin-binding protein associated with an ATP-binding cassette transporter. This is the first report on the identification and characterisation of a protein triggering chemotaxis towards the macromolecule pectin as well as its assimilation.

Formation of a single polar flagellum by two distinct flagellar gene sets in Sphingomonas sp. strain A1.

Gram-negative Sphingomonas sp. strain A1, originally identified as a non-motile and aflagellate bacterium, possesses two sets of genes required for flagellar formation. In this study, we characterized the flagellar genes and flagellum formation in strain A1. Flagellar gene cluster set I contained 35 flagellar genes, including one flagellin gene (p6), where the gene assembly structure resembled that required for the formation of lateral flagella in gammaproteobacteria. The set II flagellar genes were arranged in eight shorter clusters with 46 flagellar genes, including two flagellin genes (p5 and p5') and flhF, which is required for polar flagella. Our molecular phylogenetic analysis of the bacterial flagellins also demonstrated that, in contrast to p5 and p5', p6 was categorized as a lateral flagellin group. The motile phenotype appeared in strain A1 cells when they were subcultured on semisolid media. The motile strain A1 cells produced a single flagellum at the cell pole. DNA microarray analyses using non-motile and motile strain A1 cells indicated that flagellar formation was accompanied by increased transcription of both flagellar gene sets. The two flagellins p5 and p6 were major components of the flagellar filaments isolated from motile strain A1 cells, indicating that the polar flagellum is formed by lateral and non-lateral flagellins.

Sphingomonas sp. is a novel cell culture contaminant.

A novel contaminant was isolated from Madin Darby Bovine Kidney (MDBK) cells. The organism was unable to grow on standard microbiological media by conventional techniques, but grew well in Dulbecco's Modified Eagle's Medium (DMEM) containing high glucose concentration. The organism formed a white biofilm on the bottom without any signs of turbidity. Upon genome sequence analysis of 16 S rDNA, the contaminant was identified as Sphingomonas sp. Shah, a member of the group α-Proteobacteria. Neutral red dye uptake method confirmed clear cytotoxic potential of the bacterium on A-549 cells. The organism was capable of invading and infecting different mammalian cell lines: MDBK, ZZ-R, 293-T, A549, and HeLa cells. Infected cells showed a variety of cytopathic effects including vacuolation at perinuclear area, cytoplasmic granulation and membrane blebbing. Microscopic analysis of the infected cells revealed the presence of cytoplasmic vacuoles harboring motile organisms. Apparently local serum preparations seem to be the source of this contamination, which is imperceptibly passed on from one culture passage to the other and ultimately leading to serious cytopathic manifestations.

[Using flow cytometry to explore the changes of Sphingomonas sp. GY2B bacterial surface characteristics in the process of degrading phenanthrene].

The first step of biodegradation is the contact of microorganism and pollutants, in order to examine the influence of phenanthrene on Sphingomonas sp. GY2B's surface properties during its degrading process, the bacteria was cultivated at different conditions, and detected by flow cytometry combined with fluorescent dyes for its surface changes. The results indicated that, the membrane structure had been certainly damaged during the degrading process, leading to an increased membrane permeability. Moreover, the destruction of bacteria membrane integrity became more serious with a higher pollutant concentration. At the concentration of 300 mg x L(-1), the ratio of stained bacterial cells/unstained cells was 12.44 after cultured for 60 h, while at 100 mg x L(-1) and 1.2 mg x L(-1), the ratios were 1.95 and 1.11, respectively. The results of fourier transform infrared (FT-IR) absorbance spectroscopy detection, the discrimination of death, injured and intact cells, and Zeta potential detection further verified the bacterial cell surface permeability changes. Flow cytometry combined with fluorescent dye propidium iodide was used to monitor the changes of bacterial membrane integrity on single-cell level which exhibited a good potential for exploring the changes of bacterial surface properties during the degrading progress and more deeply for investigating the degradation mechanism.

Immobilization of growing Sphingomonas sp. HXN-200 to gelatin microspheres: efficient biotransformation of N-Cbz-pyrrolidine and N-Boc-pyrrolidine into hydroxypyrrolidine derivatives.

Sphingomonas sp. HXN-200 bacteria were immobilized onto gelatin and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres in an aqueous system for industrial scale biohydroxylation. Scanning electron microscopy and confocal laser scanning microscopy were used to confirm the bacterial immobilization. The gelatin microsphere-immobilized cells successfully transformed N-benzyloxycarbonyl-pyrrolidine and N-tert-butoxycarbonyl-pyrrolidine into R-N-benzyloxycarbonyl-3-hydroxypyrrolidine and R-N-tert-butoxycarbonyl-3-pyrrolidinol at various substrate concentrations showing an improved conversion yield and product efficiency with respect to the freely suspended cells system at each reaction interval time. Additionally, in the immobilized cells system, it showed a faster reaction rate as compared to the freely suspended cells system for reaching the same product concentration. The inhibition effect due to substrate and product concentrations was significantly lower after immobilizing the bacteria onto the microspheres, which is beneficial for continuous reaction. The efficient whole-cell biocatalytic process was feasibly conducted in a 500mL preparative scale bioreactor and the conversion yield of N-benzyloxycarbonyl-pyrrolidine did not decrease after consecutive repeated use, suggesting that the microsphere-cells immobilization system was stable and reusable.

Rhamsan gum production by Sphingomonas sp. CGMCC 6833 using a two-stage agitation speed control strategy.

Varying the agitation speed could greatly affect rhamsan gum production by Sphingomonas sp. CGMCC 6833. Batch fermentations at agitation speeds of 400, 600, 800, and 1,000 rpm were therefore carried out. The time course of specific cell growth rate, specific glucose consumption rate, and specific rhamsan gum formation rate was subsequently determined. Based on the results, a novel two-stage agitation speed control strategy was developed. From 0 to 13 H, the high specific cell growth and glucose consumption rates were achieved by setting the agitation speed of the fermenter at 800 rpm. From 13 H onward to the end of fermentation, the glucose consumption rate and specific cell growth rate were high at the agitation speed of 600 rpm. Using this method, the maximum concentration and productivity of rhamsan gum reached 21.63 ± 1.76 g L(-1) and 0.338 ± 0.028 g L(-1)  H(-1) , respectively, which were both higher than the optimum results obtained at constant agitation speeds.

Colonization and modulation of host growth and metal uptake by endophytic bacteria of Sedum alfredii.

Sedum alfredii Hance is a Zn and Cd co-hyperaccumulating plant species found in an old mining area in China. Four bacterial strains, Burkholderia sp. SaZR4, Burkholderia sp. SaMR10, Sphingomonas sp. SaMR12 and Variovorax sp. SaNR1, isolated from surface-sterilized S. alfredii plants were used to investigate their endophytic nature and root colonization patterns and effects on phytoextraction of Zn and Cd. Laser scanning confocal microscopy revealed that gfp-tagged SaZR4, SaMR12, and SaNR1 cells formed biofilms on roots and that SaZR4 and SaMR12 cells could invade root tissues. SaMR10 showed the lowest total population associated with S. alfredii and little effect on plant growth and phytoextraction. SaZR4 significantly promoted Zn-extraction but not Cd-extraction. SaMR12 and SaNR1 significantly promoted plant growth in substrates supplemented with Zn or Cd and phytoextraction of Zn and Cd. Together, this study have shown that the four native endophytic bacteria differently colonize the host plants and modulate metal uptake and growth of host plant, and that SaMR12 and SaNR1 strains are promising assistants of S. alfredii plants for phytoremediation of Zn/Cd-contaminated soil.

Coenzyme Q(10) production by immobilized Sphingomonas sp. ZUTE03 via a conversion-extraction coupled process in a three-phase fluidized bed reactor.

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR.

Impact of matrix properties on the survival of freeze-dried bacteria.

BACKGROUND: Disaccharides are, in general, the first choice as formulation compounds when freeze-drying microorganisms. Although polysaccharides and other biopolymers are considered too large to stabilise and interact with cell components in the same beneficial way as disaccharides, polymers have been reported to support cell survival. In the present study we compare the efficiency of sucrose and the polymers Ficoll, hydroxyethylcellulose, hydroxypropylmethylcellulose and polyvinylalcohol to support the survival of three bacterial strains during freeze drying. The initial osmotic conditions were adjusted to be similar for all formulations. Formulation characterisation was used to interpret the impact that different compound properties had on cell survival. RESULTS: Despite differences in molecular size, both sucrose and the sucrose-based polymer Ficoll supported cell survival after freeze drying equally well. All formulations became amorphous upon dehydration. Scanning electron microscopy and X-ray diffraction data showed that the discerned differences in structure of the dry formulations had little impact on the survival rates. The capability of the polymers to support cell survival correlated with the surface activity of the polymers in a similar way for all investigated bacterial strains. CONCLUSION: Polymer-based formulations can support cell survival as effectively as disaccharides if formulation properties of importance for maintaining cell viability are identified and controlled.

Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures.

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

Factors influencing the electrokinetic dispersion of PAH-degrading bacteria in a laboratory model aquifer.

Despite growing interest in the electro-bioremediation of contaminated soil it is still largely unknown to which degree weak electric fields influence the fate of contaminant-degrading microorganisms in the sub-surface. Here we evaluate the factors influencing the electrokinetic transport and deposition of fluorene-degrading Sphingomonas sp. LB126 in a laboratory model aquifer exposed to a direct current (DC) electric field (1 V cm(-1)) typically used in electro-bioremediation measures. The influence of cell size, cell membrane integrity, cell chromosome contents (all assessed by flow cytometry), cell surface charge and cell hydrophobicity on the spatial distribution of the suspended and matrix-bound cells after 15 h of DC-treatment was evaluated. In presence of DC the cells were predominantly mobilised by electroosmosis to the cathode with an apparent velocity of 0.6 cm h(-1), whereas a minor fraction only of the cells augmented was mobilised to the anode by electrophoresis. Different electrokinetic behaviour of individual cells could be solely attributed to intra-population heterogeneity of the cell surface charge. In the absence of DC by contrast, a Gaussian-type distribution of bacteria around the point of injection was found. DC had no influence on the deposition efficiency, as the glass beads in presence and absence of an electric field retained quasi-equal fractions of the cells. Propidium iodide staining and flow cytometry analysis of the cells indicated the absence of negative influences of DC on the cell wall integrity of electrokinetically mobilised cells and thus point at unchanged physiological fitness of electrokinetically mobilised bacteria.

Isolation and characterization of marine bacterial strain degrading fucoidan from Korean Undaria pinnatifida Sporophylls.

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct lowmolecular- mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer Lfucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an alpha-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.

Sphingomonas molluscorum sp. nov., a novel marine isolate with antimicrobial activity.

An aerobic, Gram-negative, yellow-pigmented, non-motile bacterium, designated strain KMM 3882T, was isolated from a marine bivalve (Anadara broughtoni) collected from Peter the Great Bay, Sea of Japan, and was subjected to phenotypic and phylogenetic analyses. Strain KMM 3882T was found to exert a remarkable inhibitory activity against a number of Gram-positive micro-organisms. Phylogenetic analysis based on 16S rRNA gene sequences placed strain KMM 3882T within the genus Sphingomonas, as an independent lineage adjacent to Sphingomonas dokdonensis DS-4T and Sphingomonas panni DSM 15761T. Strain KMM 3882T showed the highest 16S rRNA gene sequence similarity to Sphingomonas dokdonensis DS-4T (97.3 %); similarities of 96.5-96.7 % were obtained with Sphingomonas pituitosa DSM 13101T, Sphingomonas azotifigens NBRC 15497T, Sphingomonas asaccharolytica NBRC 15499T, Sphingomonas trueperi DSM 7225T and Sphingomonas panni DSM 15761T. Chemotaxonomically, strain KMM 3882T contained sphingoglycolipid, C(16 : 0) and C(18 : 1) as predominant fatty acids and 2-OH C(14 : 0) as a major 2-hydroxy fatty acid, confirming the affiliation of strain KMM 3882T with the genus Sphingomonas. On the basis of phylogenetic analysis, DNA-DNA hybridization and physiological and biochemical characterization, strain KMM 3882T should be classified as representing a novel species of the genus Sphingomonas, for which the name Sphingomonas molluscorum sp. nov. is proposed. The type strain is KMM 3882T (=An 18T=NRIC 0685T=JCM 14122T=CIP 109223T).

Simultaneous biodegradation of methyl parathion and carbofuran by a genetically engineered microorganism constructed by mini-Tn5 transposon.

A genetically engineered microorganism (GEM) capable of simultaneous degrading methyl parathion (MP) and carbofuran was successfully constructed by random insertion of a methyl parathion hydrolase gene (mpd) into the chromosome of a carbofuran degrading Sphingomonas sp. CDS-1 with the mini-transposon system. The GEM constructed was relatively stable and cell viability and original degrading characteristic was not affected compared with the original recipient CDS-1. The effects of temperature, initial pH value, inoculum size and alternative carbon source on the biodegradation of MP and carbofuran were investigated. GEM cells could degrade MP and carbofuran efficiently in a relatively broad range of temperatures from 20 to 30 degrees C, initial pH values from 6.0 to 9.0, and with all initial inoculation cell densities (10(5)-10(7) CFU ml(-1)), even if alternative glucose existed. The optimal temperature and initial pH value for GEM cells to simultaneously degrade MP and carbofuran was at 30 degrees C and at pH 7.0. The removal of MP and carbofuran by GEM cells in sterile and non-sterile soil were also studied. In both soil samples, 50 mg kg(-1) MP and 25 mg kg(-1) carbofuran could be degraded to an undetectable level within 25 days even if there were indigenous microbial competition and carbon sources effect. In sterile soil, the biodegradation rates of MP and carbofuran were faster, and the decline of the inoculated GEM cells was slower compared with that in non-sterile soil. The GEM constructed in this study was potential useful for pesticides bioremediation in natural environment.

Slow-release inoculation allows sustained biodegradation of gamma-hexachlorocyclohexane.

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (gamma-hexachlorocyclohexane [gamma-HCH]). Slow-release inoculation of Sphingomonas sp. gamma 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding gamma-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.

Sphingomonas yunnanensis sp. nov., a novel gram-negative bacterium from a contaminated plate.

A Gram-negative bacterium, YIM 003T, which was isolated from a contaminated plate in the laboratory, was subjected to a polyphasic taxonomic study. The organism had short-rod-shaped, motile cells, formed yellow-pigmented colonies on ISP2 medium and its optimum growth pH was 7.0-7.5. The major respiratory lipoquinone was ubiquinone Q-10. The phosphate-containing lipids detected in strain YIM 003T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and one unidentified phospholipid. The major fatty acids were C(18 : 1)omega7c (59.8 %), C(16 : 0) (9.9 %), ai-C(17 : 0) (5.3 %), i-C(17 : 0) (4.4 %) and C(14 : 0) 2-OH (15.8 %). The G+C content of the genomic DNA was 67.5 mol%. Strain YIM 003T exhibited levels of 16S rRNA gene sequence similarity of 98.2 % to Sphingomonas phyllosphaerae FA2T and 98.0 % to Sphingomonas adhaesiva DSM 7418T but showed less than 97.0 % similarity with respect to other species with validly published names. The DNA-DNA relatedness values of the isolate with S. phyllosphaerae FA2T and S. adhaesiva DSM 7418T were 59 and 26 %, respectively. The phenotypic characteristics and genotypic data indicate that strain YIM 003T should be distinguished from S. phyllosphaerae FA2T and S. adhaesiva DSM 7418T. Therefore, on the basis of the polyphasic taxonomic data presented, a novel species of the genus Sphingomonas, Sphingomonas yunnanensis sp. nov., is proposed, with the type strain YIM 003T (=CCTCC AB 204064T=KCTC 12346T).

Toxic effect of biosurfactant addition on the biodegradation of phenanthrene.

The effect of the biosurfactant rhamnolipid on phenanthrene biodegradation and cell growth of phenanthrene degraders was investigated. To compare the effect of rhamnolipid addition, two bacterial strains, 3Y and 4-3, which were isolated from a diesel-contaminated site in Korea, were selected. Without the biosurfactant, large amounts of phenanthrene were degraded with both strains at neutral pH, with higher rates of phenanthrene degradation when the cell growth was higher. Upon the addition of 240 mg/L rhamnolipid, the phenanthrene degradation and optical density were reduced, with this inhibitory effect similar for both 3Y and 4-3. To explain this inhibition, the cell growths of both strains were monitored with various concentrations of rhamnolipid, which showed significant toxic effects toward strain 3Y, but was nontoxic toward 4-3. Combining the inhibitory and toxicity results with regard to the biodegradation, different mechanisms can be suggested for each strain. In the biodegradation experiments, the toxicity of rhamnolipid itself mainly was responsible for the inhibitory effect in the case of 3Y, whereas the toxicity of solubilized phenanthrene or the increased toxicity of rhamnolipid in the presence of solubilized phenanthrene could have resulted in the inhibitory effect in the case of 4-3. This study demonstrated that the effectiveness of biosurfactant-enhanced biodegradation can be significantly different depending on the strain, and the toxicity of the biosurfactant should be considered as an important factor.

Sphingomonas phyllosphaerae sp. nov., from the phyllosphere of Acacia caven in Argentina.

Two bacterial strains (FA1 and FA2(T)) were isolated from the phyllosphere of a leguminous tree, Acacia caven, in central Argentina. The strains were Gram-negative, strictly aerobic, rod-shaped, motile and formed yellow-pigmented colonies on nutrient agar. The two-primer RAPD patterns of the two strains were identical, suggesting that they belong to the same species. The complete 16S rRNA gene sequences of the two strains were obtained and comparisons demonstrated that they cluster phylogenetically with the species of the genus Sphingomonas sensu stricto. Strain FA2(T) was most closely related (97.6 %) to Sphingomonas adhaesiva. 16S rRNA gene sequence similarities to all other established Sphingomonas species ranged from 94.4 % (to Sphingomonas echinoides) to 97.6 % (to S. adhaesiva). Strains FA1 and FA2(T) were catalase-positive and oxidase-negative. Aesculin was hydrolysed, gelatin and urea were not. beta-Galactosidase was produced. From 51 compounds tested 21 were used as single sources of carbon. The major respiratory lipoquinone was ubiquinone-10. The predominant cellular fatty acids were 16 : 0, 18 : 1omega7c and 16 : 1omega7c (from summed feature 3). Hydroxy fatty acids 14 : 0 2-OH and 15 : 0 iso 2-OH were present as well (from summed feature 4). The polar lipids detected in strain FA2(T) were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and two unidentified phospholipids. The DNA G+C content of strain FA2(T) was 61 mol%. DNA-DNA hybridization experiments showed 27.6 % relatedness between strain FA2(T) and S. adhaesiva DSM 7418(T). Based upon phenotypic and molecular evidence, a novel species of the genus Sphingomonas is proposed, Sphingomonas phyllosphaerae sp. nov., with strain FA2(T) (=LMG 21958(T)=CECT 5832(T)) as the type strain.

Biodegradation kinetics of volatile hydrophobic organic compounds in cultures with variable fractional volumes.

An extension of the models developed by Guha and Jaffé (Biotechnol Bioeng [1996] 50:693-699) to describe the phenanthrene biodegradation kinetics for the cultures with variable fractional volumes is presented. Batch experiments were conducted with a culture capable of degrading the phenanthrene using a single culture vessel from which samples were withdrawn over time to monitor the disappearance of phenanthrene. For accurate measurement of phenanthrene concentrations, a sampling procedure designed for quantifying the sorption of phenanthrene onto glassware was also introduced. The Monod parameters were estimated by nonlinear regression analyses of simultaneous solutions to the substrate utilization/volatilization and Monod equations for growth of the cell mass. The results demonstrate that the models were able to be extended to phenanthrene-degrading cultures with variable fractional volumes. When the ratio between sampling volume and volume of the culture medium was relatively small, the parameters obtained were similar to those which would be obtained using constant fractional volumes of culture medium. It was also found that the model's fit to the phenanthrene disappearance data in this study were better than those obtained by Guha and Jaffé, implying that the sorption process of phenanthrene during the sampling period could significantly affect the measurement of phenanthrene concentrations. Failing to account for these losses led to less accurate measurements of substrate concentrations, which in turn resulted in a poor estimation of the parameters. The findings of this study reduce considerably the experimental work necessary in the estimation of Monod kinetic parameters for the purpose of modeling.