Isolation, Identification, and Characterization of an Efficient Siderophore Producing Bacterium From Heavy Metal Contaminated Soil.

An efficient siderophore producing strain, YQ9, was isolated from heavy metal contaminated soil and identified as Burkholderia vietnamiensis. To the best of our known, the strain owns the highest siderophore producing capacity among genus Burkholderia with 96.6% siderophore unit. Moreover, B. vietnamiensis YQ9 has good adaptability to different pH values, temperatures, NaCl, and Fe3+ concentrations. In addition, the minimum inhibitory concentration (MIC) of heavy metals and antibiotics were also tested. It was found that the MIC values of strain YQ9 to several major soil heavy metal pollutants, such as Pb2+, Zn2+, Cu2+, and Cd2+ reached 3000, 5000, 4500, and 1000 µmol·L-1, respectively. And YQ9 was sensitive to 4 of 8 test antibiotics, including rifampicin, kanamycin, doxycycline hyclate, and gentamicin (25, 25, 30, and 30 µg·mL-1, respectively). Strain YQ9 also owns the ability to produce indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and dissolve phosphorus. The IAA production capacity was 6.93 mg·L-1, the ACC deaminase activity was 8.71 µmol α-KA·(h·mg)-1, and the phosphorus dissolving capacity of YQ9 was 104.05 mg·L-1. The traits were excellent, and the strain was qualified as a candidate for microbial reinforcement of phytoremediation in soil contaminated by heavy metals.

New protocol for the R134a cryogen spray cooling assisted 1064-nm laser lipolysis.

Laser lipolysis is a promising body contouring technology. However, the skin tissue could be thermally damaged owing to the laser energy absorption by water, which limits the lipolysis efficiency. To protect skin tissue and improve the lipolysis effect, cryogen spray cooling is introduced and synergized with the pulsed laser irradiation aiming to propose a new therapy protocol. By simulating heat conduction in the skin after spray cooling assisted laser lipolysis, the temperature distribution in the skin tissue was obtained to analyze the tissue damage by the Arrhenius integral. After parameter optimization according to the damage threshold of skin and adipose tissue, a new protocol with high laser intensity and short time was proposed including 150-ms R134a spray cooling with spray distance of 30 mm, and 100 ms 1064 nm laser irradiation with energy density of 20 J/cm2, with a relaxation for 9.75 s. This cycle of 10 s can be repeated 90 to 150 times for a total of 15 to 25 min. Compared with previous treatment procedure, new protocol can increase the fat dissolution depth from 2 to 4.5 mm beneath the dermis with same order laser fluence.

Isolation, characterization, and evaluation of a high-siderophore-yielding bacterium from heavy metal-contaminated soil.

Heavy metal-resistant siderophore-producing bacteria (SPB) with plant growth-promoting traits can assist in phytoremediation of heavy metal-contaminated soil. We isolated siderophore-producing bacteria from Pb and Zn mine soil in Shangyu, Zhejiang, China. The isolate with the highest siderophore production, strain SX9, was identified as Burkholderia sp. Burkholderia sp. SX9 produced catecholate-type siderophore, with the highest production at a pH range of 6.0 to 8.0, a temperature range of 20 to 30 °C and NaCl concentration below 2%. Siderophore production was highest without Fe3+ and became gradually lower with increasing Fe3+ concentration. Minimal inhibitory concentrations (MIC) of Pb2+, Zn2+, Cu2+, and Cd2+ were 4000, 22000, 5000, and 2000 µmol L-1, respectively. The strain had a strong metal solubilization ability: the contents of Cu2+, Zn2+, and Cd2+ in the supernatant were 47.4%, 133.0%, and 35.4% higher, respectively, in strain SX9-inoculated cultures than in the not inoculated controls. The siderophore produced by strain SX9 could combine with Fe3+, Zn2+, and Cd2+ with good effectiveness. The plant growth-promoting traits of the strain included indole acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, and phosphate solubilization capability. Compared to the uninoculated growth medium and SX9 culture supernatant, the germination rate of Lolium perenne seeds was higher when inoculated with strain SX9 culture. In the experiment of seed germination, adding bacterial culture or supernatant could alleviate the toxicity of heavy metals to L. perenne seed germination. Under Cu2+ and Zn2+ stress, strain SX9 promoted the germination rate. Taken together, Burkholderia sp. SX9 had properties beneficial in the microbial enhancement of phytoremediation of soil contaminated with heavy metals.

Detection of N2O-producing fungi in environment using nitrite reductase gene (nirK)-targeting primers.

Fungal denitrification has been increasingly investigated, but its community ecology is poorly understood due to the lack of culture-independent tools. In this work, four pairs of nirK-targeting primers were designed and evaluated for primer specificity and efficiency using thirty N2O-producing fungal cultures and an agricultural soil. All primers amplified nirK from fungi and soil, but their efficiency and specificity were different. A primer set, FnirK_F3/R2 amplified ∼80 % of tested fungi, including Aspergillus, Fusarium, Penicillium, and Trichoderma, as compared to ∼40-70 % for other three primers. The nirK fragments of fungal and soil DNA amplified by FnirK_F3/R2 were phylogenetically related to denitrifying fungi in the orders Eurotiales, Hypocreales, and Sordariales; and clone sequences were also distributed in the clusters of Chaetomium, Metarhizium, and Myceliophthora that were uncultured from soil in our previous work. This proved the wide-range capability of primers for amplifying diverse denitrifying fungi from environment. However, our primers and recently-developed other primers amplified bacterial nirK from soil and this co-amplification of fungal and bacterial nirK was theoretically discussed. The FnirK_F3/R2 was further compared with published primers; results from clone libraries demonstrated that FnirK_F3/R2 was more specifically targeted on fungi and had broader taxonomical coverage than some others.

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.

Cloning of a dibutyl phthalate hydrolase gene from Acinetobacter sp. strain M673 and functional analysis of its expression product in Escherichia coli.

A dibutyl phthalate (DBP) transforming bacterium, strain M673, was isolated and identified as Acinetobacter sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl, dipentyl, dihexyl, di(2-ethylhexyl), di-n-octyl, and dinonyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. During growth in Luria-Bertani medium, M673 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. One DBP hydrolase gene containing an open reading frame of 1,095 bp was screened from a genomic library, and its expression product hydrolyzed various dialkyl phthalates to the corresponding monoalkyl phthalates.

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.

[Application of biogas fermentation residue in Ziziphus jujuba cultivation].

With field experiment, this paper studied the effects of applying biogas fermentation residue (dregs and slurry) on jujube growth, its fruit quality, and soil fertility. The results showed that biogas fermentation residue could enhance the disease-resistance of jujube plant and its fruit, and improve fruit quality and soil fertility. Compared with applying chemical fertilizers (the control), biogas fermentation residue increased the contents of jujube fruit coarse fiber, vitamin C, amino acids, Fe and P by 27.69%, 24.85%, 19.81%, 10.89% and 5.26%, and of soil organic carbon, total nitrogen and mineral nitrogen by 42.65%, 37.61% and 35.26%, respectively. The soil pH was decreased from 8.75 to 8.21. Biogas fermentation residue could also increase the amount of soil microorganisms. The microbial biomass-C and biomass-N were 59.44% and 56.06% higher than the control, respectively. It was suggested that biogas fermentation residue application could bring better economic and environmental benefits for Z. jujuba cultivation, and also, provide a new approach for no-pollution production of jujube.