Isolation and Molecular Characterization of Heavy Metal-Resistant Azotobacter chroococcum from Agricultural Soil and Their Potential Application in Bioremediation

Pollution of soil with heavy metals, herbicides, antibiotics and other chemicals is known to have a negative effect on microbial activities. Therefore, the aim of this study was to isolate cultures of Azotobacter sp. from polluted and unpolluted soils and to study the effect of these pollutants on their growth. A total of 120 Azotobacter sp. were isolated from soils irrigated with wastewater (contaminated soils) and groundwater (uncontaminated soils). These isolates were screened for resistance to heavy metals, herbicide and antibiotics. Also, the soils from which the cultures were isolated were analyzed for the concentrations of Zn²⁺, Cd²⁺, Cu²⁺, Pb²⁺ and Mn²⁺ they contained. Contaminated soil showed high levels of heavy metals as compared to uncontaminated soil. The size of the Azotobacter population in contaminated soil was lower than that in uncontaminated soil. Of the Azotobacter isolates, 64 that were recovered from contaminated soil exhibited high resistance to heavy metals (Hg²⁺, Cd²⁺, Cu²⁺, Cr³⁺, Co²⁺, Ni²⁺, Zn²⁺ and Pb²⁺) and herbicide 2,4-D compared to 56 isolates from uncontaminated soil. Also, isolates from contaminated soil showed high resistance to chloramphenicol, nitrofurantoin and co-trimoxazole compared to those isolated from uncontaminated soil. The majority of Azotobacter isolates from contaminated soil showed multiple-resistance to different metal ions and antibiotics. All isolates failed to grow at pH less than 6. Salt concentration (5%) was found to be inhibitory to all isolates. The most potent isolates from contaminated soil that showed multiresistance to all substances tested were identified on the basis of morphological and biochemical characteristics, and 16S rRNA as A. chroococcum. These resistant isolates could be employed in contaminated soils and/or bioremediation.     

固氮粪产碱菌谷氨酰胺合成酶的分离纯化及其特性

联合固氮细菌粪产碱菌（Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓ）Ａ１５０１菌体经超声破碎后,无细胞粗提液以ＰＥＧ－６０００分级沉淀,丙酮沉淀,再经蓝琼脂糖（ＢｌｕｅＳｅｐｈａｒｏｓｅＣＬ－６８）亲和层析分离、纯化。获得的纯谷氨酰胺合成酶（ＧＳ）在ＳＤＳ－ＰＡＧＥ和４－３０％梯度ＰＡＧＥ上均呈均一的一条带。ＧＳ亚基及整酶分子量分别为５５ｋＤ和６４５ｋＤ,亚基由４５６个氨基酸残基组成。ＧＳ的Ｋｍ值,在以Ｇｌｕ为氮源的介质中培养时分别为２０ｍｍｏｌ／Ｌ（Ｇｌｕ）,５０ｍｍｏｌ／Ｌ（ＡＴＰ）和４５ｍｍｏｌ／Ｌ（ＮＨ~＋_４）;在以ＮＨ~＋_４为氮源的介质中培养时则分别为７０ｍｍｏｌ／Ｌ（Ｇｌｕ）,４９ｍｍｏｌ／Ｌ（ＡＴＰ）和８０ｍｍｏｌ／Ｌ（ＮＨ~＋_４）,表明ＮＨ~＋_４培养下形成高度腺苷化的ＧＳ对Ｇｌｕ及ＮＨ~＋_４的亲和力有所下降。     

N2和NH4^＋培养下粪产碱菌固氮酶钼铁蛋白的合成及其特性

应用柱层析和制备电泳分别将N_2和 NH_4~ 培养的粪产碱菌固氮酶钼铁蛋白(Af 1和Af 1)分离并纯化,两者理化性质十分相似。分子量分别为226 kD和 222 kD;α亚基和β亚基分子量分别为57 kD和60 kD;氨基酸种类相同,总残基数分别为1790和1750;每分子Af 1和Af 1均含有2个原子Mo和32个原子 Fe;金属原子簇的氧化还原当量数为6。Af 1的比活性为 1477 nmolC_2H_4 mg~(-1)protein min~(-1),Af 1无活性;分子结构两者有差异。     

Specific and sensitive detection of Alcaligenes species from an agricultural environment

A quantitative real‐time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10²–10⁴ times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.     

Synthesis and Characterization of Eugenia uniflora L. Silver Nanoparticles and L-Cysteine Sensor Application

L-Cysteine (Cys) is a non-essential sulfur-containing amino acid, crucial for protein synthesis, detoxification, and several metabolic functions. Cys is widely used in the agricultural, food, cosmetic, and pharmaceutical industries. So, a suitable sensitive and selective sensing approach is of great interest, and a low-cost sensor would be necessary. This article presents silver nanoparticles (EuAgNPs) synthesized by a green synthesis method using Eugenia uniflora L. extracts and photoreduction. The nanoparticles were characterized by UV/VIS, transmission electron microscopy, high-performance liquid chromatography (HPLC), FTIR, and Zeta potential. With the addition of Cys in the EuAgNPs solution, the terminal thiol part of L-cysteine binds on the surface of nanoparticles through Ag−S bond. The EuAgNPs and CysAgNPs coexist until flavonoids bound the amino group of Cys, enhancing the red color of solutions. The EuAgNPs provided selectivity to detect Cys among other amino acids, and its detection limit was found to be 3.8 nM. The sensor has the advantages of low-cost synthesis, fast response, high selectivity, and sensitivity.     

Molecular characterization of early colonizer bacteria from wastes in a steel plant

Aims: Forty‐nine bacteria isolated from four newly‐produced waste samples of a steel industry, which had a high content of CaO, MgO, Cr and P₂O₅, were characterized molecularly and phenotypically by susceptibility testing against heavy metals. Methods and Results: Phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to nine genera, Pseudomonas, Micrococcus, Acinetobacter, Bacillus, Dietzia, Kocuria, Diaphorobacter, Staphylococcus and Brevibacillus. Besides, some isolates could be affiliated to species: M. luteus, Ac. junii, Ac. schindleri, B. cereus, K. marina, D. nitroreducens and Staph. warneri. The bacteria that were characterized are taxonomically diverse, and Pseudomonas and Micrococcus predominated. Fingerprinting BOX‐PCR revealed high genomic heterogeneity among the isolates. Among the heavy metal compounds Zn, Ni, Pb and Cu were least toxic to the bacterial isolates, whereas Ag inhibited all isolates at 0·001 mmol l^?1. Conclusions: Heterotrophic bacteria, affiliated with several phylogentic groups, were able to colonize different wastes of a steel industry. Significance and Impact of the Study: This study extends our knowledge of the early colonizers bacteria populating siderurgic environments. Some of these bacteria could have potential for recycling siderurgic waste for steel production.     

极端污染环境草甘膦抗性菌株的分离、鉴定及特性

[目的]筛选高抗草甘膦菌株并对其进行鉴定和特性研究.[方法]从草甘膦极端污染土壤中分离高抗草甘膦菌株,并检测其草甘膦耐受能力,最适生长pH和抗生素抗性.通过生理生化特征和分子生物学特征的测定对该菌株进行鉴定.[结果]从草甘膦极端污染土壤中分离到一株高抗草甘膦的菌株SL06500,该菌株最高耐受草甘膦浓度为500 mmol/L,并且在200～500 mmol/L之间,菌株生长迅速,最适生长pH为4.0,具有氨苄青霉素、卡那霉素、四环素和氯霉素抗性.用16S rDNA的通用引物,经PCR扩增、测序得到SL06500的16S rDNA序列,该序列在GenBank的登录号为EU006066.将此序列经NCBI Blast进行核苷酸比对发现SL06500与无色杆菌属(Achromobacter)和产碱杆菌属(Alcaligenes)的Identity值均为99%.按照1994年版伯杰氏鉴定细菌学手册的命名规则,结合生理生化指标测定的结果,将菌株命名为木糖氧化产碱杆菌木糖氧化亚种SL06500 (Alcaligenes xylosoxidans subsp.xylosoxidans SL06500).[结论]该菌株的较高草甘膦抗性和嗜药性的特点值得我们进行进一步的研究.更重要的是,这是首次关于木糖氧化产碱杆菌木糖氧化亚种草甘膦抗性的报道.     

Isolation and Characterization of A Metal-resistant Pseudomonas Aeruginosa Strain

Summary The use of microorganisms to remove heavy metals from industrial effluent is an area of extensive research and development. Attempts have been made to isolate and characterize metal-resistant microorganisms from treated oil mill industry effluent wastewater samples. The metal-resistant organisms that showed values of minimum inhibitory concentration towards metals (Cd, Cr, Ni and Pb) ranging from 100 to 800 ppm level were screened. A potent metal-resistant organism, isolate BC15 from the wastewater samples was tentatively identified as Pseudomonas sp. Detailed analysis of morphological, biochemical and 16S rDNA sequence of the isolate revealed that it is closely related to Pseudomonas aeruginosa (94%). Pseudomonas BC15 was capable of absorbing 93% Ni, 65% Pb, 50% Cd and 30% Cr within 48 h from the medium containing 100 mg of each heavy metal per liter. The multiple metal tolerance of this strain was also associated with resistance to antibiotics such as ampicillin, tetracycline, chloramphenicol, erythromycin, kanamycin and streptomycin.     

Morphological and Molecular Data Reveal the Presence of the Invasive Artemia franciscana in Margherita di Savoia Salterns (Italy)

Introduced populations of the American invasive Artemia franciscana have been reported in Mediterranean countries except for Italy. A recent sampling at Margherita di Savoia revealed the presence of mating pairs in a saltwork known to host only parthenogens. An integrated approach, based on scanning electron microscopy of four morphological traits, discriminant analysis of 13 morphometric characters and 16S rRNA PCR‐RFLP profiles of eight endonucleases was implemented for the identification of the invader. Patterns of variability in all assayed markers provided congruent and solid evidence that the allochthonous species is A. franciscana. Native parthenogens are still predominant (∼98.4%) in this Italian site but they can be rapidly outcompeted by A. franciscana, as it occurred in similar cases throughout Europe and elsewhere. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Isolation of Bdellovibrio sp. from Wastewater and Their Potential Application in Control of Salmonella paratyphi in Water

The problem of the increasing resistance of bacteria to conventional antibiotics gives the bacteria Bdellovibrio a great utility as a potential alternative source of antibiotics. Therefore, the preliminary goal of the present study was isolation and identification of antibiotic-resistant bacteria used as prey organisms for isolated Bdellovibrio sp., by xylose lysine desoxycholate (XLD) agar from different types of water in the Taif area, Saudi Arabia, and also to investigate water quality. Four antibiotic-resistant isolates of Salmonella sp. which were susceptible to Bdellovibrio were identified by morphological, biochemical and 16S rRNA characterization as Salmonella paratyphi. Seventeen strains of Bdellovibrio sp. were isolated from sewage wastewater using isolated S. paratyphi as prey bacteria by a double-layer plate. Only one of them causing a large plaque after 48 h of incubation at 37°C was designated Bdellovibrio AOA12. The shape of Bdellovibrio was confirmed by morphological characterization and electron microscopy. Bdellovibrio could lyse four antibiotic-resistant Gram-negative bacterial strains of Salmonella paratyphi but could not lyse Gram-positive bacteria such as Staphylococcus aureus and Bacillus cereus. The kinetic lysis of the Bdellovibrio as predator to four isolates of antibiotic-resistant Salmonella paratyphi as prey organisms was demonstrated. The results suggest that it may be possible to utilize Bdellovibrio to control antibiotic-resistant S. paratyphi in water.     

Isolation and Characterization of Thermophilic Bacteria from Geothermal Areas in Turkey and Preliminary Research on Biotechnologically Important Enzyme Production

In this study, the isolation, identification and characterization of the thermophilic bacteria from different hot springs in Turkey were carried out by conventional (morphological, physiological and biochemical tests) and molecular methods (fatty acid methyl esters, GTG5-PCR and 16S rRNA sequencing). These thermophilic bacteria were then tested for their capability to produce enzymes such as lipase, protease, amylase and cellulase. O20 strain is a novel species according to identification studies. All of its isolates were capable of producing industrially valuable enzymes based on screening. In fact, most of them could produce at least two of these enzymes.     

一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

摘要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16S rRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌（Escherichia coli）BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70 mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA     

Isolation and characterization of <Emphasis Type="Italic">Pseudoalteromonas ruthenica</Emphasis> (SBT033), an EPS-producing biofilm bacterium from the seawater intake point of a tropical power station

Biofilm formation in bacteria is closely linked with production of exopolysaccharides (EPS). This study examined the quantitative variations in EPS production and biofilm-forming ability among bacteria isolated from the seawater intake point of a power station located on the east coast of India. Of the 233 isolates obtained from the intake site, 71 bacterial isolates displayed different colony morphological characteristics. Thirteen isolates that produced wide and thick mucoid colonies were further tested for their ability to attach and form biofilms by microtitre plate assay and confocal microscopy. EPS production among the selected bacterial isolates ranged from 826 to 1838 μg ml⁻¹. Strain SBT033, which produced the maximum amount of EPS also displayed the maximum biofilm-forming ability among the 13 isolates. This strain was selected for further characterization using biochemical and molecular methods. The pale orange-pigmented isolate was a Gram negative, aerobic, short rod-shaped and grew well only in the presence of 2% NaCl. On the basis of phenotypic characteristics the isolate SBT033 is shown to belong to the genus Pseudoalteromonas. Analysis of 16S rRNA of the isolate revealed 99% homology with Pseudoalteromonas ruthenica.