固定化Ralstonia metallidurans CH34降解苯酚的研究*

将既能耐抗重金属又能降解苯酚的细菌Ralstonia m etalliduransCH34固定化以提高其降酚效率。首先通过正交实验,得到了固定化该菌种的最优制备条件,然后对固定化细胞的降酚效果进行了研究。结果表明,固定化R.m etalliduransCH34的降酚效果明显优于游离细胞;抗重金属毒性方面也有较大提高;在加入额外碳源(甲苯,柠檬酸)情况下,固定化R.m etalliduransCH34进行苯酚降解时所受影响明显要小于游离态菌。     

Ralastonia metallidurans CH34苯酚降解特性的研究

RalstoniametalliduransCH34是从一家锌工厂的沉积物中分离筛选到的一株细菌。对其降解苯酚的特性进行了研究。结果表明R.metalliduransCH34具有很高的降解苯酚的能力,其降解苯酚的速率常数为0.33,降解苯酚的最适条件为pH7.0,温度30℃,装液量20%(v/v)。在高浓度重金属存在的条件下,R.metalliduransCH34仍保持较高的苯酚降解活力。柠檬酸钠、琥珀酸则能促进其对苯酚的降解。     

固定化Ralstonia metallidurans CH34在三相流化床中降解苯酚的研究

通过探索固定化细菌Ralstonia metallidurans CH34在三相流化反应器中降解苯酚的反应条件,对固定化细胞处理工业废水进行模拟研究,以期提高R.metallidurans CH34降解苯酚的能力和效率。结果表明,固定化R.metallidurans CH34在三相流化反应器中明显提高了降解苯酚的能力,耐抗金属性也有较大的提高,而且能够在模拟工业废水中批次培养3-4次,其降酚能力退化并不明显。这为R.metallidurans CH34实际应用提供了可靠的基础。     

竹炭固定化施氏假单胞菌Pseudomonas stutzeri ZH-1降解苯酚的研究

通过竹炭固定化以加强施氏假单胞菌Pseudomonas stutzeri ZH-1对苯酚的降解能力。采用正交试验优化竹炭对菌株ZH-1固定化条件,冷场发射扫描电镜（SEM）观察固定化后的菌株在竹炭上的附着情况,对比固定化菌和游离菌的降解性能并对固定化菌做重复利用性能测试。结果显示,竹炭固定化P. stutzeri ZH-1的最适条件为竹炭1 g,接种量5%(体积分数),吸附时间24 h;SEM显示菌附着在竹炭表面和内部空隙中;竹炭固定化后菌株ZH-1对苯酚的降解率显著增加（P<0.05）,处理48 h时降解率增加15%;竹炭固定化菌ZH-1经10轮重复利用后仍有很高的苯酚降解性能,48 h时降解率增加173%（P<0.05）。竹炭固定化菌ZH-1在去除含苯酚类有机废水中具有较好的应用前景。     

高效苯酚降解菌细胞固定化方法与条件的研究

含酚废水是一种难降解有机废水,对环境污染非常严重。目前常利用细菌处理含酚废水。但利用细菌处理含酚废水存在一些缺点,为此将1株高效苯酚降解菌进行细胞固定化。采用正交实验设计方法确定了该菌株固定化的最佳条件,并且考察了该固定化细胞降解苯酚的最佳条件。实验表明:该菌株的固定化细胞降解苯酚能力和耐受苯酚能力均大于游离细胞,经36 h可将1 800 mg/L苯酚降解完全。其降解苯酚的最适温度为30℃,最佳pH值为5~9。     

Pseudomonas stutzeri JFL2012复合固定化及降酚性能研究

采用正交实验法对假单胞菌进行复合固定化,并对其降酚过程建立动力学方程。研究发现,菌株Pseudomonas stutzeri JFL2012进行复合固定化,最佳固定化条件为w_{(聚乙烯醇)}=10%,w_(生物炭)=2.5%,w_(包菌量)=0.3%,交联时间4 h。苯酚降解过程符合Haldane底物抑制方程。     

固定化白腐真菌对多种染料脱色的研究

白腐真菌F17 Schizophyllum sp．对不同结构的多种染料具有较强的脱色能力。聚酯无纺布是该菌固定化的最佳载体。利用正交实验,确定了该菌株固定化细胞制备的最优操作条件。与游离菌相比,固定化菌不仅提高其脱色能力,而且在pH值和染料浓度的变化情况下,仍保持稳定的脱色率。固定化菌对染料脱色后的紫外可见光谱分析表明,可见光区吸收峰消失,紫外区的光吸收峰有所增加,染料结构发生了变化。     

Ralstonia eutropha CH34酯酶基因的克隆和序列分析

采用含α－乙酸萘酯和固兰RR的表面琼脂法从Ralstonia eutropha CH34的基因文库中筛选酯酶基因estA,对含有est的１.7kb DNA片段的核甘酸序列分析表明,该基因全长８２５bp,编码由２７５个氨基酸组成的ＥstＡ蛋白,分子量为30785D,经推导氨基酸序列的同源性分析,发现EstA与参与芳香化合物代谢中间位裂解途径的水解酶有很高的同源性。     

固定化白腐真菌对多种染料脱色的研究*

白腐真菌F17Schizophyllumsp.对不同结构的多种染料具有较强的脱色能力。聚酯无纺布是该菌固定化的最佳载体。利用正交实验,确定了该菌株固定化细胞制备的最优操作条件。与游离菌相比,固定化菌不仅提高其脱色能力,而且在pH值和染料浓度的变化情况下,仍保持稳定的脱色率。固定化菌对染料脱色后的紫外可见光谱分析表明,可见光区吸收峰消失,紫外区的光吸收峰有所增加,染料结构发生了变化。     

新型固定化硝化细菌和好氧反硝化细菌处理氨氮废水

以改性沸石、聚乙烯醇、海藻酸钠作为固定化载体材料,硼酸和氯化钙作为交联剂,采用吸附-包埋-交联法将硝化细菌和好氧反硝化细菌复合固定化制备成微生物小球.通过复合菌配比实验,考察其对氨氮的去除率以及亚硝酸盐和硝酸盐的累积量;对制成的固定化小球做四因素三水平的正交实验,考察不同条件下对氨氮的去除率.结果表明,硝化细菌和好氧反硝化细菌配比为3:2时,氨氮去除率最高达82.32%,亚硝酸盐和硝酸盐的累积量为0.032mg·L^-1和0.053 mg·L^-1;通过正交实验,确定沸石投加量为2g·100mL^-1、温度为30℃、pH值为7.5、振荡速度为130r·min^-1时,对氨氮达到最好的去除效果,去除率达90.31%,此法制得的小球机械性能和吸水性能良好.     

The iron-containing superoxide dismutase of Ralstonia metallidurans CH34

The iron-containing superoxide dismutase (Fe-SOD) of Ralstonia metallidurans CH34 was purified and characterised as a homodimer of 2 x 21500 Da containing one iron atom per monomer and exhibiting all the characteristics of the prokaryotic Fe-SODs except for a higher isoelectric point. The protein was 2-fold overexpressed in the presence of selenite, zinc or paraquat. R. metallidurans CH34 was suggested to contain a gene encoding for a manganese-containing SOD located in the inducible chromate resistance operon. Whatever the culture conditions used in this study, including the presence of chromate, only a Fe-SOD, genetically distinct from the putative Mn-SOD, was detected. This Fe-SOD seems to be the only active superoxide dismutase expressed in R. metallidurans CH34.     

Mobilization of Selenite by Ralstonia metallidurans CH34

Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.     

Mobilization of selenite by Ralstonia metallidurans CH34

Ralstonia metallidurans CH34 (formerly Alcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metallidurans CH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.     

Crystal structure of the oxidized form of the periplasmic mercury-binding protein MerP from Ralstonia metallidurans CH34

In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom. The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases. We purified MerP from R.metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0A resolution. Superposition with structures of other metal-binding proteins shows that the global structure of R.metallidurans CH34 oxidized MerP follows the general topology of the whole family. The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP. Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction. The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae. Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer. We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.     

Characteristics of zinc transport by two bacterial cation diffusion facilitators from Ralstonia metallidurans CH34 and Escherichia coli

CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, bound Zn2+, Co2+, Cu2+, and Ni2+ but not Mg2+, Mn2+, or Cd2+, as shown by metal affinity chromatography. Histidine residues were involved in the binding of 2 to 3 mol of Zn2+ per mol of CzcD. ZitB transported 65Zn2+ in the presence of NADH into everted membrane vesicles with an apparent Km of 1.4 microM and a Vmax of 0.57 nmol of Zn2+ min(-1) mg of protein(-1). Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn2+ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.     

CzcE from Cupriavidus metallidurans CH34 is a copper-binding protein

CzcE is encoded by the most distal gene of the czc determinant that allows Cupriavidus metallidurans CH34 to modulate its internal concentrations of cobalt, zinc and cadmium by regulation of the expression of the efflux pump CzcCBA. We have overproduced and purified CzcE. CzcE is a periplasm-located dimeric protein able to bind specifically 4 Cu-equivalent per dimer. Spectrophotometry and EPR are indicative of type II copper with typical d-d transitions. Re-oxidation of fully reduced CzcE led to the formation of an air stable semi-reduced form binding both 2 Cu(I) and 2 Cu(II) ions. The spectroscopic characteristics of the semi-reduced form are different of those of the oxidized one, suggesting a change in the environment of Cu(II).     

Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation

Cupriavidus metallidurans CH34 is a β-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (β-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.