不同富集方法分离多环芳烃降解菌的比较研究

多环芳烃是一类普遍存在的环境污染物。本研究探讨了普通富集法,固定化富集法以及巴斯德消毒后富集法三种途径从相同红树林土壤中分离菲降解茵的差异。通过平板培养和变性梯度凝胶电泳两种方法分析分离结果。上述方法分别获得以鞘氨醇单胞茵、分枝杆菌以及红球茵为优势菌群的群落,表明分离方法对多环芳烃降解菌多样性的研究是一种重要的影响因素。     

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.     

Decolorization of 1-aminoanthraquinone-2-sulfonic acid by Sphingomonas xenophaga

Sphingomonas xenophaga QYY from sludge samples could effectively decolorize 1-aminoanthraquinone-2-sulfonic acid (ASA-2), one kind of anthraquinone dye intermediate, under aerobic conditions. More than 98% of ASA-2 could be removed within 120 h at the dye concentration from 200 mg l⁻¹ to 1,000 mg l⁻¹ due to oxidative degradation. The strain converted ASA-2 to 2-(2′-hydroxy-3′-amino-4′-sulfo-benzoyl)-benzoic acid, 2-(2′-amino-3′-sulfo-6′-hydroxy-benzoyl)-benzoic acid, o-phthalic acid and 2-amino-3-hydroxy-benzenesulfonic acid, which were identified using HPLC-MS and NMR. A possible initial decolorization pathway was proposed according to these metabolites. The decolorization of ASA-2 by cells in the basal salt medium was induced by ASA-2, and was due to soluble cytosolic enzymes. Combined initial decolorization pathway and the analysis of decolorization enzyme(s), the major enzyme responsible for ASA-2 decolorization was a NADH-dependent oxygenase.     

Improvement in Production and Quality of Gellan Gum by Sphingomonas paucimobilis Under High Dissolved Oxygen Tension Levels

The effect of agitation rate and dissolved oxygen tension (DOT) on growth and gellan production by Sphingomonas paucimobilis was studied. Higher cell growth of 5.4 g l⁻¹ was␣obtained at 700 rpm but maximum gellan (15 g l⁻¹) was produced at 500 rpm. DOT levels above 20% had no effect on cell growth but gellan yield was increased to 23 g l⁻¹ with increase in DOT level to 100%. Higher DOT levels improved the viscosity and molecular weight of the polymer with change in acetate and glycerate content of the polymer.     

Degradation of diphenyl phthalate by <Emphasis Type="Italic">Sphingomonas chungbukensis</Emphasis>

More than 80% of diphenyl phthalate (DPP) at 100 mg l⁻¹ was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30°C within 48 h. The maximum specific degradation rate was 5 mg DPP l⁻¹ h⁻¹. It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.     

Aromatic metabolism in Rhizobium trifolii-catechol 1,2-dioxygenase

Catechol 1,2-dioxygenase has been purified 46-fold from cells of Rhizobium trifolii TA1 grown on benzoate plus glucose. The dioxygenase had a molecular weight of 107,000 and a sub-unit molecular weight of 59,000. The enzyme had a K _m of 2 M for catechol and also cleaved 4-methylcatechol. The dioxygenase contained 2 g atoms of Fe³⁺ per mole of enzyme which could be removed by treatment with 1,10-phenanthroline, resulting in a complete loss of activity; reactivation of the enzyme occurred specifically with Fe³⁺.     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Implication of two glutathione S-transferases in the optimal metabolism of m-toluate by Sphingomonas yanoikuyae B1

A putative glutathione S-transferase (GST) gene (bphK) was identified in the meta-cleavage operon for the degradation of m-toluate by Sphingomonas yanoikuyae B1. Disruption of bphK resulted in the loss of GST activity against 1-chloro-2,4-dinitrobenzene and a much increased lag time of the mutant strain MB3 (bphK::Km) following subculture into m-toluate medium. In contrast, an increased lag time was not observed when MB3 was grown on biphenyl or m-xylene and MB3 showed normal growth on m-toluate when complemented with a subclone containing the bphK gene only. Furthermore, an additional GST activity was detected in MB3. The induction timing of this second GST activity coincided with the beginning of the exponential growth phase of MB3 on m-toluate, reached maximal activity within three hours, and then dropped sharply to the basal level. Thus, it is apparent that BphK and/or the second GST are necessary for optimal growth of B1 on m-toluate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioaugmentation of Bromoamine Acid Degradation with Sphingomonas xenophaga QYY and DNA Fingerprint Analysis of Augmented Systems

One high-effective bromoamine acid (1-amino-4-bromoanthraquinone-2-sulfonic acid, BAA) degrading strain was isolated previously with the ability to use BAA as sole source of carbon and nitrogen. It was identified as Sphingomonas xenophaga QYY by 16S rDNA sequence analysis and physio-biochemical tests. In this study, bioaugmentation of BAA degradation with suspended and immobilized cells of strain QYY was investigated. The optimal degradation conditions were as follows: temperature 30 °C, pH 6.0–7.0, 150 rev min⁻¹ and the immobilized cells maintained degradation activity to BAA after 60 days storage at 4 °C. The structure of BAA was evidently changed according to the analysis of total organic carbon removal of BAA (about 50%) and the UV–VIS spectra changes during the biodegradation. Bioaugmented systems exhibited stronger abilities degrading BAA than the non-bioaugmented control ones. And microbial community dynamics of augmented systems was revealed by amplified ribosomal DNA restriction analysis (ARDRA), a modern DNA fingerprint technique. The results indicated that the microbial community dynamics was substantially changed throughout the augmentation process. This study suggests that it is feasible and potentially useful to enhance BAA degradation using bioaugmentation with the immobilized cells of BAA-degrading bacterium.     

Novel organization of catechol meta-pathway genes in Sphingomonas sp. HV3 pSKY4 plasmid

Sphingomonas sp. strain HV3 (formerly Pseudomonas sp. HV3), which degrades aromatics and chloroaromatics, harbors a mega-plasmid, pSKY4. A sequenced 4 kb fragment of the plasmid reveals a novel gene organization for catechol meta-pathway genes. The putative meta operon starts with the cmpF gene encoding a 2-hydroxymuconic semialdehyde hydrolase. The gene has a 6 bp overlap with the previously characterized ring-cleavage gene, catechol 2,3-dioxygenase, cmpE. Downstream of cmpE is a 429 bp open reading frame of unknown function. Gene cmpC, encoding a 2-hydroxymuconic semialdehyde dehydrogenase, starts 44 bp further downstream. It has the highest homology to 2-hydroxymuconic semialdehyde dehydrogenases of dmp and xyl pathways and to XylC from the marine oligotroph Cycloclasticus oligotrophus. The gene organization is different from other known meta pathways. This is the first report of organization of plasmid-encoded meta-pathway genes in the genus Sphingomonas.     

Physiological Basis of the Difference in Net Photosynthetic Rate of Leaves between Two Maize Strains

Significant differences in net photosynthetic rate (P _N) of leaves between two maize (Zea mays L.) strains (Shuang 105 and 40×44) grown in the field were observed. At several growth stages, P _N of 40×44 was higher than that of Shuang 105 (from 10.3 to 32.5 %). Moreover, the strain 40×44 had a higher plant height, larger leaf area, lower chlorophyll content, and higher photochemical efficiency of photosystem 2 (PS2) (F_v/F_m and F/F_m) than strain Shuang 105. Shuang 105, which showed lower P _N, had lower stomatal conductances (g _s) but slightly higher intercellular CO₂ concentrations (C _i) than those of 40×44. Hence the differences in P _N between the two strains did not result from the difference in g _s, but probably from that in light reaction system.     

The role of the Sphingomonas species UG30 pentachlorophenol-4-monooxygenase in p-nitrophenol degradation

Pentachlorophenol-4-monooxygenase is an aromatic flavoprotein monooxygenase which hydroxylates pentachlorophenol and a wide range of polyhalogenated phenols at their para position. The PCP-degrading Sphingomonas species UG30 was recently shown to mineralize p-nitrophenol. In this study, the UG30 pcpB gene encoding the pentachlorophenol-4-monooxygenase gene was cloned for use to study its potential role in p-nitrophenol degradation. The UG30 pcpB gene consists of 1614 bp with a predicted translational product of 538 amino acids and a molecular mass of 59,933 Da. The primary sequence of pentachlorophenol-4-monooxygenase contained a highly conserved FAD binding site at its N-terminus associated with a beta alpha beta fold. UG30 has been shown previously to convert p-nitrophenol to 4-nitrocatechol. We observed that pentachlorophenol-4-monooxygenase catalyzed the hydroxylation of 4-nitrocatechol to 1,2,4-benzenetriol. About 31.2% of the nitro substituent of 4-nitrocatechol (initial concentration of 200 microM) was cleaved to yield nitrite over 2 h, indicating that the enzyme may be involved in the second step of p-nitrophenol degradation. The enzyme also hydroxylated p-nitrophenol at the para position, but only to a very slight extent. Our results confirm that pentachlorophenol-4-monooxygenase is not the primary enzyme in the initial step of p-nitrophenol metabolism by UG30.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Isolation and identification of a carbazole degradation gene cluster from Sphingomonas sp.JS1

The carbazole degrading bacterium JS1 was isolated from carbazole polluted soil and identified as Sphingomonas sp. bacterium based on its 16S rDNA gene. The car gene cluster located in the genome of JS1 was isolated by PCR and its presence verified by Southern hybridization. Sequence analysis of the car gene cluster showed that the arrangement of elements in JS1 was different from that of Pseudomons sp. CA10 and Nocardioides aromaticivorans IC177, but car gene cluster and neighboring regions were nearly identical to that of Sphingomonas sp. KA1 and Sphingomonas sp.GTIN11. Each element of the car gene cluster was expressed in E. coli upon IPTG induction. The amount of CaBb protein expressed was higher than CarBa and the ratio of these two proteins was 1:1.5. CarC expression level was detected using anti-CarC antibody. The result showed that carbazole degrading proteins were induced by the substrate carbazole. The quantity of CarC at 0.5 mg/ml carbazole was five times more than that at 0.1 mg/ml. Meiying Yang and Wenming Li have the equal contribution for this work.     

Enhanced Degradation of a Mixture of Polycyclic Aromatic Hydrocarbons by a Defined Microbial Consortium in a Two-Phase Partitioning Bioreactor

Biological treatment methods are effective at destroying polycyclic aromatic hydrocarbons (PAHs), and some of the highest rates of PAH degradation have been achieved using two-phase-partitioning bioreactors (TPPBs). TPPBs consist of a cell-containing aqueous phase and a biocompatible and immiscible organic phase that partitions toxic and/or recalcitrant substrates to the cells based on their metabolic demand and on maintaining the thermodynamic equilibrium of the system. In this study, the degradation of a 5-component mixture of high and low molecular weight PAHs by a defined microbial consortium of Sphingomonas aromaticivorans B0695 and Sphingomonas paucimobilis EPA505 in a TPPB was examined. The extremely low aqueous solubilities of the high molecular weight (HMW) PAHs significantly reduce their bioavailability to cells, not only in the environment, but in TPPBs as well. That is, in the two-phase system, the originally selected solvent, dodecane, was found to sequester the HMW PAHs from the cells in the aqueous phase due to the inherent high solubility of the hydrophobic compounds in this solvent. To circumvent this limitation, the initial PAH concentrations in dodecane were increased to sufficient levels in the aqueous phase to support degradation: LMW PAHs (naphthalene, phenanthrene) and fluoranthene were degraded completely in 8 h, while the HMW PAHs, pyrene and benzo[a]pyrene, were degraded by 64% and 11%, at rates of 42.9 mg l⁻¹ d⁻¹ and 7.5 mg l⁻¹ d⁻¹, respectively. Silicone oil has superior PAH partitioning abilities compared to dodecane for the HMW PAHs, and was used to improve the extent of degradation for the PAH mixture. Although silicone oil increased the bioavailability of the HMW PAHs and greater extents of biodegradation were observed, the rates of degradation were lower than that obtained in the TPPB employing dodecane.     

Biodegradation of triphenylmethane dye Malachite Green by <Emphasis Type="Italic">Sphingomonas paucimobilis</Emphasis>

Triphenylmethane dyes belong to the most important group of synthetic colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. Sphingomonas paucimobilis, was isolated from the soil sample collected from contaminated sites of textile industry located in KsarHellal, Tunisia, and it was able to decolorize Malachite Green (MG) dye (50 mg/l) within 4 h under shaking condition (pH 9 and temperature 25°C). The effect of inoculum size, dye concentration, temperature and initial pH of the solution were studied. The results obtained from the batch experiments revealed the ability of the tested bacteria to remove dye. UV–Vis spectroscopy and FTIR analysis of samples before and after decolorization confirmed the ability of the tested strain to decolorize MG. In addition, the phytotoxicity study revealed the degradation of MG into non-toxic product by S. paucimobilis.     

Biochemical characterization and phylogenetic analysis of UDP-glucose dehydrogenase from the gellan gum producer <Emphasis Type="Italic">Sphingomonas elodea</Emphasis> ATCC 31461

Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications. In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily. The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG from S. elodea and UDPG-DHs from Novosphingobium, Zymomonas, Agrobacterium, and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH of 37°C and 8.7, respectively. The estimated apparent K _m values for UDP-glucose and NAD⁺ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis of S. elodea cell wall polysaccharides.     

Efficiency of the EPS emulsifier produced by <Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> in different hydrocarbon bioremediation assays

Ochrobactrum anthropi strain AD2 was isolated from the waste water treatment plant of an oil refinery and was identified by analysis of the sequence of the gene encoding 16S rDNA. This bacterium produced exopolysaccharides in glucose nutrient broth media supplemented with various hydrocarbons (n-octane, mineral light and heavy oils and crude oils). The exopolysaccharide AD2 (EPS emulsifier) synthesized showed a wide range of emulsifying activity but none of them had surfactant activity. Yield production varied from 0.47 to 0.94 g of EPS l⁻¹ depending on the hydrocarbon added. In the same way, chemical composition and emulsification activity of EPS emulsifier varied with the culture conditions. Efficiency of the EPS emulsifier as biostimulating agent was assayed in soil microcosms and experimental biopiles. The AD2 biopolymer was added alone or combined with commercial products frequently used in oil bioremediation such as inorganic NPK fertilizer and oleophilic fertilizer (S200 C). Also, its efficiency was tested in mixture with activated sludge from an oil refinery. In soil microcosms supplemented with S200 C + EPS emulsifier as combined treatment, indigenous microbial populations as well as hydrocarbon degradation was enhanced when compared with microcosms treated with NPK fertilizer or EPS emulsifier alone. In the same way EPS emulsifier stimulated the bioremediation effect of S200 C product, increasing the number of bacteria and decreasing the amount of hydrocarbon remained. Finally, similar effects were obtained in biopile assays amended with EPS emulsifier plus activated sludge. Our results suggest that the bioemulsifier EPS emulsifier has interesting properties for its application in environment polluted with oil hydrocarbon compounds and may be useful for bioremediation purposes.     

Structural characterization of the exopolysaccharide PS-EDIV from <Emphasis Type="Italic">Sphingomonas pituitosa</Emphasis> strain DSM 13101

Members of the bacterial genus Sphingomonas are known to produce highly viscous polysaccharides in solution. The exopolysaccharide PS-EDIV was produced by Sphingomonas pituitosa strain DSM 13101, purified using centrifugation, and precipitation and its structure was elucidated by 1D and 2D NMR techniques and chemical microderivatization combined with various mass spectrometric techniques. The following repeating unit of the polysaccharide could be identified: In addition, the polysaccharide also contains acetyl and glyceryl groups whose exact positions were not determined. PS-EDIV is similar in structure to a known exopolysaccharide but differs in being the first bacterial polysaccharide in which two different glucuronic acids are combined. It caused a high viscosity of the culture broth after cultivation for 48 h, although a gelation was not observed.