Ralstonia eutropha菌株镉抗性调节基因CzcR的克隆与序列测定

G-细菌镉抗性决定子是一个编码4个蛋白的CzcCBAD操纵子,参照GenBank已登录Czc基因序列进行引物设计,利用PCR技术,从可在含350mg/LCdCl2的培养基上生长的抗镉菌株Ralstoniaeutropha质粒中,扩增出长度约为1200bp的革兰氏阴性细菌镉抗性系统中的抗性调节基因CzcR,然后将其亚克隆到pGEM-T-easy载体上,并转化至受体菌中,构建重组质粒,采用碱性裂解法提取质粒DNA后,经EcoRI酶切分析和核苷酸序列分析,其与GenBank中登录的CzcR基因序列相似性高达98%,显示其具有正确的CzcR基因核苷酸序列。镉抗性基因的获得将大大推动对微生物抗重金属机理的研究,并为进一步构建耐镉基因工程菌,高效净化重金属污染环境打下坚实的基础。     

Role of Acinetobacter sp. in arsenite As(III) oxidation and reducing its mobility in soil

Microbial arsenite oxidation was observed by Acinetobacter sp. XS21, this strain oxidised arsenite As(III) up to 80?mM within 48–72?h of incubation. The present strain XS21 oxidised As(III) at a very high concentration in a shorter interval of time than any of the previous reported microbes. Further, XS21 was applied to the soil to observe its ability in reducing the mobility of As(III), and we found that Acinetobacter sp. XS21 efficiently removed arsenite from soluble-exchangeable fraction and removed 70% of the arsenite as compared to control. This feature makes it a potential candidate for bioremediation. Arsenic-resistant bacteria with strong As(III)-oxidising ability may have potential to improve bioremediation of As(III)-contaminated sites. To understand their basis of resistance and transformation we found the As(III) oxidase gene using degenerate primer and amplified ～550?bp of aioA gene. Amplified aioA gene sequence exhibiting 52% identity in terms of gene and deduced protein sequence to Uncultured bacterium, and Achromobacter sp. arsenite oxidase of larger subunit. Arsenite oxidase, an enzyme, was also observed in this isolate, which may provide a resistance and transforming ability. This bacterium was identified as Acinetobacter sp., by sequencing 16s rRNA gene sequence analysis.     

As(V)-reduction to As(III) by arsenic-resistant Bacillus spp. bacterial strains isolated from low-contaminated sediments of the Oliveri-Tindari Lagoon,Italy

Five arsenic-resistant bacterial strains designated MT1, MT2, MT3, V1 and V2 were isolated from sediments of the Oliveri-Tindari Lagoon (Italy), which comprises six small lakes whose sediments contain low arsenic concentrations. Phylogenetic analysis of the 16S rRNA gene sequences assigned them to the genus Bacillus. Bacillus sp. strain MT3 showed higher tolerance to As(III) and As(V), as indicated by minimum inhibitory concentrations of 14 and 135 mmol^?1, respectively. Bacillus sp. strain V1 showed growth inhibition at 14 mmol^?1 in the presence of As(III) and at 68 mmol^?1 in the presence of As(V), whereas the arsenic resistance of Bacillus sp. strain MT1 was 10 and 27 mmol^?1 for As(III) and As(V), respectively. The strains Bacillus spp. MT2 and V2 showed low levels of As(III) and As(V) resistance, as it was unable to grow at concentrations>7 and 14 mmol^?1, respectively. The isolated arsenic-resistant Bacillus spp. strains were able to reduce As(V) to As(III), especially Bacillus spp. strain MT3. This study suggests that the isolated bacterial strains play a role in the arsenic biogeochemical cycle of arsenic-poor sediments in the Oliveri-Tindari Lagoon.     

假单胞菌反式氯双烯内酯异构酶基因克隆和序列分析

从土壤中分离得到一株降解2,4-二氯酚能力较强的假单胞菌菌株GT241-1,从该菌株中克隆出参与降解2,4-二氯酚的反式氯双烯内酯异构酶基因(dcpE).克隆策略是采用Southern杂交对其邻近基因进行定位后构建基因组文库,再用斑点杂交从基因文库中筛选目的转化子.经序列测定得知,dcpE基因编码区1062bp.核苷酸和推测的编码氨基酸序列分析表明,dcpE与已在GenBank登记的相关基因有一定的差异.图5表1参12     

Vehicle-Induced Lead and Cadmium Contamination of Roadside Soil and Plants in Italy

Lead and cadmium concentrations in spontaneous vegetation and in soil sampled at various distances (0–208 m) from several motorways throughout Italy were measured. Lead and cadmium concentrations appear to be correlated to distance from the motorway and to traffic levels. A comparison with data from a remote and unpolluted site shows that foliar lead concentrations can be up to 40 times higher, and foliar cadmium concentrations up to 3 times higher. Plants behave differently in their accumulation of lead and cadmium; conifers present the highest levels. Also the various receptors and compartments of the roadside ecosystem differ in their accumulation of lead and cadmium; soil and bark have consistently higher concentrations, while the leaves and twigs of trees have consistently lower levels. the data recorded by ad hoc introduced bio-accumulating plants (Lolium multiflorum L.) are consistent with those of the spontaneous vegetation. the concentrations of both lead and cadmium in Lolium vary from June to November, following in part the fluctuation in traffic density.     

In situ enhanced bioremediation of dichlorvos by a phyllosphere Flavobacterium strain

A bacterium capable of degrading dichlorvos was isolated from the rape phyllosphere and designated YD4. The strain was identified as Flavobacterium sp., based on its phenotypic features and 16S rRNA gene sequence. Strain YD4 was able to utilize dichlorvos as the sole source of phosphorus. In situ enhanced bioremediation of dichlorvos by YD4 was hereafter studied. Chlorpyrifos and phoxim could also be degraded by this strain as the sole phosphorus source. A higher degradation rate of dichlorvos was observed after spraying YD4 onto the surface of rape leaves when compared to the sterilized-YD4 and water-treated samples. The results indicated that pesticide-degrading epiphytic bacterium could become a new way for in situ phyllosphere bioremediation where the hostile niche is unsuitable for other pesticide-degrading bacteria isolated from soil and water.     

Uptake of zinc, cadmium and phosphorus by arbuscular mycorrhizal maize (Zea mays L.) from a low available phosphorus calcareous soil spiked with zinc and cadmium

In a multifactorial pot experiment, maize (Zea mays L.) with or without inoculation with the arbuscular mycorrhizal (AM) fungus Glomus mosseae BEG167 was grown in a sterilized soil spiked with three levels of zinc (0, 300 and 900 mg Zn kg⁻¹ soil) and three levels of cadmium (0, 25 and 100 mg Cd kg⁻¹ soil). At harvest after 8 weeks of growth, the proportion of root length of inoculated plants colonized decreased with increasing Zn or Cd additon, and was 56% in the absence of both metals and was reduced significantly to 27% in the presence of the higher levels of both metals. Mycorrhizal plants had higher biomass than non-mycorrhizal controls except at the highest soil level of Cd. Cadmium had more pronounced effects on plant biomass than did Zn at the levels studied and the two metals showed a significant interaction. The data suggest that mycorrhizal inoculation increased plant growth with enchancement of P nutrition, perhaps increasing plant tolerance to Zn and Cd by a dilution effect. AM inoculation also led to higher soil solution pH after harvest, possibly reducing the availability of the metals for plant uptake, and lowered the concentrations of soluble Zn and Cd in the soil solution, perhaps by adsorption onto the extrametrical mycelium.     

The role of sulphur in cadmium(II) ions detoxification demonstrated in in vitro model: Dionaea muscipula Ell.

Fertilization by phosphorus and also nitrogen fertilizers are the main source of cadmium soil contamination. Cadmium is relatively easily taken up by plants and then consequently enters their food chain. We investigated whether the cultivation medium with or without sulphates supplement affects the synthesis of protective low molecular mass thiols such as cysteine, reduced glutathione, oxidized glutathione and phytochelatins (PC2) in Dionaea muscipula treated with cadmium(II) ions at 0, 1, 5, 10, 25, 50, 100, 250, 500 and 1,000 μM for 6 weeks. The plants cultivated in the presence of sulphates showed higher cadmium tolerance due to faster growth and lesser number of necrosis hallmarks. Further, we utilized liquid chromatography with electrochemical detection for determination of the thiols in plant tissues. In the case of the plants cultivated on media supplemented with sulphates for 6 weeks the average PC2 level was 2,830 ng/g of fresh weight (FW). However, the plants cultivated on media without the presence of sulphates on average contained 1,160 ng PC2/g FW. Results obtained showed the positive effect of sulphur supplementation in cadmium detoxification processes in plants. In addition to thiol content, we also determined level of majority secondary metabolite of Venus flytrap, naphthoquinone plumbagin. Generally, the presence of sulphates in the media enhanced the protective mechanism and did not affect directly the synthesis of secondary metabolite plumbagin.     

Cadmium accumulation characteristics of floricultural plant Cosmos bipinnata*

The artificially high soil cadmium (Cd) concentration screening method was used to screen Cd-hyperaccumulators from floricultural plants. Among the five species of floricultural plants screened, Cosmos bipinnata showed the characteristics of Cd-accumulators. A pot experiment was conducted to further study Cd accumulation characteristics of C. bipinnata. The results showed that the biomass, chlorophyll content, superoxide dismutase activity, peroxidase activity and soluble protein content of C. bipinnata first increased and later decreased with the increase in soil Cd concentration, but the carotenoid content and catalase activity of C. bipinnata reduced. Cd contents in roots, stems, leaves and shoots of C. bipinnata increased with increasing soil Cd concentration. When the soil Cd concentration was 50?mg?kg^?1, the Cd content in shoots was up to 112.62?mg?kg^?1, which was higher than the Cd-hyperaccumulator critical value. The root and shoot bioconcentration factors exceeded 1 in various Cd treatments, but the translocation factors were less than 1. When the soil Cd concentration was 50?mg?kg^?1, the Cd accumulation in shoots achieved the maximum of 224.30?μg plant^?1. Therefore, considering the tolerance and accumulation of Cd, C. bipinnata is a Cd-accumulator that could be used to remediate Cd-contaminated urban soil.     

皮氏罗尔斯通氏菌株DX-T3-01的耐镉性能及镉富集机理

从江西德兴铜矿土壤中分离筛选到一株具有较强耐镉(Cd)能力的细菌DX-T3-01,经生理生态及16S rRNA分析进行鉴定,研究其对不同浓度Cd2+的耐受特性及对重金属Zn2+、Cu2+、Ni2+的耐受性,进一步利用扫描电镜-能谱仪(SEM-EDX)和红外光谱(FTIR)探讨菌株对Cd2+累积去除的机理.经鉴定,耐镉细菌DX-T3-01为皮氏罗尔斯通氏菌(Ralstonia pickettii);Cd2+最高耐受浓度(MTC)为16 mmol L-1,最低抑制浓度(MIC)为4 mmol L-1,并可耐受一定浓度的Zn2+、Cu2+;菌体积累Cd2+后细胞表面有沉淀物附着并有镉元素检出,对Cd2+的累积主要依靠细胞壁上—PO43-、胺基中的—C—N—、—M—O(O—M—O)、C=O和酰胺基(—CO—NH—)基团.图5参16     

一株菲降解菌的特性及相关降解基因的克隆

从石油污染土壤中分离到一株菲降解菌2F5-2.根据该菌株生理生化特征和16S rDNA序列相似性分析,将其初步鉴定为鞘氨醇杆菌属(Sphingobium sp.).该菌株在10 h内对100 mg/L的菲的降解率为100%.降解菲的最适温度为30℃,最适pH为7.对降解途径的初步研究显示,该菌株通过水杨酸途径降解菲.克隆了编码芳香烃双加氧酶α亚基的基因phdA,它与菌株Sphingomonas sp.P2、Sphingobium yanoikuyae B1、Sphingomonas sp.ZP1中phdA的同源性分别为97.9%、98%和100%,表明该基因具有保守性.图6参16     

Large plasmids governing multiple resistances to heavy metals: A genetic approach∗

Gram negative bacteria classified as Alcaligenes eutrophus and carrying large resistance plasmids (generally two) were found in various industrial sites highly contaminated by heavy metals (Zn⁺⁺, Cu⁺⁺, Co⁺⁺,...). These strains were detected by DNA hybridization with a probe made with a 9kb fragment (ccz⁺ fragment) encoding for resistances to Cd⁺⁺, Co⁺⁺ and Zn⁺⁺, and cloned from plasmid pMOL30. This plasmid was isolated from the representative strain A. eutrophus CH34 which harbours the plasmids pMOL30 (240 kb) and pMOL28 (165 kb). Phenotypes related to pMOL28 and pMOL30 include the tolerance to Cd⁺⁺, Co⁺⁺, Cr0₄ ⁼, Cu⁺⁺, Hg⁺⁺, Ni⁺⁺, Pb⁺⁺ and Zn⁺⁺. The described genetic properties of these plasmids refer to some cloned or mapped functions and to some plasmid rearrangements. Plasmid pMOL85 (250 kb) which is related to pMOL30 was also described. Its host (A. eutrophus DS185) was isolated from a zinc desert. pMOL85 can efficiently self transfer in plasmidfree derivatives.     

Release of Cd immobilized by soil constituents and its bioavailability

Release of Cd immobilized by soil constituents (clay, humic acids, bacterial cells) in vitro and under soil conditions and its bioavailability to lettuce were studied. The most resistant to extraction with 0.02 M EDTA in vitro as well as under soil conditions was cadmium immobilized by humic acids during decomposition of contaminated plant residues but Cd‐dead cells when 0.1 M NaNO₃ was the extractant.

Cadmium addition (3 mg kg^‐1) to soil and its form were without effect on plant growth and amount of metal accumulated in roots. Tops of plants grown in soil supplemented with Cd‐resting cells contained significantly less metal then other ones.     

成团泛菌(Pantoea agglomerans)对镉胁迫下蒌蒿耐受性的影响

为探讨功能微生物对镉(Cd)胁迫下蒌蒿耐受性的影响,以成团泛菌(Pantoea agglomerans)J2为对象,通过盆栽试验,研究不同Cd处理水平下J2菌对蒌蒿生长、生理、Cd富集转运与根际微生态的影响。结果表明:J2菌可显著促进蒌蒿株高、地上部干重、地下部干重、总生物量及根冠比的增加。在J2菌的作用下,蒌蒿叶绿素a、叶绿素b和总叶绿素含量以及超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著增加,丙二醛(MDA)含量明显减少。J2菌可显著促进蒌蒿对Cd的富集转运,蒌蒿地上部、地下部与总Cd含量均显著增加,地上部生物富集系数(BCF)明显大于地下部,转运系数(TF)相比于对照增加了7.5%~37.4%。J2菌可显著增加蒌蒿根际土壤细菌、放线菌数量及微生物总量,明显减少真菌数量。研究表明成团泛菌J2对蒌蒿生长、生理、Cd富集转运与根际微生态可产生积极影响,在一定程度上可提高蒌蒿植株对Cd的耐受性。     

Heavy metal concentration in soil and woody plants in a quarry

This study determined the concentration of three heavy metals zinc (Zn), lead (Pb), and cadmium (Cd) in soil and in a woody plant species, Milicia excelsa, at Ishiagu quarry, Nigeria. The highest soil concentrations of Zn, Pb, and Cd in soil were obtained at 1?m from the quarry site. In M. excelsa, the highest concentrations of Zn, Pb, and Cd were 3.12–9.1, 3.9–6.01, and 0.51–1.12?mg?kg^?1, respectively. There were significant positive correlations between Cd and Zn (r?=?0.963) and Cd and Pb (r?=?0.974) in plants as well as between Cd and Pb (r?=?9.84) in soil. The level of Cd in soil reflected significant pollution compared to average global concentrations in soils.     

Biodegradation of anthracene and phenanthrene by bacteria isolated from oil-contaminated soil of Bangladesh

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants entering into the environment through natural and anthropogenic activities. Owing to their toxicity to various life forms including humans, detoxification of PAHs is crucial to reduce their effects on cells. In this study, we have isolated two bacteria capable of degrading two common PAHs, anthracene and phenanthrene, from contaminated soil samples by using selective enrichment culture supplemented with test PAHs as the sole source of carbon and energy. The isolated bacteria were identified and affiliated as Pseudomonas aeruginosa strain KD and Stenotrophomonas maltophilia strain RC based on their 16s rRNA gene sequences. The degradation of anthracene and phenanthrene was estimated indirectly by measuring the decolourisation extent of a redox indicator, 2, 6-dichlorophenolindophenol, incorporated into PAH-supplemented mineral salt media. In the case of anthracene, ≥90% decolourisation was recorded at 20 and 48 days for P. aeruginosa and S. Maltophilia, respectively. On the other hand, ≥94% decolourisation was recorded at 56 and 52 days for P. aeruginosa and S. maltophilia, respectively during the utilisation of phenanthrene.     

Project Tocoen. the fate of selected pollutants in the environment. Part XIX. the phytotoxicity of organic and inorganic pollutants‐cadmium. The effect of cadmium on the growth of germinating maize plants

The effect of increasing cadmium concentration (10, 100 and 1000 μmol dm^‐3) on the growth, leaf area, content of assimilation pigments, cadmium content and the regulatory ability of the tissue of maize plants was investigated.

The results obtained document, already after 6 days, a significant decrease of dry weight, reduction of leaf area, chlorophyll a and b as well as carotenoids in plants grown in the nutrient solution containing 1000 μmol dm ^‐3 of cadmium. A highly significant inhibition of growth, leaf area and assimilation pigments in plants growing in the nutrient solutions with 100 and 10 μmol dm^‐3 of cadmium was registered after 17 days of cultivation. In plants growing in the nutrient solution containing 100 μmol dm^‐3 of cadmium a demonstrable reduction of the content of assimilation pigments was registered after 11 days of culture.

With increasing cadmium concentration in the nutrient solution as well as in the plant tissue after 6 and 17 days of culture both the range of the regulatory zone and the extent of optimum pH increased into the acid region. The pH values of the isoelectric point decreased with increasing cadmium content in the solution.     

The effect of two different biochars on remediation of Cd-contaminated soil and Cd uptake by Lolium perenne

Biochar can be widely used to reduce the bioavailability of heavy metals in contaminated soil because of its adsorption capacity. But there are few studies about the effects of biochar on cadmium uptake by plants in soil contaminated with cadmium (Cd). Therefore, an incubation experiment was used to investigate the effects of rice straw biochar (RSBC) and coconut shell biochar (CSBC) on Cd immobilization in contaminated soil and, subsequently, Cd uptake by Lolium perenne. The results showed that the microbial counts and soil enzyme activities were significantly increased by biochar in Cd-contaminated soil, which were consistent with the decrease of the bioavailability of Cd by biochar. HOAc-extractable Cd in soil decreased by 11.3–22.6% in treatments with 5% RSBC and by 7.2–17.1% in treatments with 5% CSBC, respectively, compared to controls. The content of available Cd in biochar treatments was significantly lower than in controls, and these differences were more obvious in treatment groups with 5% biochar. The Cd concentration in L. perenne reduced by 4.47–26.13% with biochar. However, the biomass of L. perenne increased by 1.35–2.38 times after adding biochar amendments. So, Cd uptake by whole L. perenne was augmented by RSBC and CSBC. Accordingly, this work suggests that RSBC and CSBC have the potential to be used as a useful aided phytoremediation technology in Cd-contaminated soil.

     

Plant diversity reduces the effect of multiple heavy metal pollution on soil enzyme activities and microbial community structure

It is unclear whether certain plant species and plant diversity could reduce the impacts of multiple heavy metal pollution on soil microbial structure and soil enzyme activities. Random amplified polymorphic DNA (RAPD) was used to analyze the genetic diversity and microbial similarity in planted and unplanted soil under combined cadmium (Cd) and lead (Pb) pollution. A metal hyperaccumulator, Brassica juncea, and a common plant, Festuca arundinacea Schreb, were used in this research. The results showed that microorganism quantity in planted soil significantly increased, compared with that in unplanted soil with Cd and Pb pollution. The order of microbial community sensitivity in response to Cd and Pb stress was as follows: actinomycetes>bacteria>fungi. Respiration, phosphatase, urease and dehydrogenase activity were significantly inhibited due to Cd and Pb stress. Compared with unplanted soil, planted soils have frequently been reported to have higher rates of microbial activity due to the presence of additional surfaces for microbial colonization and organic compounds released by the plant roots. Two coexisting plants could increase microbe population and the activity of phosphatases, dehydrogenases and, in particular, ureases. Soil enzyme activity was higher in B. juncea phytoremediated soil than in F. arundinacea planted soil in this study. Heavy metal pollution decreased the richness of the soil microbial community, but plant diversity increased DNA sequence diversity and maintained DNA sequence diversity at high levels. The genetic polymorphism under heavy metal stress was higher in B. juncea phytoremediated soil than in F. arundinacea planted soil.     

Calanoid copepod eggs in sea-bottom muds. IV. Effects of some environmental factors on the hatching of resting eggs

During cadmium uptake by the shore crab Carcinus maenas (L.), cadmium appears in both haemolymph and urine within 1 h, and comes into equilibrium with the haemolymp after some 60 h. The equilibrium concentration for haemolymph cadmium is always considerably less than that of the external medium, whether the latter is close to background level or at the artificially high concentration of 2.2 mg l^-1. Despite earlier evidence that cadmium entering the haemolymph is mainly bound to protein, current results show most of this to be freely labile and quickly displaced by cadmium taken up subsequently. There is some evidence to suggest that cadmium is passed on from the haemolymph to the hepatopancreas. A large proportion of whole-body cadmium is adsorbed onto the exoskeleton and gills. The comparatively rapid efflux of ¹⁰⁹Cd from loaded crabs over a period of 1 week is mainly due to desorption of this cadmium from the outer body surface.