Cloning and expression of delta-1-pyrroline-5-carboxylate dehydrogenase in Escherichia coli DH5α improves phosphate solubilization.

A primary cDNA library of Penicillium oxalicum I1 was constructed using the switching mechanism at the 5' end of the RNA transcript (SMART) technique. A total of 106 clones showed halos in tricalcium phosphate (TCP) medium, and clone I-40 showed clear halos. The full-length cDNA of clone I-40 was 1355 bp with a complete open reading frame (ORF) of 1032 bp, encoding a protein of 343 amino acids. Multiple alignment analysis revealed a high degree of homology between the ORF of clone I-40 and delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH) of other fungi. The ORF expression vector was constructed and transformed into Escherichia coli DH5α. The transformant (ORF-1) with the P5CDH gene secreted organic acid in medium with TCP as the sole source of phosphate. Acetic acid and α-ketoglutarate were secreted in 4 and 24 h, respectively. ORF-1 decreased the pH of the medium from 6.62 to 3.45 and released soluble phosphate at 0.172 mg·mL(-1) in 28 h. Expression of the P. oxalicum I1 p5cdh gene in E. coli could enhance organic acid secretion and phosphate-solubilizing ability.

[Construction of cDNA library of Aspergillus niger H1 and screening of phosphate-dissolving related gene].

OBJECTIVE: To obtain phosphate-dissolving genes from cDNA library of Aspergillus niger H1. METHODS: The double-stranded cDNA was synthesized using switching mechanism at 5'end of RNA transcript technique and ligated to the vector pBluescript II SK (+). We transformed recombinant plasmid into E. coli HST08, resulting in a primary cDNA library. We screened clones with phosphate-dissolving activities on the insoluble phosphate medium and blasted the sequence in National Center for Biotechnology Information (NCBI). To study the phosphate dissolving mechanisms of the cloned gene, we analyzed the changes of the pH value, the soluble phosphate content and the production of organic acids in the insoluble phosphate liquid medium inoculated with the clones harboring the phosphate-dissolving gene. RESULTS: A cDNA library of A. niger H1 was successfully constructed. Titer tests showed that the content of constructed A. niger H1 cDNA library reached 5.65 x 10(6) cfu/mL, in which the percentage of recombinant clones was 99.15%. We screened 61 clones with phosphate-dissolving activities on the solid medium with insoluble phosphate. The corresponding gene in one of these clones was identified. The full length cDNA of clone H-54 was 839 bp, encoding a predicted protein with 180 amino acid residues. The expression of phosphate-dissolving gene in E. coli enhanced organic acids secretion and improved the phosphate solubilizing activity. Formic acid and acetic acid were found in 12 h, and malic acid and alpha-ketoglutarate were secreted in 24 h. The clone H-54 decreased the pH value of medium from 6.32 to 3.93 and released soluble phosphate up to 0.105 mg/mL in 36 h. CONCLUSION: We had obtained a phosphate-dissolving gene designated psgA from Aspergillus niger H1.

Adsorption and degradation of triazophos, chlorpyrifos and their main hydrolytic metabolites in paddy soil from Chaohu Lake, China.

Triazophos and chlorpyrifos are organophosphorus pesticides (OPs), and their primary hydrolytic metabolites are 1-phenyl-3-hydroxy-1,2,4-triazole (BZC) and 3,5,6-trichloro-2-pyridinol (TCP). In this study, the adsorption and degradation of triazophos, chlorpyrifos, BZC and TCP were investigated in paddy soil from Chaohu Lake, China. Adsorption tests demonstrated that the adsorption of these compounds to soils could be described by the Freundlich equation. Moreover, chlorpyrifos displayed the highest affinity for adsorption, followed by triazophos, BZC and TCP. Degradation of these compounds in non-sterile soil followed first-order exponential decay kinetics, and the half-life (t(1/2)) of these contaminants ranged from 8.40 to 44.34 d. Sterilization of soil decreased the degradation rate, indicating that microorganisms played a significant role in the degradation of these compounds. The values of t(1/2) and K(oc) were fitted to obtain models that could predict the leaching potential of the contaminants from soil. Compared to their parent compounds, BZC and TCP showed high potential for leaching into groundwater. The inoculation of OPs-degrading bacterium (Diaphorobacter sp. GS-1) removed 95.38%, 100% and 100% of triazophos, chlorpyrifos and BZC in paddy soil after 21 d, respectively. The pollution risk of triazophos, chlorpyrifos and BZC could be greatly decreased by inoculating soil with Diaphorobacter sp. GS-1, which decreases the t(1/2) of the contaminants.

[Screening and application of phosphate-dissolving microorganism suitable for corn production].

OBJECTIVE: To obtain phosphate-dissolving strains which can be suitable for corn production. METHODS: We screened phosphate-dissolving strains from soil samples. We conducted the plate experiment and soil experiment in laboratory, pot experiment in greenhouse and field experiment to test the insoluble phosphate dissolving ability of the strains. RESULTS: Fifteen strains of phosphate-dissolving fungi were isolated from soil samples, including 9 Penicillium oxalicum, 2 Penicillium variabile, 1 Penicillium aculeatum, 1 Trichoderma viride, 1 Aspergillus niger, and 1 Aspergillus nidulans. The 15 strains could obviously drop the pH in culture media. Five strains (Z15+, ZQ3, ZI1, Zh and Z30) reduced the pH value from 7.0 to below 2.0 in 18 h. The plate experiment and soil experiment in laboratory indicated that these 5 strains could propagate in plates and soil by using the root secretion of corn as sole carbon source. The pot experiment inoculating with the 5 strains was conducted in greenhouse by planting corn. The result demonstrated that the content of available phosphorous was increased remarkably with strains ZI1 and Zh. The content of available phosphorous at 49th day increased 28.05% and 37.04%, respectively, than initial content in strains ZI1 and Zh treatment. The dry matter yield of corn significantly increased 26.04% and 20.21% than the control, respectively. Then strains ZI1 and Zh were used in field experiment as microbial inoculums. The yield of corn treated with strain Zh increased 13.22% than that of the control, reaching 10873.05 kg/ha. However, the yield of corn treated with strain ZI1 presented no significant difference with the control. CONCLUSION: The phosphate-dissolving strain Zh obtained in this experiment was identified as Aspergillus nidulans, which is suitable for corn production.

[Isolation and identification of a novel phosphate-dissolving strain P21].

Phosphate-dissolving microorganisms can be applied for better use of insoluble phosphorus as fertilizer., A phosphate-dissolving strain P21 was isolated from soil samples in China. The isolate was identified as Erwinia herbicola var. ananas, based on its 16Sr DNA sequence and physiological characteristics. Its activity was measured in solid media as well as liquid media using different phosphate sources including tricalium phosphate, hydroxyapatite, ferric phosphate, aluminium phosphate, zinc phosphate, and rock phosphates. E. herbicola could strongly dissolve 1206.20 mg tricalium phosphate and 529.67 mg hydroxyapatite in per liter liquid media. The strain showed high phosphate-dissolving ability for rock phosphates from Jinning and Kunyang in Yunnan province, Yaan in Sichuan province and Jinping in Jiangsu province with the capacity of 6.64 mg, 78.46 mg, 67.07 mg and 65.24 mg soluble phosphate respectively per liter medium, whereas the phosphate-dissolving ability to the rest of the eight rock phosphates was weak. According to the experiments, the phosphate-dissolving ability of E. herbicola was specific to different rock phosphates, and phosphate-dissolving ability of E. herbicola was not directly related to pH reduction of liquid media.

An alkaline and surfactant-tolerant lipase from Trichoderma lentiforme ACCC30425 with high application potential in the detergent industry.

Alkaline lipases with adaptability to low temperatures and strong surfactant tolerance are favorable for application in the detergent industry. In the present study, a lipase-encoding gene, TllipA, was cloned from Trichoderma lentiforme ACCC30425 and expressed in Pichia pastoris GS115. The purified recombinant TlLipA was found to have optimal activities at 50 °C and pH 9.5 and retain stable over the pH range of 6.0-10.0 and 40 °C and below. When using esters of different lengths as substrates, TlLipA showed preference for the medium length p-nitrophenyl octanoate. In comparison to commercial lipases, TlLipA demonstrated higher tolerance to various surfactants (SDS, Tween 20, and Triton X100) and retained more activities after incubation with Triton X100 for up to 24 h. These favorable characteristics make TlLipA prospective as an additive in the detergent industry.

Transformation of inorganic P fractions of soil and plant growth promotion by phosphate-solubilizing ability of Penicillium oxalicum I1.

The solubilization of tricalcium phosphate is often considered as the standard for screening of most phosphate-solubilizing microorganisms (PSMs). However, usually the effect of large-scale application of PSM on the promotion of crop growth varies. This study presents an efficient method for screening and testing phosphate-solubilizing fungus that enhance plant growth. A fungus Penicillium oxalicum I1 (P-I1) was isolated and identified that had high ability of phosphate-solubilization and could utilize maize root exudates as sources, and propagate well in vitro and in soil. P-I1 excreted oxalic acid and reached 593.9 µg/ml, and the pH value was decreased from 6.90 to 1.65 in 26 h. The amount of P-I1 increased by 48-fold in 28 d and was maintained for 49 d in soil. PSM showed selectivity on the transformation of the different forms of phosphorus, a wide range of insoluble phosphates, such as Ca8H2(PO4)6·5H2O, AlPO4, FePO4, and Ca10(PO4)6(OH)2, were converted to soluble CaHPO4in soil, and CaHPO4was also inhibited from being converted into insoluble phosphate by P-I1. The Ca2-P content reached 27.11 µg/g soil on day 28 at 20°C, which increased by 110.32%, and plant growth promotion was tested and verified, the results showed that maize yield increased remarkably than control after inoculated P-I1, maize yield increased maximum by 14.47%. The data presented that P-I1 appear attractive for exploring their plant growth-promoting activity and potential field application.

[Isolation, identification of a pyrene-degrading strain Mycobacterium sp. M11 and its degrading characteristics].

A bacterial strain M11 using phenanthrene, anthracene, fluorethene,pyrene as a sole carbon and energy sources was isolated from soils contaminated with polycyclic aromatic hydrocarbons. Strain M11 was identified as Mycobacterium sp. according to the results of morphology, physiology and the phylogenetical analyses of 16S rDNA sequence. The degradation rate of pyrene (at levels of 50 mg/L, 100 mg/L and 200 mg/L) by strain M11 was 76.9%, 91.8% and 79.23% within 16 d in broth assay. Mycobacterium sp. M11 showed a strong ability to grow from pH 5 to pH 9 in liquid minimal medium containing pyrene 100 mg/L. Mycobacterium sp. M11 possessed aromatic-ring dioxygenase genes, which are highly homologous to the known nidAB genes from pyrene-degrading Mycobacteria.

[Screening of two phenanthrene-utilizing and high-effective phosphorus-accumulating bacteria and their effects on phosphorus-accumulating characteristics].

In this study, two phenanthrene-utilizing and high-effective P-accumulating bacteria Y11 and Y4-2 were isolated from sludge samples of Taihu Lake using plate culture and blue-colored colonies methods. Strains Y11 and Y4-2 were identified as Acinetobacter sp. according to the results of morphology, physiology and the phylogenetical analyses of 16S rDNA sequences. The two strains showed high effective P-accumulating ability in plate and broth cultures. Y11 could grow well from 10 degrees C to 35 degrees C and pH from 6 to 9, Y4-2 could grow well from 10 degrees C to 35 degrees C and pH from 6 to 8. Phenanthrene could be used as sole carbon and energy sources. In our experiment, high phosphorus concentration in broth culture had no negative effect on the growth of strain Y11, however the growth of strain Y4-2 was slightly affected. Under cultivation condition of 30 degrees C, 170 r/min, 1% inoculation (D600 = 0.4), the accumulation of phosphorus was various by strains and cultivation concentration of phosphorus as well: In 2 mg/L phosphorus concentration medium, the highest phosphorus-accumulation ratio of strains Y11 and Y4-2 were 96.13% and 94.65%, the phosphorus concentration decreased from 2 mg/L to 0.08 mg/L and 0.11 mg/L, respectively. In 5 mg/L phosphorus concentration medium, the highest phosphorus-accumulation ratio of strains Y11 and Y4-2 were 95.94% and 71.19%, the phosphorus concentration decreased from 5 mg/L to 0.20 mg/L and 1.44 mg/L, respectively. In 8 mg/L phosphorus concentration medium, the highest phosphorus-accumulation ratio of strains Y11 and Y4-2 were 71.24% and 47.81%, the phosphorus concentration decreased from 8 mg/L to 2.30 mg/L and 4.18 mg/L, respectively. For the P removal of Yunnan Dianchi water sample containing 1.01 mg/L phosphorus, Y11 and Y4-2 were successful to decrease the phosphorus concentration from 1.01 mg/L to 0.06 mg/L in 6 h and 48 h, respectively. The results indicated that Acinetobacter sp.Y11 and Y4-2 possess high ability of phosphorus removal and suitability to various water environments. Y11 strain adapt to all kinds of eutrophic waterbody even polluted with phenanthrene, Y4-2 adapt to pH < or = 8.0 eutrophic waterbody with phenanthrene pollution.