To reveal the impact of mining on bacterial ecology around mining area,bacterial community and geochemical characteristics about Dabaoshan Mine(Guangdong Province,China)were studied.By amplified ribosomal DNA restrict...To reveal the impact of mining on bacterial ecology around mining area,bacterial community and geochemical characteristics about Dabaoshan Mine(Guangdong Province,China)were studied.By amplified ribosomal DNA restriction analysis and phylogenetic analysis,it is found that mining pollution greatly impacts the bacterial ecology and makes the habitat type of polluted environments close to acid mine drainage(AMD)ecology.The polluted environment is acidified so greatly that neutrophil and alkaliphilic microbes are massively dead and decomposed.It provided organic matters that can make Acidiphilium sp.rapidly grow and become the most bacterial species in this niche.Furthermore,Acidithiobacillus ferrooxidans and Leptospirillum sp.are also present in this niche.The amount of Leptospirillum sp.is far more than that of Acidithiobacillus ferrooxidans,which indicates that the concentration of toxic ions is very high.The conclusions of biogeochemical analysis and microbiological monitor are identical. Moreover,because the growth of Acidithiobacillus ferrooxidans and Leptospirillum sp.depends on ferrous iron or inorganic redox sulfur compounds which can be supplied by continual AMD,their presence indicates that AMD still flows into the site.And the area is closer to the outfalls of AMD,their biomasses would be more.So the distinction of their biomasses among different areas can help us to find the effluent route of AMD.展开更多
In order to solve the citrus peel resource waste problem protopectinase-overproducing strain CD-01 for pectin production and minimize the drawbacks of chemical extraction of pectin, a was isolated from a pit soil dump...In order to solve the citrus peel resource waste problem protopectinase-overproducing strain CD-01 for pectin production and minimize the drawbacks of chemical extraction of pectin, a was isolated from a pit soil dumped with perished orange in Changde City, Hunan Province of China. The strain CD-01 had the same morphology and 28S rRNA gene sequence （FJ184995） as that of Aspergillus niger （ATCC 64028）. It was thus identified and named as Aspergillus niger CD-01. The fermentation condition was optimized based on L9（34） orthogonal experimental design and the variances analyses. The results show that the optimal condition for producing pectin is as follows： time 36 h, temperature 35 ℃, pH 5, and urea as the nitrogen source. Under this condition, the pectin yield can reach up to 24.5%. This shows a great potential of Aspergillus niger CD-01 in pectin extraction from citrus.展开更多
Eleven acid mine drainage （AMD） samples were obtained from southeast of China for the analysis of the microbial communities diversity, and the relationship with geochemical variables and spatial distance by using a ...Eleven acid mine drainage （AMD） samples were obtained from southeast of China for the analysis of the microbial communities diversity, and the relationship with geochemical variables and spatial distance by using a culture-independent 16S rDNA gene phylogenetic analysis approach and multivariate analysis respectively. The principle component analysis （PCA） of geochemical variables shows that eleven AMDs can be clustered into two groups, relative high and low metal rich （RHMR and RLMR） AMDs. Total 1691 clone sequences are obtained and the detrended correspondence analysis （DCA） of operational taxonomic units （OTUs） shows that, ~,-Proteobacteria, Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes and Nitrospirae are dominant species in RHMR AMDs. In contrast, a-Proteobacteria, fl-Proteobacteria, Planctomycetes and Bacteriodetes are dominant species in RLMR AMD. Results also show that high-abundance putative iron-oxidizing and only putative sulfur-oxidizing microorganisms are found in RHMR AMD. Multivariate analysis shows that both geochemical variables （r=0.429 3, P=-0.037 7） and spatial distance （r=0.321 3, P=-0.018 1） are significantly positively correlated with microbial community and pH, Mg, Fe, S, Cu and Ca are key geochemistry factors in shaping microbial community. Variance partitioning analysis shows that geochemical variables and spatial distance can explain most （92%） of the variation.展开更多
Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse re...Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse reflectance Fourier transform infrared spectroscopy （FT-IR） and sulfur K-edge X-ray absorption near edge structure spectroscopy （XANES）. The results show that the presence of S. metallicus effectively enhances the dissolution of the mineral. The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching. The pyrite in the concentrate facilitates zinc dissolution in the early stage, but has hindrance role in the late stage for the formation of jarosite. Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process, and the accumulation ofjarosite is mainly responsible for the decline of leaching efficiency.展开更多
In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A,...In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.展开更多
A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was...A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens （99.54%）. After that, an optimization of culture conditions was carried out for the improvement of a-amylase production. Response surface methodology （RSM） was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaC1 and CaCl2. Three variables （wheat bran, cottonseed extract, and starch）, which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design （BBD）. The optimal concentrations estimated for each variable related to the maximum of amylase activity （86 kU/mL） were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaC12. The fermentation using optimized culture medium allowed a significant increase in amylase production （by 3-fold）. The improvement in the a-amylase production after optimization process can be considered adequate for large-scale applications.展开更多
High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastori...High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.展开更多
A bacterial strain that was capable of degrading organic sulfur (dibenzothiophene) was isolated by enrichment techniques from the petroleum-contaminated soil collected from Zhongyuan Oil Field. The strain is named ZYX...A bacterial strain that was capable of degrading organic sulfur (dibenzothiophene) was isolated by enrichment techniques from the petroleum-contaminated soil collected from Zhongyuan Oil Field. The strain is named ZYX and is gram-positive. This strain undergoes bacilus-coccus morphological change, and forms yellow-pigment glossy circular colonies with 1.5 mm in diameter on average after 2 d incubation on Luria-Bertani(LB) plates. The full-length of 16S rDNA sequence of strain ZYX was determined and analyzed. Strain ZYX is found most relative with the genus of Arthrobacter. The similarity values between ZYX and Arthrobacter sp. P2 is 99.53%. The main morphological, biochemical and physiological features of strain ZYX accord with those of Arthrobacter. It is found that the optimal initial pH for growth is about 7.0, and the optimal concentration of dibenzothiophene(DBT) for growth is 0. 10 gL. Additionally, the results show that the best carbon source and nitrogen source are glycerol and glutamine, respectively.展开更多
Acid mine drainage(AMD)has become a widespread environmental issue and its toxicity can cause permanent damage to the ecosystem.However,there are few studies focusing on the formation of AMD under moderately thermophi...Acid mine drainage(AMD)has become a widespread environmental issue and its toxicity can cause permanent damage to the ecosystem.However,there are few studies focusing on the formation of AMD under moderately thermophilic conditions,hence we employed X-ray diffraction(XRD),scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS)and 16S rRNA sequencing to study the dissolution of pyrite and bornite by a moderate thermophilic consortium,and explored the role of free and attached microorganisms in the formation of AMD.The consortium mainly comprised Acidithiobacillus caldus,Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans.The results indicated that total iron in pyrite solution system reached 33.45 g/L on the 12th day,and the copper dissolution rate of bornite dissolution reached 91.8%on the 24th day.SEM results indicated that the surfaces of pyrite and bornite were significantly corroded by microorganisms.XRD and XPS results showed that ore residues contained jarosite,and the dissolving residue of bornite contained elemental sulfur.The dominant bacterial genus in pyrite dissolution was A.caldus,and L.ferriphilum in bornite dissolution.To sum up,microbes significantly accelerated the mineral dissolution process and promoted the formation of AMD.展开更多
The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tr...The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tricalcium phosphate (TCP) as sole phosphorus (P) source. The strain YC is identified as Stenotrophomonas maltophilia (S. maltophilia) based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis. The results show that the strain S. maltophilia YC can solubilize TCP and release soluble P in NBRIP growth medium. A positive correlation between concentration of soluble P and population of the isolate and a negative correlation between concentration of soluble P and pH in the culture medium are observed from statistical analysis results. Moreover, gluconic acid is detected in the culture medium by HPLC analysis. It indicates that the isolate can release gluconic acid during the solubilizing experiment, which causes acidification of the culture medium and then TCP solubilization. S. maltophilia YC has a maximal TCP solubilizing capability when using maltose as carbon source and ammonium nitrate as nitrogen source, respectively, in NBRIP growth medium.展开更多
The energy metabolism structure of microbial community plays an important role in the process of biohydrometallurgy.In this article,an artificial microbial community composed of three strains(Acidithiobacillus ferroox...The energy metabolism structure of microbial community plays an important role in the process of biohydrometallurgy.In this article,an artificial microbial community composed of three strains(Acidithiobacillus ferrooxidans,Leptospirillum ferriphilum and Acidithiobacillus thiooxidans)was used to leach three kinds of chalcopyrites with different iron-sulfur ratios.After 36 d of leaching,the chalcopyrite with iron-sulfur ratio of about 1:1 achieved the highest copper extraction(69.62%).In the early stage,iron oxidizing bacteria predominated,and the expression of rus and rio was 8 times higher than that in the late stage.In the late stage,sulfur oxidizing bacteria predominated,and the expression of tetH and HdrAB was 4 times higher than that in the early stage.Furthermore,the three bioleaching systems above were added with elemental sulfur(3 g/L);the chalcopyrite with iron-sulfur ratio of about 2:1 achieved the highest copper extraction(80.63%).The results suggest that the energy metabolism structure of the microbial community could be changed by changing the iron-sulfur ratio during the leaching process for improving the leaching efficiency of chalcopyrite.展开更多
Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is ...Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.展开更多
The effect of L-cysteine in different concentrations on the bioleaching of Ni-Cu sulfide was studied with an extremely thermophilic archaea,Acidianus manzaensis. It is found that adding certain amounts of L-cysteine t...The effect of L-cysteine in different concentrations on the bioleaching of Ni-Cu sulfide was studied with an extremely thermophilic archaea,Acidianus manzaensis. It is found that adding certain amounts of L-cysteine to the bioleaching system of Ni-Cu sulfide largely enhances the leaching rate. X-ray diffraction (XRD) patterns show the change of bioleached solid residues and the effect of L-cysteine on the surface charges of minerals. Zeta potential and IR spectra of mineral surface show that the interaction between L-cysteine and mineral leads to the formation of metal complex,which is propitious to the bioleaching of Ni-Cu sulfide by Acidianus manzaensis.展开更多
PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from...PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.展开更多
The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pa...The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.展开更多
Electrochimcal behaviors of rusticyanin （Rus.） isolated from Acidithiobacillus ferrooxidans were investigated through Rus.-ZnS-QDs/L-Cys/Au electrode. The cyclic voltammetric results indicate that rusticyanin immobi...Electrochimcal behaviors of rusticyanin （Rus.） isolated from Acidithiobacillus ferrooxidans were investigated through Rus.-ZnS-QDs/L-Cys/Au electrode. The cyclic voltammetric results indicate that rusticyanin immobilized on the surface of Rus.-ZnS-QDs/L-Cys/Au electrode can undergo a direct quasi-reversible electrochemical reaction. The immobilized rusticyanin is not denatured and still retains its activity in the temperature range of 19-43 ℃. The reduction ability of the protein increases and its oxidation ability becomes weak with the increase of pH from 6.0 to 7.8. Fe^2＋ ions in the solution can promote the electron transfer kinetics of the immobilized rusticyanin and make its peak potentials （φp） markedly move negatively.展开更多
One bioleaching bacterium, named as strain DXS, was isolated from acid mine drainages （AMDs） of Dongxiangshan Mine of Hami, Xinjiang Province, China. The strain DXS is gram-negative and rod-shaped with a size of （0...One bioleaching bacterium, named as strain DXS, was isolated from acid mine drainages （AMDs） of Dongxiangshan Mine of Hami, Xinjiang Province, China. The strain DXS is gram-negative and rod-shaped with a size of （0.40±0.05） μm x （1.3±0.5） μm. The optimal temperature and pH for growth are 30 ℃ and pH 2.0, respectively. It can grow autotrophically by using ferrous iron, elemental sulfur and NaS203 as sole energy sources. In the phylogenetic tree, strain DXS has similarity with Acidithiobacillus ferrooxidans type strain ATCC 23270 with 99.57% sequence similarity. The cloning and sequencing of Iro protein gene （iro） and tetrathionate hydrolase gene （tth） reveal that strain DXS is completely identical in iro gene sequence to A. ferrooxidans LY （DQ166841）, and almost identical in tth gene sequene to .4. ferrooxidans （AB259312） （only two nucleotides change）. The bioleaching experiments of marmatite and pyrite reveal that the leached zinc and iron concentrations reach 3.01 g/L and 2.75 g/L, respectively. The strain has a well potential application in industry bioleaching.展开更多
Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase ...Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supematant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50 × 10^4. Its maximum activity appears at pH 5 and 55 ℃. The chitinase is stable at pH 4.0-7.5 and below 45 ℃.展开更多
The biosorption mechanism of Cr （Ⅳ） ions on Synechococcus sp. biosorbent was studied by analyzing the biosorption kinetics as well as speciation change and bond formation during the biosorption process. The kinetic...The biosorption mechanism of Cr （Ⅳ） ions on Synechococcus sp. biosorbent was studied by analyzing the biosorption kinetics as well as speciation change and bond formation during the biosorption process. The kinetics study shows that the adsorption process of Cr （Ⅳ） consists of a very fast stage in the first several minutes, in which more than half of the saturation adsorption is attained, and a slower stage that approximately follows the first order kinetic model, basically Freundlich isotherm models were observed. Comparative studies of FT-LR spectra of K2Cr2O7, free cells of Synechococcus sp., and Cr-bound cells of Synechococcus sp show that the speciation of chromium that binds to the cells ofSynechococcus sp. is Cr （Ⅲ）, instead of Cr （Ⅳ）, and the carboxylic, alcoholic, amido and amino groups may be involved in the binding of Cr （Ⅲ）. Integrative analyses of the surface electric potential, the effect of pH value on adsorption behavior of Cr （Ⅵ）, and the results of FT-IR show that the biosorption of Cr （Ⅵ） follows two subsequent steps, biosorption of Cr2O7 ^2- by electrostatical force at the protonated active sites and reduction of Cr2O7^2- to Cr^3＋ by the reductive groups on the surface of the biosorbents.展开更多
The enhancement of chalcopyrite bioleaching with an enriched microbial community by acidified seawater was studied,and the enhancing mechanism was analyzed.The microbial community was enriched at the Dabaoshan mine si...The enhancement of chalcopyrite bioleaching with an enriched microbial community by acidified seawater was studied,and the enhancing mechanism was analyzed.The microbial community was enriched at the Dabaoshan mine site,and the treated ore sample had high concentrations of chalcopyrite and galena.The experimental results show that copper extraction from chalcopyrite with an enriched microbial community in seawater was promoted from 13.1%to 62.1%by acidification in comparison with that without acidification.Further analyses of the solutions,solid residues and microbial compositions by scanning electron microscopy,X-ray diffraction,Raman spectroscopy,Fourier transform infrared spectroscopy and 16 S rDNA sequencing revealed the promoting effects of acidified seawater.This acidification can increase the biodissolution of chalcopyrite to increase the concentration of iron ions and maintain the redox potential in the range of 360−410 mV.The latter produces an optimal redox environment conducive to chalcopyrite dissolution via Cu_(2)S.The adaptability of the microbial community to a high-salt environment is improved.Chloride ions at 580 mmol/L improve the leaching kinetics of chalcopyrite by increasing the porosity and noncrystallinity of the intermediate elemental sulfur.This study provides a promising way to bioleaching copper minerals using seawater for areas with freshwater shortages.展开更多
基金Project(50621063)supported by the Science Fund for Creative Research Groups of ChinaProject(2004CB619201)supported by the Major State Basic Research Development Program of China
文摘To reveal the impact of mining on bacterial ecology around mining area,bacterial community and geochemical characteristics about Dabaoshan Mine(Guangdong Province,China)were studied.By amplified ribosomal DNA restriction analysis and phylogenetic analysis,it is found that mining pollution greatly impacts the bacterial ecology and makes the habitat type of polluted environments close to acid mine drainage(AMD)ecology.The polluted environment is acidified so greatly that neutrophil and alkaliphilic microbes are massively dead and decomposed.It provided organic matters that can make Acidiphilium sp.rapidly grow and become the most bacterial species in this niche.Furthermore,Acidithiobacillus ferrooxidans and Leptospirillum sp.are also present in this niche.The amount of Leptospirillum sp.is far more than that of Acidithiobacillus ferrooxidans,which indicates that the concentration of toxic ions is very high.The conclusions of biogeochemical analysis and microbiological monitor are identical. Moreover,because the growth of Acidithiobacillus ferrooxidans and Leptospirillum sp.depends on ferrous iron or inorganic redox sulfur compounds which can be supplied by continual AMD,their presence indicates that AMD still flows into the site.And the area is closer to the outfalls of AMD,their biomasses would be more.So the distinction of their biomasses among different areas can help us to find the effluent route of AMD.
基金Projects(50621063, 50674101) supported by the National Natural Science Foundation of China
文摘In order to solve the citrus peel resource waste problem protopectinase-overproducing strain CD-01 for pectin production and minimize the drawbacks of chemical extraction of pectin, a was isolated from a pit soil dumped with perished orange in Changde City, Hunan Province of China. The strain CD-01 had the same morphology and 28S rRNA gene sequence （FJ184995） as that of Aspergillus niger （ATCC 64028）. It was thus identified and named as Aspergillus niger CD-01. The fermentation condition was optimized based on L9（34） orthogonal experimental design and the variances analyses. The results show that the optimal condition for producing pectin is as follows： time 36 h, temperature 35 ℃, pH 5, and urea as the nitrogen source. Under this condition, the pectin yield can reach up to 24.5%. This shows a great potential of Aspergillus niger CD-01 in pectin extraction from citrus.
基金Project(2010CB630901) supported by the National Basic Research Program of ChinaProject(50621063) supported by Creative Research Group of China+2 种基金Projects(51104189, 50321402, 50774102) supported by the National Natural Science Foundation of ChinaProject (1343-77341) supported by the Graduate Education Innovative Program of Central South University, ChinaProject(DOE-ER64125) supported by the Department of Energy, Office of Science under the Environmental Remediation Science Program of USA
文摘Eleven acid mine drainage （AMD） samples were obtained from southeast of China for the analysis of the microbial communities diversity, and the relationship with geochemical variables and spatial distance by using a culture-independent 16S rDNA gene phylogenetic analysis approach and multivariate analysis respectively. The principle component analysis （PCA） of geochemical variables shows that eleven AMDs can be clustered into two groups, relative high and low metal rich （RHMR and RLMR） AMDs. Total 1691 clone sequences are obtained and the detrended correspondence analysis （DCA） of operational taxonomic units （OTUs） shows that, ~,-Proteobacteria, Acidobacteria, Actinobacteria, Cyanobacteria, Firmicutes and Nitrospirae are dominant species in RHMR AMDs. In contrast, a-Proteobacteria, fl-Proteobacteria, Planctomycetes and Bacteriodetes are dominant species in RLMR AMD. Results also show that high-abundance putative iron-oxidizing and only putative sulfur-oxidizing microorganisms are found in RHMR AMD. Multivariate analysis shows that both geochemical variables （r=0.429 3, P=-0.037 7） and spatial distance （r=0.321 3, P=-0.018 1） are significantly positively correlated with microbial community and pH, Mg, Fe, S, Cu and Ca are key geochemistry factors in shaping microbial community. Variance partitioning analysis shows that geochemical variables and spatial distance can explain most （92%） of the variation.
基金Project(50974140) supported by the National Natural Science Foundation of ChinaProject(VR-09157) supported by Beijing Synchrotron Radiation Facility (BSRF) Public User Program,China
文摘Sulfur speciation transformation during bioleaching of pyrite-containing sphalerite concentrate by thermophile Sulfolobus metallicus （S. metallicus） at 65 ℃ was investigated by X-ray diffraction （XRD）, diffuse reflectance Fourier transform infrared spectroscopy （FT-IR） and sulfur K-edge X-ray absorption near edge structure spectroscopy （XANES）. The results show that the presence of S. metallicus effectively enhances the dissolution of the mineral. The yield of zinc increases from 0.5 g/L in sterile control to 2.7 g/L in bioleaching. The pyrite in the concentrate facilitates zinc dissolution in the early stage, but has hindrance role in the late stage for the formation of jarosite. Sulfur speciation analyses show that jarosite and elemental sulfur are main products in bioleaching process, and the accumulation ofjarosite is mainly responsible for the decline of leaching efficiency.
基金Project(13JJ9002)supported by Hunan Provincial Natural Science Foundation of ChinaProject(2012XK4081)supported by the Key Science Technology Plan Project of Hunan Provincial Science&Technology Department,ChinaProject(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan Province,China
文摘In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.
基金Project(31000350) supported by the National Natural Science Foundation of ChinaProject(2010CB630902) supported by the National Basic Research Program of China
文摘A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens （99.54%）. After that, an optimization of culture conditions was carried out for the improvement of a-amylase production. Response surface methodology （RSM） was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaC1 and CaCl2. Three variables （wheat bran, cottonseed extract, and starch）, which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design （BBD）. The optimal concentrations estimated for each variable related to the maximum of amylase activity （86 kU/mL） were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaC12. The fermentation using optimized culture medium allowed a significant increase in amylase production （by 3-fold）. The improvement in the a-amylase production after optimization process can be considered adequate for large-scale applications.
基金Project(31870115)supported by the National Natural Science Foundation of ChinaProject(2015JJ5006)supported by the Natural Science of Hunan Province&Changde City Joint Foundation,ChinaProjects(2015zzts268,ZY2015823)supported by the Fundamental Research Funds for the Central Universities,China
文摘High-level expression ofβ-mannanase has been reported in Pichia pastoris under control of the GAP promoter.Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature.The aim of this research was to improve the expression level ofβ-mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature.To this end,a panel of strains harboring different copy numbers ofβ-mannanase gene were obtained by multiple zeocin concentration gradients screening,the influence of gene copy number on the expression ofβ-mannanase in Pichia pastoris X33 was investigated.With the constitutive GAP promoter,the four copies strain exhibited a 4.04-fold higherβ-mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain,but an increase of the copy number above four resulted in a decrease of expression.Furthermore,the effects of culture temperature were studied in flask.The decreased culture temperature of four copies strain resulted in a 1.8-fold(26℃)and 3.5-fold(22℃)higherβ-mannanase activity compared to that at 30℃.A fed-batch strategy was successfully used for high cell-density fermentation andβ-mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.
基金Project(50621063) supported by the National Natural Science Foundation of ChinaProject(05JJ30023) supportecd by Natural Science Foundation of Hunan Province, China
文摘A bacterial strain that was capable of degrading organic sulfur (dibenzothiophene) was isolated by enrichment techniques from the petroleum-contaminated soil collected from Zhongyuan Oil Field. The strain is named ZYX and is gram-positive. This strain undergoes bacilus-coccus morphological change, and forms yellow-pigment glossy circular colonies with 1.5 mm in diameter on average after 2 d incubation on Luria-Bertani(LB) plates. The full-length of 16S rDNA sequence of strain ZYX was determined and analyzed. Strain ZYX is found most relative with the genus of Arthrobacter. The similarity values between ZYX and Arthrobacter sp. P2 is 99.53%. The main morphological, biochemical and physiological features of strain ZYX accord with those of Arthrobacter. It is found that the optimal initial pH for growth is about 7.0, and the optimal concentration of dibenzothiophene(DBT) for growth is 0. 10 gL. Additionally, the results show that the best carbon source and nitrogen source are glycerol and glutamine, respectively.
基金Projects(51934009,52074353)supported by the National Natural Science Foundation of ChinaProject(2019YFC1803600)supported by the National Key Research and Development Program of ChinaProject(2021JJ30855)supported by the Natural Science Foundation of Hunan Province,China。
文摘Acid mine drainage(AMD)has become a widespread environmental issue and its toxicity can cause permanent damage to the ecosystem.However,there are few studies focusing on the formation of AMD under moderately thermophilic conditions,hence we employed X-ray diffraction(XRD),scanning electron microscopy(SEM),X-ray photoelectron spectroscopy(XPS)and 16S rRNA sequencing to study the dissolution of pyrite and bornite by a moderate thermophilic consortium,and explored the role of free and attached microorganisms in the formation of AMD.The consortium mainly comprised Acidithiobacillus caldus,Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans.The results indicated that total iron in pyrite solution system reached 33.45 g/L on the 12th day,and the copper dissolution rate of bornite dissolution reached 91.8%on the 24th day.SEM results indicated that the surfaces of pyrite and bornite were significantly corroded by microorganisms.XRD and XPS results showed that ore residues contained jarosite,and the dissolving residue of bornite contained elemental sulfur.The dominant bacterial genus in pyrite dissolution was A.caldus,and L.ferriphilum in bornite dissolution.To sum up,microbes significantly accelerated the mineral dissolution process and promoted the formation of AMD.
基金Project(2004CB619201) supported by the Major State Basic Research and Development Program of ChinaProject(Z200515002) supported by the Key Project Foundation of the Education Department of Hubei Province, China+1 种基金Project(GCP200801) supported by the Open Research Fund of Key Laboratory for Green Chemical Process of Ministry of Education, ChinaProject(Q200811) supported by the Youths Science Foundation of Wuhan Institute of Technology, China
文摘The phosphate solubilizing characteristics of a strain YC, which was isolated from phosphate mines (Hubei, China), were studied in National Botanical Research Institute’s phosphate (NBRIP) growth medium containing tricalcium phosphate (TCP) as sole phosphorus (P) source. The strain YC is identified as Stenotrophomonas maltophilia (S. maltophilia) based upon the results of morphologic, physiological and biochemical characteristics and 16S rRNA sequences analysis. The results show that the strain S. maltophilia YC can solubilize TCP and release soluble P in NBRIP growth medium. A positive correlation between concentration of soluble P and population of the isolate and a negative correlation between concentration of soluble P and pH in the culture medium are observed from statistical analysis results. Moreover, gluconic acid is detected in the culture medium by HPLC analysis. It indicates that the isolate can release gluconic acid during the solubilizing experiment, which causes acidification of the culture medium and then TCP solubilization. S. maltophilia YC has a maximal TCP solubilizing capability when using maltose as carbon source and ammonium nitrate as nitrogen source, respectively, in NBRIP growth medium.
基金Project(2017zzts382)supported by Central South University Postgraduate Independent Exploration and Innovation,ChinaProject(2014jpkc003)supported by Central South University Graduate Excellent Course,China+1 种基金Project(2015JJ2165)supported by Hunan Provincial Natural Science Foundation of ChinaProject(165611031)supported by Central South University Fundamental Research Funds Special Funding,China。
文摘The energy metabolism structure of microbial community plays an important role in the process of biohydrometallurgy.In this article,an artificial microbial community composed of three strains(Acidithiobacillus ferrooxidans,Leptospirillum ferriphilum and Acidithiobacillus thiooxidans)was used to leach three kinds of chalcopyrites with different iron-sulfur ratios.After 36 d of leaching,the chalcopyrite with iron-sulfur ratio of about 1:1 achieved the highest copper extraction(69.62%).In the early stage,iron oxidizing bacteria predominated,and the expression of rus and rio was 8 times higher than that in the late stage.In the late stage,sulfur oxidizing bacteria predominated,and the expression of tetH and HdrAB was 4 times higher than that in the early stage.Furthermore,the three bioleaching systems above were added with elemental sulfur(3 g/L);the chalcopyrite with iron-sulfur ratio of about 2:1 achieved the highest copper extraction(80.63%).The results suggest that the energy metabolism structure of the microbial community could be changed by changing the iron-sulfur ratio during the leaching process for improving the leaching efficiency of chalcopyrite.
基金Project(31000350)supported by the National Natural Science Foundation of China
文摘Abstract： A simple optimized microplate-based method to assay endo-1,4-β-mannosidase activity was described as an improved high-throughput screening method. A series of experimental conditions were optimized. It is revealed that the optimum measurement procedure is as follows： adding 50μL of diluted enzyme sample and 50 μL substrate, incubating at 45 ℃ for exactly 5 min in micro-plate, mixing with 100 μL 3,5-dinitrosalicylic acid （DNS） reagent, maintaining at boiling point for 15 rain, cooling down to room temperature before determining the ABS value at 540 nm using an ELISA micro-plate reader. The reaction volume of the optimized microplate-assay is reduced to 200μL from 2 500 μL used in the standard β-mannanase macro-assay. The optimized micro-assay is significantly more sensitive in all of the 643 candidates during endo-1,4-β-mannosidase screening. Statistical analyses show that the sensitivity of the optimized micro-method is significantly greater than that of the macro-assay. The optimized method is convenient, fast, and cheap for high throughput enzyme screening.
基金Projects(50621063, 30400010) supported by the National Natural Science Foundation of ChinaProject(2010CB630903) supported by the National Basic Research Program of China
文摘The effect of L-cysteine in different concentrations on the bioleaching of Ni-Cu sulfide was studied with an extremely thermophilic archaea,Acidianus manzaensis. It is found that adding certain amounts of L-cysteine to the bioleaching system of Ni-Cu sulfide largely enhances the leaching rate. X-ray diffraction (XRD) patterns show the change of bioleached solid residues and the effect of L-cysteine on the surface charges of minerals. Zeta potential and IR spectra of mineral surface show that the interaction between L-cysteine and mineral leads to the formation of metal complex,which is propitious to the bioleaching of Ni-Cu sulfide by Acidianus manzaensis.
基金Project(2010CB630901)supported by the National Basic Research Program of China
文摘PCR-based DNA fingerprinting, REP-PCR(repetitive element PCR), RAPD(randomly amplified polymorphic DNA) and16 S r DNA sequence analyses were used to characterize 23 Acidithiobacillus ferrooxidans strains isolated from different environments.(GTG)5 and BOXA1 R primer were selected for REP-PCR. Twenty arbitrary primers were used for RAPD to acquire DNA profiles from A. ferrooxidans. Both RAPD and REP-PCR produce complex banding patterns and show good discriminatory ability in differentiating closely related strains of A. ferrooxidans. The strains are clustered into 4 or 5 major groups and reveal genomic diversity using(GTG)5-PCR, BOX-PCR and RAPD analysis. Phylogenetic tree based on 16 S r DNA sequences of 23 strains and related strains shows that they are clustered into two distinct groups. Twelve strains are highly related to a new Acidithiobacillus named Acidithiobacillus ferrivorans. The results indicate that PCR-based methods are effective in revealing genetic diversity among A. ferrooxidans.
基金Project(CX2012B124)supported by the Graduate Degree Thesis Innovation Program of Hunan ProvinceChina+3 种基金Project(13JJ9002)supported by the Natural Science Foundation of Hunan ProvinceChinaProject(2012XK4081)supported by the Key Science and Technology Plan of Hunan Provincial Science&Technology DepartmentChina
文摘The yeast Pichia pastoris(P. pastoris) has been used for the expression of heterologous proteins with the significant success. However, it is time-consuming to screen the high expression level of the recombinant P. pastoris directly. Thus, for β-mannanase production, developing the accurate, rapid and inexpensive screening method to substitute random screening is certainly required. A simple method based on the size of hydrolysis hole was described here, but this method was not very accurate that could only be used in preliminary screening. To further improve the accuracy, a micro-plate screening method is established, which appears to be more accurate and effective. The efficiency of this screening method is about 10 times higher than that of the general screening strategy of cultivation in shaking flasks. Two methods presented here can also be used for screening of recombinant Pichia strains with high-level expression of other heterologous protein after modification.
基金Project(2010CB630903) supported by the National Basic Research Program of ChinaProject(50621063) supported by the National Natural Science Foundation of China
文摘Electrochimcal behaviors of rusticyanin （Rus.） isolated from Acidithiobacillus ferrooxidans were investigated through Rus.-ZnS-QDs/L-Cys/Au electrode. The cyclic voltammetric results indicate that rusticyanin immobilized on the surface of Rus.-ZnS-QDs/L-Cys/Au electrode can undergo a direct quasi-reversible electrochemical reaction. The immobilized rusticyanin is not denatured and still retains its activity in the temperature range of 19-43 ℃. The reduction ability of the protein increases and its oxidation ability becomes weak with the increase of pH from 6.0 to 7.8. Fe^2＋ ions in the solution can promote the electron transfer kinetics of the immobilized rusticyanin and make its peak potentials （φp） markedly move negatively.
基金Projects(50974140, 50674101) supported by the National Natural Science Foundation of ChinaProject(2010CB630902) supported by the National Basic Research Program of China
文摘One bioleaching bacterium, named as strain DXS, was isolated from acid mine drainages （AMDs） of Dongxiangshan Mine of Hami, Xinjiang Province, China. The strain DXS is gram-negative and rod-shaped with a size of （0.40±0.05） μm x （1.3±0.5） μm. The optimal temperature and pH for growth are 30 ℃ and pH 2.0, respectively. It can grow autotrophically by using ferrous iron, elemental sulfur and NaS203 as sole energy sources. In the phylogenetic tree, strain DXS has similarity with Acidithiobacillus ferrooxidans type strain ATCC 23270 with 99.57% sequence similarity. The cloning and sequencing of Iro protein gene （iro） and tetrathionate hydrolase gene （tth） reveal that strain DXS is completely identical in iro gene sequence to A. ferrooxidans LY （DQ166841）, and almost identical in tth gene sequene to .4. ferrooxidans （AB259312） （only two nucleotides change）. The bioleaching experiments of marmatite and pyrite reveal that the leached zinc and iron concentrations reach 3.01 g/L and 2.75 g/L, respectively. The strain has a well potential application in industry bioleaching.
基金Projects(50621063, 50674101) supported by the National Natural Science Foundation of China
文摘Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supematant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50 × 10^4. Its maximum activity appears at pH 5 and 55 ℃. The chitinase is stable at pH 4.0-7.5 and below 45 ℃.
基金Project(50321402) supported by the National Natural Science Foundation of China
文摘The biosorption mechanism of Cr （Ⅳ） ions on Synechococcus sp. biosorbent was studied by analyzing the biosorption kinetics as well as speciation change and bond formation during the biosorption process. The kinetics study shows that the adsorption process of Cr （Ⅳ） consists of a very fast stage in the first several minutes, in which more than half of the saturation adsorption is attained, and a slower stage that approximately follows the first order kinetic model, basically Freundlich isotherm models were observed. Comparative studies of FT-LR spectra of K2Cr2O7, free cells of Synechococcus sp., and Cr-bound cells of Synechococcus sp show that the speciation of chromium that binds to the cells ofSynechococcus sp. is Cr （Ⅲ）, instead of Cr （Ⅳ）, and the carboxylic, alcoholic, amido and amino groups may be involved in the binding of Cr （Ⅲ）. Integrative analyses of the surface electric potential, the effect of pH value on adsorption behavior of Cr （Ⅵ）, and the results of FT-IR show that the biosorption of Cr （Ⅵ） follows two subsequent steps, biosorption of Cr2O7 ^2- by electrostatical force at the protonated active sites and reduction of Cr2O7^2- to Cr^3＋ by the reductive groups on the surface of the biosorbents.
基金Project(2022YFC2105300)supported by the National Key Research and Development Program of ChinaProjects(41802038,51774342)supported by the National Natural Science Foundation of China。
文摘The enhancement of chalcopyrite bioleaching with an enriched microbial community by acidified seawater was studied,and the enhancing mechanism was analyzed.The microbial community was enriched at the Dabaoshan mine site,and the treated ore sample had high concentrations of chalcopyrite and galena.The experimental results show that copper extraction from chalcopyrite with an enriched microbial community in seawater was promoted from 13.1%to 62.1%by acidification in comparison with that without acidification.Further analyses of the solutions,solid residues and microbial compositions by scanning electron microscopy,X-ray diffraction,Raman spectroscopy,Fourier transform infrared spectroscopy and 16 S rDNA sequencing revealed the promoting effects of acidified seawater.This acidification can increase the biodissolution of chalcopyrite to increase the concentration of iron ions and maintain the redox potential in the range of 360−410 mV.The latter produces an optimal redox environment conducive to chalcopyrite dissolution via Cu_(2)S.The adaptability of the microbial community to a high-salt environment is improved.Chloride ions at 580 mmol/L improve the leaching kinetics of chalcopyrite by increasing the porosity and noncrystallinity of the intermediate elemental sulfur.This study provides a promising way to bioleaching copper minerals using seawater for areas with freshwater shortages.
