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Biodesulfurization of alkylated forms of dibenzothiophene and benzothiophene by Sphingomonas subarctica T7b.

Sphingomonas subarctica T7b was isolated from soil in Toyotomi, Hokkaido, Japan as an organism capable of desulfurizing aromatic hydrocarbons in light gas oil (LGO) through enrichment culture. S. subarctica T7b could grow on mineral salt sulfur-free (MSSF) medium with the n-tetradecane oil phase containing dibenzothiophene (DBT), alkyl dibenzothiophenes (alkyl DBTs) or alkyl benzothiophenes (alkyl BTs) as the sole sulfur source and desulfurize these compounds, but could not utilize the tetradecane as a carbon source. This is the first report of a gram-negative bacterium which can desulfurize 4,6-dibutyl DBT and 4,6-dipentyl DBT. The desulfurized product of DBT produced by this strain was 2-hydroxybiphenyl, as in the case of other DBT-desulfurizing bacteria. S. subarctica T7b could desulfurize LGO and the sulfur content was decreased to 41% within 36 h.

Alkanes↗

Statistical approach to optimization of fermentative production of gellan gum from Sphingomonas paucimobilis ATCC 31461.

Gellan gum, a high-molecular-weight anionic linear polysaccharide produced by pure-culture fermentation from Sphingomonas paucimobilis ATCC 31461, has elicited industrial interest in recent years as a high-viscosity biogum, a suspending agent, a gelling agent, and an agar substitute in microbial media. In this paper we report on the optimization of gellan gum production using a statistical approach. In the first step, the one factor-at-a-time method was used to investigate the effect of medium constituents such as carbon and nitrogen sources; subsequently, the intuitive analysis based on statistical calculations carried out using the L16 -orthogonal array method. The design for the L16 -orthogonal array was developed and analyzed using MINITAB 13.30 software. All the fermentation runs were carried out at 30+/-2 degrees C on a rotary orbital shaker at 180 rpm for 48 h. In the second step, the effects of amino acids and gellan precursors such as uridine-5'-diphospate (UDP) and adenosine-5'-diphospate (ADP) on the fermentative production of gellan gum were studied. Media containing 4% soluble starch, 0.025% yeast extract, 1.0 mM ADP and 0.05% tryptophan gave a maximum yield of 43.6 g l(-1) starch-free gellan gum, which was significantly higher than reported values in the literature.

Bioreactors↗

Molecular identification of Sphingomonas sp. A1 alginate lyase (A1-IV') as a member of novel polysaccharide lyase family 15 and implications in alginate lyase evolution.

Sphingomonas sp. A1 (strain A1) produces three endotypes (A1-I [65 kDa], A1-II [25 kDa], and A1-III [40 kDa]) and an exotype (A1-IV [86 kDa]) alginate lyases in cytoplasm. These four enzymes cooperatively depolymerize alginate into constituent monosaccharides. In addition to the genes for these lyases, novel genes encoding hypothetical proteins homologous with A1-IV were found in the genomes of many bacteria including strain A1. One such protein, A1-IV' (90 kDa) of strain A1, was overexpressed in Escherichia coli cells, purified, and characterized. A1-IV' catalyzed the cleavage of glycosidic bonds in alginate through a beta-elimination reaction and released unsaturated di- and trisaccharides as main products, thus indicating that the enzyme is an endotype alginate lyase. A1-IV', which differed from A1-IV in some enzymatic properties, was not expressed in strain A1, suggesting that A1-IV' has no significant role in alginate metabolism. A1-IV' and other A1-IV homologs facilitate the creation of novel polysaccharide lyase family 15 based on their primary structures, implying the evolution route of alginate lyases in family PL-15.

Amino Acid Sequence↗

Selective and continuous degradation of carbazole contained in petroleum oil by resting cells of Sphingomonas sp. CDH-7.

Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1% molar conversion yield based on total CA added. The resting cells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20% (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20% (v/v) pxylene, toluene, and heptanol, however, CA degradation yields decreased.

Biodegradation, Environmental↗

A common structure of substrate shared by lignostilbenedioxygenase isozymes from Sphingomonas paucimobilis TMY1009.

A common structure of substrates of lignostilbenedioxygenases was investigated using synthesized stilbenes. Cell-free extracts of Sphingomonas paucimobilis TMY1009 degraded only trans-4-hydroxystilbene and trans-4-hydroxy-3-methoxystilbene. Other stilbenes that had no 4-hydroxyl group and had a cis structure were not substrates for lignostilbenedioxygenases. These results indicate that a 4-hydroxyl group and trans-structure is necessary for the common structure for substrates of lignostilbenedioxygenases.

Cell Extracts↗

Isolation and identification of Sphingomonas sp. that yields tert-octylphenol monoethoxylate under aerobic conditions.

Topsoil samples were collected from eight golf courses in Yamaguchi Prefecture, Japan, and enrichment cultures were carried out with a basal-salt medium containing 0.2% 4-tert-octylphenol polyethoxylate (OPPEO) as sole carbon source. OPPEO-degrading activity was detected in one of the samples, from which a strain of OPPEO-degrading bacterium was isolated. The isolated bacterium grew on a nutritionally enriched medium (NE medium) containing 0.2% OPPEO as sole carbon source, and accumulated 4-tert-octylphenol diethoxylate (OP2EO) (63%), 4-tert-octylphenol triethoxylate (OP3EO) (14%), and 4-tert-octylphenol monoethoxylate (OP1EO) (2%) after 7 d cultivation under aerobic conditions. The addition of clay mineral (vermiculite) to the medium accelerated the degradation of OP2EO (40%) and OP3EO (4%) to OP1EO (23%). This is the first report about bacteria that can degrade OPPEO to OP1EO under aerobic conditions. The strain was identified as Sphingomonas macrogoltabidus, based on the homology of a 16S rDNA sequence.

Aerobiosis↗

Identification of insertion sequence from a gamma-hexachlorocyclohexane degrading bacterium, Sphingomonas paucimobilis UT26.

Tn5-derived mutants of the gamma-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 were genetically characterized, and an endogenous insertion sequence (IS) which belongs to the IS1380 family was identified. The IS, named ISsp1, existed as multi copies in UT26, and its transposition appeared to be activated during the process of Tn5-mutagenesis. It was found that transposon mutagenesis can cause endogenous mutations.

DNA Transposable Elements↗

Estimation of kinetic parameter for the biotransformation of three-ring azaarenes by the phenanthrene-degrading strain Sphingomonas sp. LH128.

We examined the biotransformation of several azaarenes to get information on their rate of removal from contaminated soil by cometabolic degradation. Acridine, 9-methyl-acridine, phenanthridine, benzo[f]quinoline, and benzo[h]quinoline were found to be cometabolized by phenanthrene-degrading Sphingomonas sp. strain LH128. The transformation of acridine and benzo[h]quinoline was shown to be inhibited at relatively high concentrations (24.9 mg/L and 58.2 mg/L, respectively). Maximum biotransformation rates (Vmaxs), Michaelis-Menten half saturation constants (Kms), and biotransformation constants (k1s) were estimated for acridine, phenanthridine, and benzo[f]quinoline and ranged from 14.0 to 26.1 mg/g total protein/h for Vmax, 0.57 to 2.9 mg/L for Km, and 4.9 to 30.8 L/g total protein/h for k1. For 9-methyl-acridine only, a k1 of 0.6 L/g total protein/h was estimated and for benzo[h]quinoline, the highest biotransformation rate measured was 6.8 +/- 0.4 mg/g total protein/h. To our knowledge, this is the first effort to determine both Vmax and Km kinetic parameters describing the biotransformation of phenanthrene- and anthracene-analogue azaarenes. The results show that the rate of removal of azaarenes from contaminated soil will probably be rather high, although accumulation of metabolites can be expected.

Aza Compounds↗

Mineralization of soil-aged isoproturon and isoproturon metabolites by Sphingomonas sp. strain SRS2.

The aim of the study was to determine the effect of aging of the herbicide isoproturon and its metabolites monodesmethyl-isoproturon and 4-isopropyl-aniline in agricultural soil on their availability to the degrading bacterium Sphingomonas sp. strain SRS2. The 14C-ring-labeled isoproturon, monodesmethyl-isoproturon, and 4-isopropyl-aniline were added to sterilized soil and stored for 1, 49, 71, or 131 d before inoculation with strain SRS2. The availability of the compounds was estimated from the initial mineralization and the amount of 14CO2 recovered after 120 d of incubation. Aging in soil for 131 d reduced the initial mineralization of isoproturon and monodesmethyl-isoproturon and, in the case of isoproturon, also reduced the recovery of 14CO2. Initial mineralization and recovery of 14CO2 from aged 4-isopropyl-aniline were slightly reduced, but less 14CO2 was generally produced than with isoproturon or monodesmethyl-isoproturon. Thus, recovery of 14CO2 from 14C-isoproturon and 14C-monodesmethyl-isoproturon was 50.7 to 64.4% of the initially added 14C, while recovery from 14C-4-isopropyl-aniline was only 11.7 to 17.0%. Sorption measurements revealed similar Freundlich constants (K(f)) for isoproturon and monodesmethyl-isoproturon, whereas K(f) for 4-isopropyl-aniline was more than fivefold greater. The findings imply that in soil, partial degradation of isoproturon to 4-isopropyl-aniline may lead to reduced mineralization of the herbicide due to sorption of the aniline moiety.

Carbon Radioisotopes↗

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461.

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.

Amino Acids↗

Degradation of 2,4-dinitrophenol and selected nitroaromatic compounds by Sphingomonas sp. UG30.

Sphingomonas strain UG30 mineralizes both p-nitrophenol (PNP) and pentachlorophenol (PCP). Our current studies showed that UG30 oxidatively metabolized certain other p-substituted nitrophenols, i.e., p-nitrocatechol, 2,4-dinitrophenol (2,4-DNP), and 4,6-dinitrocresol with liberation of nitrite. 2,6-DNP, o- or m-nitrophenol, picric acid, or the herbicide dinoseb were not metabolized. Studies using 14C-labelled 2,4-DNP indicated that in glucose-glutamate broth cultures of UG30, greater than 90% of 103 microM 2,4-DNP was transformed to other compounds, while 8-19% of the 2,4-DNP was mineralized within 5 days. A significant portion (20-50%) of the 2,4-DNP was metabolized to highly polar metabolite(s) with one major unidentified metabolite accumulating from 5 to 25% of the initial radioactivity. The amounts of 2,4-DNP mineralized and converted to polar metabolites was affected by glutamate concentration in the medium. Nitrophenolic compounds metabolized by UG30 were also suitable substrates for the UG30 PCP-4-monooxygenase (pcpB gene expressed in Escherichia coli) which is likely central to degradation of these compounds. The wide substrate range of UG30 could render this strain useful in bioremediation of some chemically contaminated soils.

2,4-Dinitrophenol↗

Detection of mega plasmid from polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. strain KS14.

Polycyclic aromatic hydrocarbon (PAH) degrading Sphingomonas sp. strain KS14 was isolated from soil. Strain KS14 was able to utilize phenanthrene and naphthalene as sole source of carbon and energy, and mineralize phenanthrene to carbon dioxide. Strain KS14 cometabolically degraded pyrene using phenanthrene as secondary growth substrate. A large degradative plasmid (>500 kb) was detected from the strain KS14. This study suggests that this mega plasmid could be responsible for the degradation of PAH by strain KS14.

Biodegradation, Environmental↗

Microbial degradation of a single branched isomer of nonylphenol by Sphingomonas TTNP3.

The endocrine disrupting chemical nonylphenol (NP) is a technical product which consists of a complex mixture of nonylphenols with different alkyl side-chain isomers. Since the bio-degradation of each NP isomer may lead to its own range of metabolites, the isolation and identification of transformation products is very difficult. In order to overcome this difficulty, the nonylphenol isomer 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) was synthesized, and its degradation by an axenic culture of Sphingomonas TTNP3 was investigated with [ring-U-14C]-labelled and non-labelled p353NP including a time-course study. Radioactive mass balancing resulted in different polar soluble fractions, in insoluble radioactivity associated with biomass, and volatile radioactivity in the form of the mineralization product 14CO2. In the extracellular media, the presence of nonanol corresponding to the nonyl chain of the NP isomer was confirmed and its concentration was determined during the course of fermentation. No other radioactive compounds were detected beside the parent isomer. Radioactive metabolites were only found in the intracellular fraction of S. TTNP3.

Biodegradation, Environmental↗

Production and rheological characterization of biopolymer of Sphingomonas capsulata ATCC 14666 using conventional and industrial media.

This work was aimed at the production and rheological characterization of biopolymer by Sphingomonas capsulata ATCC 14666, using conventional and industrial media. The productivity reached the maximum of 0.038 g/L x h, at 208 rpm and 4% (w/v) of sucrose. For this condition, different concentrations of industrial medium were tested (2.66, 4, 6, and 8%). The best productivity was obtained using pretreated molasses 8% (w/v) (0.296 g/L x h), residue of textured soybean protein 6% (wt/v) (0.244 g/L x h) and crude molasses 8% (w/v) (0.192 g/L x h), respectively. Apparent viscosity presented similar results when compared with those in the literature for other biopolymers.

Biopolymers↗

[Analysis of the degrading products of carbofuran by Sphingomonas sp. with GC/MS and GC/FTIR].

In different degrading phases, the degrading products of carbofuran by CDS-1(Sphingomonas sp. ) were extracted by acetone. According to the analysis of gas chromatography/mass spectrometry (GC/MS), carbofuran-7-phenol was identified as the initial degrading product, and an unknown metabolite with molecular weight of 182 was determined as 2-hydroxyl-3-tertiarybutylalcohol-phenol, which was the next degrading product of carbofuran-7-phenol. According to the analysis of GC/MS and gas chromatography/Fourier transform infrared spectrum (GC/FTIR), the main component of the compounds that appeared in the final phase of degradation which could produce volatile pungent odor, was identified as carvone.

Carbofuran↗

An assessment of protein profiles from the marine oligotrophic ultramicrobacterium, Sphingomonas sp. strain RB2256.

The protein expression profile of a novel marine oligotrophic ultramicrobacterium, Sphingomonas sp. strain RB2256, was investigated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Analytical reference maps were generated from mid-log phase batches and steady-state chemostat cultures with pH 4-8 immobilised pH gradients (IPGs) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were detected by two different methods: radioactive labeling and silver staining. Protein profiles generated from analytical 2-D PAGE gels were compared and differential analysis was performed using Melanie II software. Both methods (radioactive labeling and silver staining) resulted in reproducible, high resolution gels (up to 1600 protein spots). This approach is proving to be a powerful tool for investigating the molecular basis of the unique physiology of this model oligotrophic microorganism.

Bacterial Proteins↗

Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene.

Prolyl endopeptidase (prolyl oligopeptidase, EC 3.4.21.26) was purified from Sphingomonas capsulata IFO 12533, and its gene was cloned and expressed in Escherichia coli. The recombinant enzyme was markedly inhibited by diisopropyl phosphofluoridate and hardly affected by SH reagents or metal chelators, similar to the native enzyme purified from S. capsulata. Nucleotide sequencing analysis revealed an open reading frame of 2169 bp, coding for a protein of 723 amino acids with a predicted molecular weight of 78,433. The amino acid sequence was 39.6, 45.3, 38.9, and 38.3% homologous to Flavobacterium meningosepticum, Aeromonas hydrophila, porcine brain, and human T cell prolyl endopeptidase, respectively. A region near the C-terminus and the region containing the putative catalytic triad residues were highly conserved. The enzyme was crystallized by the hanging drop vapor diffusion method, using ammonium sulfate as a precipitant.

Amino Acid Sequence↗

Separation and partial characterization of soluble fatty acid alpha-hydroxylase from Sphingomonas paucimobilus.

The crude extracts from Sphingomonas paucimobilus containing 2-hydroxy fatty acid-rich sphingolipids were found to oxidize [1-14C]-myristate to 2-hydroxymyristate in the presence of NADH. The myristate-oxidation activity was partially purified about 290-fold from the cell-free extracts by ammonium sulfate fractionation and hydroxylapatite chromatography. When laurate, myristate, and palmitate were used as the substrates, the reaction products were identified as the corresponding 2-hydroxy fatty acids by gas-chromatography (GC), HPLC, and GC-mass spectrometry. The enzyme preparation required NADH and molecular oxygen for its alpha-hydroxylation activity, suggesting that the bacterial alpha-hydroxylation is typical of monooxygenase reactions. Of fatty acids tested, myristate was the most efficient substrate.

Bacteria↗