Study on biodegradation kinetics of di-2-ethylhexyl phthalate by newly isolated halotolerant Ochrobactrum anthropi strain L1-W.

OBJECTIVE: Di-2-ethylhexyl phthalate (DEHP) pollution is one of the major environmental concerns all over the world. This research aimed at studying the biodegradation kinetics of DEHP by a newly isolated bacterial strain. Water and sediment samples were collected from Wuhan South Lake and potent bacterial isolates were screened for DEHP degradation, characterized by biochemical, physiological, morphological and 16S rDNA gene sequencing, and optimized under suitable pH, temperature, NaCl and DEHP concentrations. DEHP and its metabolites were quantified by High Performance Liquid Chromatography and their degradation kinetics were studied. RESULTS: The newly isolated bacterium was identified as Ochrobactrum anthropi strain L1-W with 99.63% similarity to Ochrobactrum anthropi ATCC 49188. It was capable of utilizing DEHP as the carbon source. The optimum growth temperature, pH, DEHP and NaCl concentration for the strain L1-W were 30 °C, 6, 400 mg/L and 10 g/L respectively. Strain L1-W was capable of degrading almost all (98.7%) of DEHP when the initial concentration was 200 mg/L within a period of 72 h. Besides, it was also found capable of degrading five other phthalates, thus making it a possible candidate for bioremediation of phthalates in the environmental settings.

Multi-approach analysis to assess the chromium(III) immobilization by Ochrobactrum anthropi DE2010.

Ochrobactrum anthropi DE2010 is a microorganism isolated from Ebro Delta microbial mats and able to resist high doses of chromium(III) due to its capacity to tolerate, absorb and accumulate this metal. The effect of this pollutant on O. anthropi DE2010 has been studied assessing changes in viability and biomass, sorption yields and removal efficiencies. Furthermore, and for the first time, its capacity for immobilizing Cr(III) from culture media was tested by a combination of High Angle Annular Dark Field (HAADF) Scanning Transmission Electron Microscopy (STEM) imaging coupled to Energy Dispersive X-ray spectroscopy (EDX). The results showed that O. anthropi DE2010 was grown optimally at 0-2 mM Cr(III). On the other hand, from 2 to 10 mM Cr(III) microbial plate counts, growth rates, cell viability, and biomass decreased while extracellular polymeric substances (EPS) production increases. Furthermore, this bacterium had a great ability to remove Cr(III) at 10 mM (q = 950.00 mg g-1) immobilizing it mostly in bright polyphosphate inclusions and secondarily on the cellular surface at the EPS level. Based on these results, O. anthropi DE2010 could be considered as a potential agent for bioremediation in Cr(III) contaminated environments.

Isolation and characterization of a chlorate-reducing bacterium Ochrobactrum anthropi XM-1.

A Gram-negative chlorate-reducing bacterial strain XM-1 was isolated. The 16S rRNA gene sequence identified the isolate as Ochrobactrum anthropi XM-1, which was the first strain of genus Ochrobactrum reported having the ability to reduce chlorate. The optimum growth temperature and pH for strain XM-1 to reduce chlorate was found to be 30 °C and 5.0-7.5, respectively, under anaerobic condition. Strain XM-1 could tolerate high chlorate concentration (200 mM), and utilize a variety of carbohydrates (glucose, L-arabinose, D-fructose, sucrose), glycerin and sodium citrate as electron donors. In addition, oxygen and nitrate could be used as electron acceptors, but perchlorate could not be reduced. Enzyme activities related to chlorate reducing were characterized in cell extracts. Activities of chlorate reductase and chlorite dismutase could be detected in XM-1 cells grown under both aerobic and anaerobic conditions, implying the two enzymes were constitutively expressed. This work suggests a high potential of applying Ochrobactrum anthropi XM-1 for remediation of chlorate contamination.

Evaluation of the removal of pyrene and fluoranthene by Ochrobactrum anthropi, Fusarium sp. and their coculture.

Fluoranthene and pyrene are polycyclic aromatic hydrocarbons of high molecular weight that are recalcitrant and toxic to humans; therefore, their removal from the environment is crucial. From hydrocarbon-contaminated soil, 25 bacteria and 12 filamentous fungi capable of growth on pyrene and fluoranthene as the sole carbon and energy source were isolated. From these isolates, Ochrobactrum anthropi BPyF3 and Fusarium sp. FPyF1 were selected and identified because they grew quickly and abundantly in both hydrocarbons. Furthermore, O. anthropi BPyF3 and Fusarium sp. FPyF1 were most efficient at removing pyrene (50.39 and 51.32 %, respectively) and fluoranthene (49.85 and 49.36 %, respectively) from an initial concentration of 50 mg L(-1) after 7 days of incubation. Based on this and on the fact that there was no antagonism between the two microorganisms, a coculture composed of O. anthropi BPyF3 and Fusarium sp. FPyF1 was formed to remove fluoranthene and pyrene at an initial concentration of 100 mg L(-1) in a removal kinetic assay during 21 days. Fluoranthene removal by the coculture was higher (87.95 %) compared with removal from the individual cultures (68.95 % for Fusarium sp. FPyF1 and 64.59 % for O. anthropi BPyF3). In contrast, pyrene removal by the coculture (99.68 %) was similar to that obtained by the pure culture of Fusarium sp. FPyF1 (99.75 %). The kinetics of removal for both compounds was adjusted to a first-order model. This work demonstrates that the coculture formed by Fusarium sp. FPyF1 and O. anthropi BPyF3 has greater potential to remove fluoranthene than individual cultures; however, pyrene can be removed efficiently by Fusarium sp. FPyF1 alone.

Colonization by endophytic Ochrobactrum anthropi†Mn1 promotes growth of Jerusalem artichoke.

The Ochrobactrum anthropi Mn1 strain, taxonomically identified using 16S ribosomal DNA sequence, was isolated from roots of Jerusalem artichoke. Its endophytic colonization was investigated microscopically using green fluorescent protein introduced by vector pHC60. The strain entered Jerusalem artichoke tissues through the root, and was localized in the roots and stems. The plant growth-promoting (PGP) effects of O. anthropi Mn1 were assessed in greenhouse as well as field trials with different nitrogen supplies. Only under moderate to ample nitrogen supply, could O. anthropi Mn1 promoted growth of host plant. The PGP effects of the strain were symbiotic nitrogen fixation, root morphological optimization and enhanced nutrient uptake. We hypothesize that the symbiotic interspecies interaction might be quorum sensing related.

Utilization of palm oil decanter cake as a novel substrate for biosurfactant production from a new and promising strain of Ochrobactrum anthropi 2/3.

A biosurfactant-producing bacterium, isolate 2/3, was isolated from mangrove sediment in the south of Thailand. It was evaluated as a potential biosurfactant producer. The highest biosurfactant production (4.52 g/l) was obtained when the cells were grown on a minimal salt medium containing 25 % (v/v) palm oil decanter cake and 1 % (w/v) commercial monosodium glutamate as carbon and nitrogen sources, respectively. After microbial cultivation at 30 °C in an optimized medium for 96 h, the biosurfactant produced was found to reduce the surface tension of pure water to 25.0 mN/m with critical micelle concentrations of 8.0 mg/l. The stability of the biosurfactant at different salinities, pH and temperature and also its emulsifying activity was investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pH and salt concentrations. The biosurfactant obtained was confirmed as a glycolipid type biosurfactant by using a biochemical test, fourier-transform infrared spectroscopy, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance polyaromatic hydrocarbons solubility.

Bioremediation of Cr(VI) and immobilization as Cr(III) by Ochrobactrum anthropi.

Bioremediation of Cr(VI) through reduction relies on the notion that the produced Cr(III) may be precipitated or efficiently immobilized. However, recent reports suggest that soluble organo-Cr(III) complexes are present in various chromate-reducing bacterial systems. This work was designed to explore the factors that affect the immobilization of Cr(III) in the Ochrobactrum anthropi system. X-ray absorption fine structure analysis on the cell debris clearly verified that coordination of Cr(III) occurs on the surfaces via the chelating coordination with carboxyl- and amido-functional groups. However, competitive coordination experiments of Cr(III) revealed that the small molecules such as amino acids and their derivatives or multicarboxyl compounds hold stronger coordination ability with Cr(III) than with cell debris. We speculate that it is the preferential coordination of Cr(III) to the soluble organic molecules in the bacterial culture medium that inhibits effective immobilization of Cr(III) on the cells. On the basis of this understanding, a strategy with two-step control of the medium was proposed, and this achieved successful immobilization of Cr(VI) as Cr(III) by O. anthropi and Planococcus citreus in 5-50 L pilot-scale experiments.

Microbial conversion of 5-sulfoisophthalic acid into 5-hydroxyisophthalic acid by Ochrobactrum anthropi S9.

5-Hydroxyisophthalic acid-producing microorganisms were isolated from enrichment cultures using 5-sulfoisophthalic acid as a sulfur source. One bacterium, Ochrobactrum anthropi S9, had the highest 5-sulfoisophthalic acid-degrading activity, and stoichiometrically formed 5-hydroxyisophthalic acid, a raw material for polymer synthesis. Under optimum culture conditions, 1.3 mM 5-hydroxyisophthalic acid accumulated in the medium by 60 h. The addition of Na(2)SO(4), L: -methionine or L: -cysteine at 2 mM inhibited the conversion of 5-sulfoisophthalic acid. O. anthropi S9 cells converted 5-sulfoisophthalic acid, benzenesulfonic acid, 3-sulfobenzoic acid, 4-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid and naphthalene-2-sulfonic acid into the corresponding hydroxylated compounds.

Evaluation of Ochrobactrum anthropi TRS-2 and its talc based formulation for enhancement of growth of tea plants and management of brown root rot disease.

AIM: To evaluate Ochrobactrum anthropi TRS-2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. METHODS AND RESULTS: Ochrobactrum anthropi TRS-2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV-18, T-17, HV-39, S-449, UP-3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, beta-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7.0 log(10) CFU g(-1), after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. CONCLUSION: Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.

[Sorption and microbial degradation of glyphosphate in soil suspensions].

Sorption and microbial destruction of glyphosphate, the active agent of the herbicide Groundbio, in suspensions of sod-podzolic and gray forest soils has been studied. According to the values of the adsorptive capacity (3560 and 8200 mg/kg, respectively) and the Freundlich constants (Kf, 15.6 and 18.7, respectively), these soils had a relatively high sorption capacity as related to the herbicide. Sorbed glyphosphate is represented by extractable and bound (inextractable) fractions. After long-term incubation of sterile suspensions, the ratio of these fractions reached 2 : 1 for sod-podzolic soil and 1 : 1 for gray forest soil. Inoculation of a native suspension of sod-podzolic soil with cells of a selected degrader strain Ochrobactum anthropi GPK 3 resulted in a 25.4% decrease in the total glyphosphate content (dissolved and extractable), whereas in a noninoculated suspension, the loss did not exceed 5.5%. The potential for the use of a selected bacterial strain for intensification of the glyphosphate destruction processes in soil systems is demonstrated for the first time.

Effect of glutaraldehyde treatment on stability of permeabilized Ochrobactrum anthropi SY509 in nitrate removal.

For practical application, the stability of permeabilized Ochrobactrum anthropi SY509 needs to be increased, as its half-life of enzymatic denitrification is only 90 days. As the cells become viable after permeabilization treatment, this can cause decreased activity in a long-term operation and induce breakage of the immobilization matrix. However, the organic solvent concentration causing zero cell viability was 50%, which is too high for industrial application. Thus, wholecell immobilization using glutaraldehyde was performed, and 0.1% (v/v) glutaraldehyde was determined as the optimum concentration to maintain activity and increase the half-life. It was also found that 0.1% (v/v) glutaraldehyde reacted with 41.9% of the total amine residues on the surface of the cells during the treatment. As a result, the half-life of the permeabilized cells was increased from 90 to 210 days by glutaraldehyde treatment after permeabilization, and no cell viability was detected.

A semi-quantitative GeLC-MS analysis of temporal proteome expression in the emerging nosocomial pathogen Ochrobactrum anthropi.

BACKGROUND: The alpha-Proteobacteria are capable of interaction with eukaryotic cells, with some members, such as Ochrobactrum anthropi, capable of acting as human pathogens. O. anthropi has been the cause of a growing number of hospital-acquired infections; however, little is known about its growth, physiology and metabolism. We used proteomics to investigate how protein expression of this organism changes with time during growth. RESULTS: This first gel-based liquid chromatography-mass spectrometry (GeLC-MS) temporal proteomic analysis of O. anthropi led to the positive identification of 131 proteins. These were functionally classified and physiochemically characterized. Utilizing the emPAI protocol to estimate protein abundance, we assigned molar concentrations to all proteins, and thus were able to identify 19 with significant changes in their expression. Pathway reconstruction led to the identification of a variety of central metabolic pathways, including nucleotide biosynthesis, fatty acid anabolism, glycolysis, TCA cycle and amino acid metabolism. In late phase growth we identified a number of gene products under the control of the oxyR regulon, which is induced in response to oxidative stress and whose protein products have been linked with pathogen survival in response to host immunity reactions. CONCLUSION: This study identified distinct proteomic profiles associated with specific growth points for O. anthropi, while the use of emPAI allowed semi-quantitative analyses of protein expression. It was possible to reconstruct central metabolic pathways and infer unique functional and adaptive processes associated with specific growth phases, thereby resulting in a deeper understanding of the physiology and metabolism of this emerging pathogenic bacterium.

The effects of salinity and other factors on nitrite reduction by Ochrobactrum anthropi 49187.

The nitrite reductase (NIR) gene was cloned from Ochrobactrum anthropi 49187 and found to contain an open reading frame of 1131 nucleotides, encoding a polypeptide of 376 amino acids. The O. anthropi NIR gene encodes a copper-type dissimilatory reductase based on sequence homology with other genes. The polypeptide product is predicted to form a trimeric holoenzyme of 37 kDa subunits based on molecular weight estimates of extracts in activity gels. Expression of the enzyme is up-regulated by nitrate, presumably through the intermediate nitrite, and its activity is influenced by inhibitors. Salinity enhances the activity of existing NIR enzyme, but appears to decrease the expression of new enzyme.

L-selective amidase with extremely broad substrate specificity from Ochrobactrum anthropi NCIMB 40321.

An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.

Expression of glutathione S-transferase and peptide methionine sulphoxide reductase in Ochrobactrum anthropi is correlated to the production of reactive oxygen species caused by aromatic substrates.

Peptide methionine sulphoxide reductase (MsrA) and glutathione S-transferases (GSTs) are considered as detoxification enzymes. In the xenobiotics-degrading bacterium Ochrobactrum anthropi the two enzymes are co-induced by toxic concentrations of aromatic substrates such as phenol and 4-chlorophenol. In aerobic organisms, degradation of aromatic substrates by mono- and dioxygenases leads to a generation of oxidative stress that causes the occurrence of reactive oxygen species (ROS). A capillary electrophoretic method, using the intracellular conversion of dihydrorhodamine-123 into rhodamine-123, was developed to measure the content of ROS in the bacteria. The presence of toxic concentrations of the aromatic substrate 4-chlorophenol, an inducer of GST and MsrA, leads to a significant increase in the production of ROS. These results strongly suggest that GST and MsrA enzymes are part of the bacterial defence mechanism against particular oxidative stress conditions. As oxidative stress is known to be present predominantly close to the cytoplasmic membrane, we investigated the subcellular distribution of both MsrA and GST enzymes in this bacterium grown in the presence of 4-chlorophenol. By Western blotting, MsrA and GST was assayed in the cytoplasm as well as in the periplasm. Moreover, immunolocalisation by colloidal gold immunoelectron microscopy identified the two proteins associated with the cell envelope.

The effects of NaCl and some heavy metals on the denitrification activity of Ochrobactrum anthropi.

Ochrobactrum anthropi is a well-known Gram-negative bacterium, with the ability to degrade atrazine, urea-formaldehyde and chlorophenols. Investigation were made of the nitrate and nitrite reduction capacities of the strain in succinate and glucose media, and the tolerance of its denitrification to NaCl and some heavy metals. Succinate proved to be a better carbon source to drive denitrification by O. anthropi. Batch fermentation studies in anaerobic succinate medium indicated reduction capacities of 85.4 +/- 9.1 and 48.6 +/- 5.2 mgh(-1)g(-1) dry cell for NO(3) (-) and NO(2) (-), respectively. The nitrite accumulation of the cells revealed that O. anthropi is a group C denitrifying bacterium. Its growth in DSM 1 broth containing NaCl up to 40 g l(-1) demonstrates that O. anthropi belongs in the group of moderately halophilic bacteria. Despite the fact that 42.5 g NaCl l(-1) caused 50% growth inhibition in DSM 1 broth, the cells in the stationary phase readily tolerated NaCl concentrations up to 100 g l(-1). Complete denitrification was achieved in test media containing 30 g NaCl l(-1) after 1 week and the nitrate reductase retained its activity up to 100 g NaCl l(-1). The cells were tolerant to Hg, Zn, Pb, Cu and Ni, and N(2) was producted at tolerated concentrations of the metal in the cases of Hg and Pb.

Population analysis of a binary bacterial culture by multi-parametric flow cytometry.

To study the degradation of a xenobiotic that requires a mixed culture it is essential to monitor the proportions and to control the population dynamics of the component strains. For these purposes fluorochromising techniques and multi-parametric flow cytometry were used to follow Rhodococcus erythropolis K2-3 and Ochrobactrum anthropi K2-14, both of which are needed to degrade 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Although the two strains can grow in constant proportions in mixed cultures on other substrates, 2,4-DB could not be degraded as a sole substrate in a continuous process and R. erythropolis K2-3 was clearly impaired in the binary mixture. Addition of a second, easily assimilable substrate (xylitol) in appropriate concentrations (empirically determined) helped this strain survive, and thus facilitated complete degradation of the xenobiotic. This combination of substrates was found to stabilise the growth of R. erythropolis K2-3 and, consequently promoted the action of O. anthropi K2-14. Thus, the two organisms became established in constant proportions in a continuous process until reaching steady state. Consequently, multiplication and cell division activities of the two components of the binary culture were high and reached similar values to those attained when they are grown in pure culture.

Physical map and gene survey of the Ochrobactrum anthropi genome using bacterial artificial chromosome contigs.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes research aimed at determining the genome structure of Ochrobactrum anthropi, an opportunistic human pathogen that has potential applications in biodegradation of hazardous organic compounds. A BAC library for O. anthropi was constructed that provides a 70-fold genome coverage based on an estimated genome size of 4.8 Mb. The library contains 3072 clones with an average insert size of 112 kb. High-density colony filters of the library were made, and a physical map of the genome was constructed using a hybridization without replacement strategy. In addition, 1536 BAC clones were fingerprinted with HindIII and analyzed using IMAGE and Fingerprint Contig software (FPC, Sanger Centre, U.K.). The FPC results supported the hybridization data, resulting in the formation of two major contigs representing the two major replicons of the O. anthropi genome. After determining a reduced tiling path, 138 BAC ends from the reduced tile were sequenced for a preliminary gene survey. A search of the public databases with the BLASTX algorithm resulted in 77 strong hits (E-value < 0.001), of which 89% showed similarity to a wide variety of prokaryotic genes. These results provide a contig-based physical map to assist the cloning of important genomic regions and the potential sequencing of the O. anthropi genome.