Biodegradation of dimethyl phthalate by Sphingomonas sp. isolated from phthalic-acid-degrading aerobic granules

This study isolated nine strains of aerobic phenol-degrading granules. These isolates (I1–I9) were characterized using 16S rRNA gene sequencing, with γ-Proteobacteria as the dominant strains in the aerobic granules. While most strains demonstrated either high phenol-degrading capabilities or auto-aggregation capabilities, three isolates, I2, I6, and I8 showed both features. These findings contradict the previous view that auto-aggregation and phenol degradation are mutually exclusive in aerobic granules. Strains I2 and I8 independently formed single-culture aerobic granules except for I3. Anti-microbial activity test results indicated that strains I2 and I8 inhibited growth of strain I3. However, co-culturing I3 with I2 or I8 helped to form granules.     

好氧条件下Sphingomonas sp．XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp．XJ1对邻苯二甲酸丁酯（DBP）进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4．79min和5．11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonassp．XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJI的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

好氧条件下Sphingomonassp.XJ1降解DBP途径的研究

在三角瓶中采用Sphingomonas sp.XJ1对邻苯二甲酸丁酯(DBP)进行好氧降解,以考察DBP的降解途径。分别对降解16h、32h和40h的DBP样品进行代谢产物分析,可判定保留时间为4.79min和5.11min所对应的代谢产物分别为原儿茶酸和邻苯二甲酸。由此可知,菌株Sphingomonas sp.XJ1对DBP的降解遵循DBP好氧生物降解途径的一般途径。即在菌株XJ1的作用下,DBP首先水解为MBP,继而水解为PA,经由PCA最终完全降解为CO2和H2O。     

Parametric study of diethyl phthalate biodegradation

Summary The effects of temperature, dissolved oxygen, and other environmental parameters under both aerobic and anaerobic conditions were investigated using one aerobic and one facultative strain isolated from wastewater treatment plant sludge. Among other results, we found that low dissolved oxygen levels and low temperatures decreased the rate of DEP degradation and the growth rate, and that the facultative strain was much less affected by the lower DO concentrations than the aerobic strain.     

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.     

Biodegradation of diethyl phthalate in soil by a novel pathway

Biodegradation of diethyl phthalate (DEP) has been shown to occur as a series of sequential steps common to the degradation of all phthalates. Primary degradation of DEP to phthalic acid (PA) has been reported to involve the hydrolysis of each of the two diethyl chains of the phthalate to produce the monoester monoethyl phthalate (MEP) and then PA. However, in soil co-contaminated with DEP and MeOH, biodegradation of the phthalate to PA resulted in the formation of three compounds, in addition to MEP. These were characterised by gas chromatography-electron ionisation mass spectrometry and nuclear magnetic resonance as ethyl methyl phthalate, dimethyl phthalate and monomethyl phthalate, and indicated the existence of an alternative pathway for the degradation of DEP in soil co-contaminated with MeOH. Transesterification or demethylation were proposed as the mechanisms for the formation of the three compounds, although the 7:1 ratio of H(2)O to MeOH means that transesterification is unlikely.     

Isolation of hexachlorocyclohexane-degrading Sphingomonas sp. by dehalogenase assay and characterization of genes involved in gamma-HCH degradation

Aim: To screen and identify bacteria from contaminated soil samples which can degrade hexachlorocyclohexane (HCH)‐isomers based on dechlorinase enzyme activity and characterize genes and metabolites. Methods and Results: Dechlorinase activity assays were used to screen bacteria from contaminated soil samples for HCH‐degrading activity. A bacterium able to grow on α‐, β‐, γ‐ and δ‐HCH as the sole carbon and energy source was identified. This bacterium was a novel species belonging to the Sphingomonas and harbour linABCDE genes similar to those found in other HCH degraders. γ‐Pentachlorocyclohexene 1,2,4‐trichlorobenzene and chlorohydroquinone were identified as metabolites. Conclusions: The study demonstrates that HCH‐degrading bacteria can be identified from large environmental sample‐based dehalogenase enzyme assay. This kind of screening is more advantageous compared to selective enrichment as it is specific and rapid and can be performed in a high‐throughput manner to screen bacteria for chlorinated compounds. Significance and Impact of the Study: The chlorinated pesticide HCH is a persistent and toxic environmental pollutant which needs to be remediated. Isolation of diverse bacterial species capable of degrading all the isomers of HCH will help in large‐scale bioremediation in various parts of the world.     

Aromatic hydrocarbon degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to grow on a wide variety of aromatic compounds including biphenyl, naphthalene, phenanthrene, toluene, m-, and p-xylene. In addition, the initial enzymes for degradation of biphenyl have the ability to metabolize a wide variety of different polycyclic aromatic hydrocarbons. The catabolic pathways for the degradation of both the monocyclic and polycyclic aromatic hydrocarbons are intertwined, joining together at the level of (methyl)benzoate and catechol. Both upper branches of the catabolic pathways are induced when S. yanoikuyae B1 is grown on either class of compound. An analysis of the genes involved in the degradation of these aromatic compounds reveals that at least six operons are involved. The genes are not arranged in discrete pathway units but are combined in groups with genes for the degradation of both classes of compounds in the same operon. Genes for multiple dioxygenases are present perhaps explaining the ability of S. yanoikuyae B1 to grow on a wide variety of aromatic compounds. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram

Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C9 alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.     

Aerobic degradation of polychlorinated biphenyls by Alcaligenes sp. JB1: metabolites and enzymes

In contrast to the degradation of penta-and hexachlorobiphenyls in chemostat cultures, the metabolism of PCBs by Alcaligenes sp. JB1 was shown to be restricted to PCBs with up to four chlorine substituents in resting-cell assays. Among these, the PCB congeners containing ortho chlorine substituents on both phenyl rings were found to be least degraded. Monochloro-benzoates and dichlorobenzoates were detected as metabolites. Resting cell assays with chlorobenzoates showed that JB1 could metabolize all three monochlorobenzoates and dichlorobenzoates containing only meta and para chlorine substituents, but not dichlorobenzoates possessing an ortho chlorine substituent. In enzyme activity assays, meta cleaving 2,3-dihydroxybiphenyl 1,2-dioxygenase and catechol 2,3-dioxygenase activities were constitutive, whereas benzoate dioxygenase and ortho cleaving catechol 1,2-dioxygenase activities were induced by their substrates. No activity was found for pyrocatechase II, the enzyme that is specific for chlorocatechols. The data suggest that complete mineralization of PCBs with three or more chlorine substituents by Alcaligenes sp. JB1 is unlikely.Abbreviations PCB polychlorinated biphenyls - CBA chlorobenzoate - D di - Tr tri - Te tetra - Pe penta- - H hexa     

Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1

The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined. Received: 12 November 1996 / Received revision: 21 February 1997 / Accepted: 22 February 1997     

Chlorophenol and nitrophenol metabolism by Sphingomonas sp UG30

Sphingomonas sp UG30 is a pentachlorophenol (PCP)-degrading bacterial strain capable of degrading several nitrophenolic compounds, including p-nitrophenol (PNP), 2,4-dinitrophenol (2,4-DNP), p-nitrocatechol and 4,6-dinitro-o-cresol (DNOC). The ability to degrade both chlorophenolic and nitrophenolic compounds is probably not restricted to UG30, but may also be possessed by other pentachlorophenol-degrading Sphingomonas spp. The interesting question arises as to whether there is any point of convergence between the initial pathways of PCP and nitrophenol degradation in these microorganisms. There is some experimental evidence that PCP-4-monooxygenase is involved in metabolism of both p-nitrophenol and 2,4-dinitrophenol. Further studies are needed to confirm this and to examine the role(s) of other PCP-degrading enzymes in nitrophenol metabolism by this microorganism. In this paper, we review some of the taxonomic, biochemical, physiological and ecological properties of Sphingomonas sp UG30 with respect to biodegradation of PCP and nitrophenolic compounds. Received 19 April 1999/ Accepted in revised form 21 August 1999     

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.     

Aerobic degradation of 2-picolinic acid by a nitrobenzene-assimilating strain: Streptomyces sp. Z2

Streptomyces sp. Z2 was isolated from nitrobenzene contaminated activated sludge, which utilized nitrobenzene as a sole source of carbon, nitrogen, and energy under aerobic condition. It was found that besides nitrobenzene strain Z2 can degrade 2-picolinic acid. Strain Z2 completely degraded 2-picolinic acid with initial concentration of 500 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, and 3000 mg/L within 36 h, 50 h, 72 h, 100 h, 136 h, and 180 h, respectively. Kinetics of 2-picolinic acid degradation was described using the Andrews equation. The kinetic parameters were as follows: q_max = 3.81 h^?1, K_s = 83.10 mg/L, and K_i = 252.11 mg/L. During the biodegradation process, Z2 transformed 2-picolinic acid into a product which was identified as 6-hydroxy picolinic acid by UV–vis spectrometry, ¹H nuclear magnetic resonance spectroscopy, and mass spectrometry. 6-Hydroxy picolinic acid was then cleaved and mineralized with release of ammonia.     

Enhanced degradation and toxicity reduction of dihexyl phthalate by Fusarium oxysporum f. sp. pisi cutinase

AIMS: This research aims to investigate the efficiency of two lipolytic enzymes--fungal cutinase and yeast esterase--upon the biodegradation of dihexyl phthalate (DHP). METHOD AND RESULTS: During the enzymatic degradation of DHP dissolved in methanol, several degradation products were detected and their time-course changes were monitored using GC/MS. The DHP-degradation rate of cutinase was surprisingly high; i.e. almost 70% of the initial DHP (500 mg l(-1)) was decomposed within 4.5 h. Although the same amount of esterase was employed, more than 85% of the DHP remained after 3 days. Almost all the DHP was converted by cutinase into 1,3-isobenzofurandione (IBF), whereas hexyl methyl phthalate and IBF were abundantly produced by esterase. In addition, the toxicities of the DHP-degraded products by esterase were evaluated using various recombinant bioluminescent bacteria, which caused oxidative and protein damage, whereas the hydrolysis products from cutinase never caused any cellular damage in the methanol-containing reaction system. CONCLUSIONS: Cutinase starts to act as a DHP-degrader much earlier and faster than esterase, with high stability in ester-hydrolytic activity, therefore a plausible approach to the practical application of cutinase for DHP degradation in the DHP-contaminated environments may be possible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the enhanced degradation and detoxification of DHP using Fusarium oxysporum f. sp. pisi cutinase.     

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 microM inhibited the activity by a competitive manner with the inhibition constant, K(I) of 7.07+/-1.36 microM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 microM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v(max)(app), and increasing the apparent Michaelis-Menten constant, K(M)(app). The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.     

Effect of rhamnolipids on degradation of anthracene by two newly isolated strains, Sphingomonas sp. 12A and Pseudomonas sp. 12B

Anthracene is a PAH that is not readily degraded, plus its degradation mechanism is still not clear. Thus, two strains of bacteria-degrading bacteria were isolated from longterm petroleum-polluted soil and identified as Sphingomonas sp. 12A and Pseudomonas sp. 12B by a 16S rRNA sequence analysis. To further enhance the anthracene-degrading ability of the two strains, the biosurfactants produced by Pseudomonas aeruginosa W3 were used, which were characterized as rhamnolipids. It was found that these rhamnolipids dramatically increased the solubility of anthracene, and a reverse-phase HPLC assay showed that the anthracene degradation percentage after 18 days with Pseudomonas sp. 12B was significantly enhanced from 34% to 52%. Interestingly, their effect on the degradation by Sphingomonas sp. 12A was much less, from 35% to 39%. Further study revealed that Sphingomonas sp. 12A also degraded the rhamnolipids, which may have hampered the effect of the rhamnolipids on the anthracene degradation.     

Denitrifying degradation of dimethyl phthalate

Results of batch experiments on the denitrifying degradation of dimethyl phthalate (DMP) was most favorable at pH 7–9 and 30–35°C. DMP was first degraded to monomethyl phthalate (MMP), which was in turn degraded to phthalate before complete mineralization. There was no fatty acid residue in the mixed liquor throughout the experiments. The maximum specific degradation rates were 0.32 mM/(gVSS·h) for DMP, 0.19 mM/(gVSS·h) for MMP, and 0.14 mM/(gVSS·h) for phthalate. About 86% of available electron in DMP was utilized for denitrification; the remaining 14% was presumable conserved in the new biomass with an estimated yield of 0.17 mg/mg DMP. Based on 16S rDNA analysis, the denitrifying sludge was mainly composed of β-subdivision and α-subdivision of Proteobacteria (33 and 5 clones out of a total of 43 clones, respectively), plus some Acidobacteria. Using a primer set specifically designed to amplify the denitrification nirK gene, 10 operational taxonomy units (OTUs) were recovered from the clone library. They clustered into a group in the α-subdivision of Proteobacteria most closely related to denitrifier Bradyrhizobium japonicum USDA110 and several environmental clones.     

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.