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Objective: In order to provide more natural antifouling compounds, marine bacterium Pseudomonas putida isolated from the sponge Haliclona sp. was explored to test its anti-diatom compounds., Methods: The strain was identified by colonial morphology, scanning electron microscope (SEM) and 16S rDNA sequence analysis. The separation procedure was guided by bioactive (Anti-diatom) and chemical (TLC, DAD-HPLCand 1H NMR) analysis, and their structures were elucidated by spectrographic techniques. The anti-diatom activity of all purified compounds was assayed., Results: Strain 272 isolated from the sponge Haliclona sp. was identified as Pseudomonas putida. Six diketopiperazine compounds were isolated from the culture of this strain and their structures were determined as cyclo(Leu-Pro) (1), cyclo (Leu-Ala) (2), cyclo(Phe-Ala) (3), cyclo(Val-Tyr) (4), cyclo(Ala-Tyr) (5), cyclo(Ala-Trp) (6); Compounds 3 and 6 displayed significant anti-diatom activity with the inhibitory rate of 50% and 85% at the concentration of 50 microg/mL, respectively., Conclusion: The anti-diatom compounds isolated from marine bacterium Pseudomonas putida were cyclo (Phe-Ala) and cyclo (Ala-Trp).

Two aerobic denitrifying bacteria were isolated from the sludge of municipal wastewater treatment plant by enrichment, preliminary screening with BTB culture medium and the denitrification potential test and identified through 16S rDNA sequence analysis. Then they were bioaugmented to oligotrophic biofilm system respectively, aiming to enhance the denitrification capacity. The two strains were identified as Pseudomonas aeruginosa and Pseudomonas putida respectively. They could remove 78% or 82% of the total nitrogen in the simulate wastewater when existed alone. And the total nitrogen removal efficiency of the biofilm system reached 68% and 64% after bioaugmentation with two trains, increased by 47% and 43% compared to the control, respectively. Meanwhile, the ammonia nitrogen was nearly 100% removed. It can be concluded that two aerobic denitrifiers can enhance the denitrification of biofilm system significantly under oligotrophic conditions and will not inhibit the nitrification process, therefore can help biofilm system achieve simultaneous nitrification and denitrification.

To obtain efficient halotolerant petroleum-degrading bacteria, 39 bacteria strains were isolated from 30 petroleum contaminated saline soil samples in Yellow River Delta, an important base of petroleum production in China. One bacterium (strain BM38) was found to efficiently degrade crude oil in highly saline environments based on a series of liquid and soil incubation experiments. According to its morphology, physiochemical characteristics and 16S rDNA sequence analysis, this strain was identified as Pseudomonas putida. Moreover, a series of liquid incubation experiments were conducted to investigate its characteristics such as halotolerance, biosurfactants production and degrading efficiency for various hydrocarbons. The salt resistance test demonstrated that strain BM38 grew well at NaCl concentrations ranging from 0.5% to 6.0%. Petroleum degradation experiments showed that strain BM38 could degrade 73.5% crude oil after 7 days in a liquid culture medium containing 1.0% NaCl and remove more than 40% of total petroleum hydrocarbons after 40 days in the soil with 0.22% and 0.61% of salinity, these results proved that the strain was effective in removing petroleum hydrocarbons. Strain BM38 could produce a bioemulsifier in a liquid culture medium. The NaCl concentration had the significant effect on the EI24 of fermentation broth, which decreased sharply if the NaCl concentration was greater than 1.0%. However, the EI24 of BM38 was still quite high in the presence of 2.0% of NaCl, and the value was 61.0%. Furthermore, this strain was also able to grow in mineral liquid media amended with hexadecane, toluene, phenanthrene, isooctane and cyclohexane as the sole carbon sources. Among these hydracarbons, strain BM38 showed relatively high ability in degrading n-alkanes and aromatic hydracarbons. The results indicated that strain BM38 had potential for application in bioremediation of petroleum-contaminated saline soil.

One bacterial strain capable to degrade and metabolize formaldehyde as a sole carbon source was isolated from soil. Based on the results of standard morphological identification, physiological and biochemical characters, and 16S rDNA sequence analysis, the strain was identified as Pseudomonas putida. After single factor test and orthogonal test, the optimal condition for formaldehyde degradation was determined as the follows: peptone 1.2 g/L, KH2PO4 4 g/L, K2HPO4 3 g/L, MgSO4 x 7H2O 0.2 g/L, trace elements solution 0.1 mL/L, temperature 30 degrees C, pH 8. Under the optimal conditions, the strain tolerance of original formaldehyde concentration was up to 6 g/L and 86% of formaldehyde was consumed after 54 h. It completely consumed 5 g/L formaldehyde after 46 h and degraded 100% of 4 g/L formaldehyde after 35 h.

Objective: We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria that produced ACC (1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why they can promote the growth of plants., Methods: Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity., Results: In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99%. Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/microg., Conclusion: A strain producing ACC deaminase was identified and tested.

A novel strain producing an enantioselective lipolytic enzyme was isolated from soil samples, and identified as Pseudomonas putida NH33. A genomic library of P. putida NH33 was constructed and screened for esterase activity in E. coli. One positive clone was isolated, and subsequent analyses of the plasmid by restriction mapping revealed a 4.7kb DNA fragment carrying esterase gene. The nucleotide sequence of the DNA was found to contain an open reading frame of 1142 nucleotides encoding esterase of 381 amino acid residues and designated PPEst. The primary structure of the esterase exhibited 35%-40% homology to those of related enzymes from various sources and 80%-90% homology to esterases from the genus Pseudomonas. Amino acid sequence deduced from the nucleotide sequence contains of the consensus active site sequence, GXSXG, of serine esterase. The PPEst fragments were cloned into the expression vector pET-22b( + ) and transformed into E. coli BL21 (DE3), and the recombinant protein fused with 6 x His at its C-terminus was purified to homogeneity by a single immobilized metal ion affinity chromatographic step. The molecular mass of the esterase was determined to be approximately 42kDa by SDS-PAGE. The purified enzyme could convert ethyl esters of 2-arylpropanoic acid to S-isomer of 2-arylpropanoic acids with an optical purity of > 99%. The result suggests that this esterase is excellent biocatalyst for synthesis of chiral pharmaceuticals. The enzyme is an novel esterase, and its nucleotide sequence has been submitted to GenBank under accession number AY896293.

The current technology for microbial hydroxylation of nicotinic acid is a procedure of resting cells transformation. Here it was reported that non-degradation of 6-hydroxynicotinic acid during hydroxylation of nicotinic acid by resting cells was due to existing of nicotinic acid in the transformation mixture, not the death of cells. Therefore it was attempted that the induced cultivation of Pseudomonas putida NA-1 for hydroxylation activity by nicotinic acid was used for accumulation of 6-hydroxynicotinic acid to establish a new technology, a combined technology of growing culture transformation and resting cells transformation. In the combination technology, when 1% of nicotinic acid was kept in cultivation medium by supplement of nicotinic acid at intervals during cultivation of Pseudomonas putida NA-1, the growing culture could consistently hydroxylate nicotinic acid without degradation of its product which was accumulated linearly in the cultivation medium with progress of cultivation; the resting cells obtained from cultivation broth after growing culture transformation still processed a very high hydroxylation activity and could be used to next resting transformation of nicotinic acid. By using of the combined technology, the inducer was saved and the production yield of 6-hydroxynicotinic acid increased above 65%.

The salinity tolerance characteristic of Pseudomonas putida DLL-1 and the environmental factors affecting the salinity tolerance of DLL-1 were investigated. The result showed that the enrichment of culture medium influenced the salt tolerance of DLL-1, in complete medium DLL-1 could survive higher salinities than in chemically defined medium. When inoculated to complete medium with 1mol/L NaCl, the least initial biomass guarantee DLL-1 surviving was 1/100(V/V) of the medium volumes. But when inoculated to chemically defined medium with 1mol/L NaCl, the least initial biomass guarantee DLL-1 surviving was 1/10(V/V) of the medium volumes. The effects of glycine betaine exogenously supplied on the salinity tolerance of DLL-1 and its osmo protection mechanisms were also studied. The results indicated that the glycine betaine present externally could promote the growth of DLL-1 under high salinity. 10mg/L of exogenous glycine betaine was sufficient to promote the growth condition of DLL-1 cells under high salinities. 150mg/L glycine betaine could support DLL-1 cells grow in chemically defined medium with 1.2mol/L NaCl. The presence of glycine betaine in chemically defined medium could reduce significantly the lag time and generation time and increase the final biomass of DLL-1 under salt stress. Compared with the control without exogenous glycine betaine, the lag time of the treatment with exogenous glycine betaine could be reduced from 24h to 6h, and the generation time from 60min to 35.7min, the final OD610 value of culture increased from 1.29 to 1.57. Under osmotic stress, DLL-1 cells could synthesis glycine betaine, trehalose and free amino acid as the main compatible solute. When exogenously supplied, DLL-1 cells accumulated exogenous glycine betaine rapidly from medium to balance the extracellular osmolality instead of the synthesis of compatible solutes.

In this paper, the aseptic eggs of Bursaphelenchus xylophilus were obtained after treated with 30% H202, and cultured with Pinus thunbergii callus. Ten B. xylophilus-carrying bacterial strains directly isolated from diseased P. thugbergii and P. massoniana in six epidemic provinces i.e., GcM6-2A Pseudomonas putida, GcM6-1A P. putida, ZpB1-2A P. putida, HeM2A Pseudomonas sp., HeM1A Pseudomonas sp., HeM142B Pseudomonas sp., GcM1-3A P. cepacia and HM3 Pantoeu sp., ZpB4-2B Staphylococcus sciuri and ZpB2-3A Enterobacter amnigenus, were collected, and the effects of axenic B. xylophilus (ABx) on their propagation were studied. The results showed that pine wood nematode (PWN) promoted the propagation of 7 bacterial strains in Pseudomonas and 1 bacterial strain in Pantoeu sp., including Pseudomonas putida, P. putida, P. putida , Pseudomonas sp., Pseudomonas sp., Pseudomonas sp., P. cepacia and Pantoeu sp., but inhibited Staphylococcus sciuri and Enterobacter amnigenus, which could explain the phenomenon that Pseudomonas was the prevailing genus of the bacteria carried by PWN, and might provide essential nutrition to the bacteria. The close relationship between PWN and bacterial strains in Pseudomonas might account for the pine wood nematode disease.

Strain screening was conducted on a large scale in order to develop a biotransforming process for 6-hydroxynicotinic acid production. A strain named as NA-1 was isolated from soil which could transform nicotinic acid into a new product showed by HPLC. The new product was identified as 6-hydroxynicotinic acid by MS and 1H-NMR. The morphology, physiological and biochemical characteristics of strain NA-1 were studied and showed that characteristics of strain NA-1 were essentially consistent with Pseudomonas putida. The analysis of 16S rDNA sequence from strain NA-1 suggested that strain NA-1 was clustered together with P. putida in phylogenetic tree and the sequence identity between stain NA-1 and Pseudomonas putida was 99%. So strain NA-1 was identified to P. putida.