Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp: ONBA-17 and deduction on its metabolic pathway

A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation.

Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp. ONBA-17 and deduction on its metabolic pathway.

A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation.

Phenotypic and phylogenetic characterization of an abamectin-degrading bacterial strain isolated from a citrus orchard.

Bacterial strain GB-01 was isolated from abamectin-contaminated soils by continuous enrichment culture. The preliminary identification of strain GB-01 as a Burkholderia species was based mainly on simple biochemical and substrate utilization tests; however, these tests alone cannot accurately differentiate all the species within the genus Burkholderia. The strain GB-01 was subjected to taxonomic analysis through a polyphasic approach, in which phenotypic, genotypic, and phylogenetic information was gathered to conclude the classification of this microbe. Phenotypic information comes from basic bacteriological tests and substrate utilization patterns using the Biolog GN2 MicroPlating system and automated miniature biochemical test kits, i.e. API 20 NE, ID 32 GN and API 50 CH, as well as analyzing the whole cell fatty acid profile. Genotypic information was gathered from whole genome DNA base composition (G+C mol%), and DNA-DNA hybridization with its closest species, while phylogenetic information was collected from the comparative analysis of 16S rRNA and recA gene sequences. The results of polyphasic analysis concluded that strain GB-01 is an atypical strain of the Burkholderia diffusa species.

Molecular cloning and characterization of S-adenosylmethionine synthetase gene from Lycoris radiata.

S-adenosylmethionine (SAM) synthetase catalyzes the synthesis of SAM, a molecule important for all cellular organisms. It is also considered to play an important role in salt tolerance of plants. Here, we cloned a Lycoris radiata (L. radiata) SAM synthetase gene LrSAMS to determine its biological function. The gene encodes a protein of 401 amino acids with a calculated molecular weight of 43.9 kDa. Amino acid sequence analysis of the deduced protein LrSAMS reveals high sequence identity to SAM synthetases from other organisms, such as Arabidopsis thaliana and Oryza sativa. The deduced LrSAMS protein contains conserved amino acids residues and sequences motifs that closely related to the function of SAM synthetase. Otherwise, the transcript levels of LrSAMS were significantly induced by NaCl treatment in L. radiata leaves, which implied that LrSAMS might play an important role in tolerance to salt stress in L.radiata. Complete ORF of LrSAMS was inserted into expression vector pET-29a(+) and transformed into Escherichia coli BL21 (DE3). The difference between the growth curve of the transgenic strain and control strain with blank vector showed that over-expressing LrSAMS could provide growth advantage to the engineered strain in high salt concentration.

Studies revealing bioremediation potential of the strain Burkholderia sp. GB-01 for abamectin contaminated soils.

Burkholderia sp. GB-01 strain was used to study different factors affecting its growth for inoculum production and then evaluated for abamectin degradation in soil for optimization under various conditions. The efficiency of abamectin degradation in soil by strain GB-01 was seen to be dependent on soil pH, temperature, initial abamectin concentration, and inoculum size along with inoculation frequency. Induction studies showed that abamectin depletion was faster when degrading cells were induced by pre-exposure to abamectin. Experiments performed with varying concentrations (2-160 mg Kg(-1)) of abamectin-spiked soils showed that strain GB-01 could effectively degrade abamectin over the range of 2-40 mg Kg(-1). The doses used were higher than the recommended dose for an agricultural application of abamectin, taking in account the over-use or spill situations. A cell density of approximately 10(8) viable cells g(-1) dry weight of soil was found to be suitable for bioremediation over a temperature range of 30-35°C and soil pH 7.5-8.5. This is the first report on bacterial degradation of abamectin in soil by a Burkholderia species, and our results indicated that this bacterium may be useful for efficient removal of abamectin from contaminated soils.

Isolation and characterization of an endosulfan-degrading strain, Stenotrophomonas sp. LD-6, and its potential in soil bioremediation.

Aerobic bacteria degrading endosulfan were isolated from contaminated sludge. One of the isolates, LD-6, was identified as Stenotrophomonas sp. The bacterium could utilize endosulfan as the sole source of carbon and sulfur. 100 mg/l endosulfan was completely degraded within 10 days, and endosulfan diol and endosulfan ether were detected as major metabolites with a slight decrease in culture pH. The results indicated that Stenotrophomonas. sp. LD-6 might degrade endosulfan by a non-oxidative pathway. Biodegradation of both isomers was relatively better at a temperature range of 25-35 degrees C, with a maximum at 30 degrees C. In addition, cell crude extract of strain LD-6 could metabolize endosulfan rapidly, and degradative enzymes were intracellular distributed and constitutively expressed. Besides, application of the strain was found to promote the removal of endosulfan in soil. This study might help with the future research in better understanding of the biodegradation.

Biotreatment of o-nitrobenzaldehyde manufacturing wastewater and changes in activated sludge flocs in a sequencing batch reactor.

o-Nitrobenzaldehyde manufacturing wastewater is characterized for being highly saline, with its TN content and dissolved organic concentrations giving rise to high COD loads. A sequencing batch reactor was established to investigate the effects of major processing variables, such as SRT and HRT, on system performance. The optimal COD (86%) and TN (40.9%) removal efficiencies were obtained at 16d (SRT) and 12 h (HRT). Design equations were developed by applying experimental data. Changes in sludge flocs were studied using a combination of methods including: chemical analysis; fluorescence in situ hybridization (FISH); and denaturing gradient gel electrophoresis profile analysis of 16S rRNA genes. Dramatic changes occurred during adaptation and ß-Proteobacteria was found to be the most prevalent population. Besides, some species affiliated with α-subclasses of Proteobacteria and Cytophaga-Flavobacterium-Bacteroides (CFB) group were also enriched. This study may help with future research in providing a better understanding of the activated sludge biotreatment.

Bioaugmentation of a sequencing batch reactor with Pseudomonas putida ONBA-17, and its impact on reactor bacterial communities.

This study demonstrates the feasibility of using Pseudomonas putida ONBA-17 to bioaugment a sequencing batch reactor (SBR) treating o-nitrobenzaldehyde (ONBA) synthetic wastewater. To monitor its survival, the strain was chromosomally marked with gfp gene. After a transient adaptation, almost 100% degradation of ONBA was obtained within 8 days as compared with 23.47% of the non-inoculated control. The bioaugmented reactor has a better chemical oxygen demand (COD) removal performance (96.28%) than that (79.26%) of the control. The bioaugmentation not only enhanced the removal capability of target compound, but shortened system start-up time. After the increase in ONBA load, performance fluctuation of two reactors was observed, and the final treating effects of them were comparable. What is more, denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes via a combination of pattern comparison and sequence phylogenetic analysis was performed to uncover changes in sludge microbial communities. Only the members of alpha, beta and gamma subdivisions of Proteobacteria were identified. To isolate ONBA-degrading relevant microorganisms, spread plate was used and four bacterial strains were obtained. Subsequent systematic studies on these bacteria characterized their traits which to some extent explained why such bacteria could be kept in the system. This study will help future research in better understanding of the bioreactor bioaugmentation.