Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Silicon and phosphorus linkage with iron via oxygen in the amorphous matrix of Gallionella ferruginea stalks

Bacterial species belonging to the genus Gallionella are Fe-oxidizing bacteria that produce uniquely twisted extracellular stalks consisting of iron-oxide-encrusted inorganic/organic fibers in aquatic environments. This paper describes the degree of crystallinity of Gallionella stalks and the chemical linkages of constituent elements in the stalk fibers. Transmission electron microscopy revealed that the matrix of the fiber edge consisted of an assembly of primary particles of approximately 3 nm in diameter. Scanning transmission electron microscopy revealed the rough granular surfaces of the fibers, which reflect the disordered assembly of the primary particles, indicating a high porosity and large specific surface area of the fibers. This may provide the surface with broader reactive properties. X-ray diffractometry, selected-area electron diffraction, and high-resolution transmission electron microscopy together showed that the primary particles had an amorphous structure. Furthermore, energy-dispersive X-ray analysis and Fourier transform infrared spectroscopy detected the bands characteristic of the vibrational modes assigned to O-H, Fe-O-H, P-O-H, Si-O-H, Si-O-Fe, and P-O-Fe bonds in the stalks, suggesting that the minor constituent elements P and Si could affect the degree of crystallinity of the fibers by linking with Fe via O. This knowledge about the mutual associations of these elements provides deeper insights into the unique inorganic/organic hybrid structure of the stalks.     

Ferric ions involved in the flower color development of the Himalayan blue poppy, Meconopsis grandis

The Himalayan blue poppy, Meconopsis grandis, has sky blue-colored petals, although the anthocyanidin nucleus of the petal pigment is cyanidin. The blue color development in this blue poppy involving ferric ions was therefore studied. We analyzed the vacuolar pH, and the organic and inorganic components of the colored cells. A direct measurement by a proton-selective microelectrode revealed that the vacuolar pH value was 4.8. The concentrations of the total anthocyanins in the colored cells were around 5mM, and ca. three times more concentrated flavonols were detected. Fe was detected by atomic analysis of the colored cells, and the ratio of Fe to anthocyanins was ca. 0.8 eq. By mixing the anthocyanin, flavonol and metal ion components in a buffered aq. solution at pH 5.0, we were able to reproduce the same blue color; the visible absorption spectrum and CD were identical to those in the petals, with Fe(3+), Mg(2+) and flavonol being essential for the blue color. The blue pigment in Meconopsis should be a new type of metal complex pigment that is different from a stoichiometric supramolecular pigment such as commelinin or protocyanin.     

Characterization of membrane foulants in a pilot-scale powdered activated carbon–membrane bioreactor for drinking water treatment

The external and internal foulants of a pilot-scale powdered activated carbon–membrane bioreactor (PAC–MBR) used for drinking water treatment were systematically examined by scanning electron microscopy (SEM), three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and energy diffusive X-ray (EDX) analysis. The results showed that external fouling, which comprised 31.68% of the total fouling, was caused by the deposition of a large amount of biological PAC on the membrane surface. Bacteria and organic matter comprised only a small fraction of the external foulants. Biologically derived proteins and polysaccharides were the major constituents of the internal foulants. EDX analysis indicated that the external and internal foulants also included inorganic elements such as Mg, Al, Si, Ca, Mn and Fe. During the operation of PAC–MBR, low flux and effective physical cleaning protocols should be adopted; proteins, polysaccharides and inorganic elements in the bioreactor should also be controlled.     

Reduction of Prussian Blue by the two iron-reducing microorganisms Geobacter metallireducens and Shewanella alga

Cyanide or cyanide-metal complexes are frequent contaminants of soil or aquifers at industrial sites, which can be released from such sites by outgassing or transport with the groundwater. They form very stable complexes with iron, which may occur in the subsurface as an insoluble blue mineral, the so-called Prussian Blue (Fe(4)[Fe(CN)(6)](3)). In this study, we show that the insoluble and colloidal Fe(III)-cyanide complex Prussian Blue can be reduced and utilized as electron acceptor by the dissimilatory iron-reducing bacteria Geobacter metallireducens and Shewanella alga strain BrY. The microbial reduction of the dark blue pigment Prussian Blue leads to the formation of a completely colourless solid mineral, presumably Prussian White (Fe(2)[Fe(CN)(6)]), which could be reoxidized through exposure to air, regaining the dark blue colour. In addition, the microorganisms were able to grow with Prussian Blue, using it as the sole electron acceptor. Geobacter metallireducens could also reduce Prussian Blue coatings on sand, which was sampled from a contaminated site.     

Cloning and expression of a Ralstonia eutropha HF39 gene mediating indigo formation in Escherichia coli

On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.     

Assessing the impact of ancient Maya animal use

Tropical forest animals are at high risk worldwide as a result of over-exploitation and forest clearing. Zooarchaeological studies of animal use by the ancient Maya of the southern lowland regions of Guatemala, Honduras, Belize, and Mexico provide long-term historical information on animal populations under conditions of human population growth and climatic change that is valuable to both archaeology and conservation biology. In this paper, zooarchaeological data from 35 chronologically defined faunal sub-samples recovered from 25 ancient Maya archaeological sites are used to assess the effects of ancient hunting on animal populations of the Maya region between the Preclassic and Colonial periods (2000 BC–AD 1697). The variations in species abundance are used as a proxy for describing changes in ancient Maya hunting practices and hunted animal populations, interpreted on the basis of hunting efficiency models from foraging ecology. A significant reduction in the proportion of large mammals, particularly Odocoileus virginianus, in zooarchaeological assemblages between the Late Classic (AD 600–850) and Terminal Classic/Postclassic periods (AD 850–1519) suggest that over-hunting during the Late Classic may have led to a reduction in availability of these animals to the ancient Maya hunters in the later periods. This finding is discussed in relation to important social and environmental variations to evaluate the impact of hunting and other factors such as forest clearance and climate on ancient animal populations in the Maya region.     

Burkholderia sp. IDO3中靛蓝合成基因的克隆表达及其合成特性

【目的】克隆和表达靛蓝合成基因,并将其用于靛蓝合成研究。【方法】对菌株Burkholderia sp.IDO3中靛蓝合成基因进行克隆和大肠杆菌异源表达,构建能合成蓝色色素的基因工程菌。利用液相色谱和质谱对产物进行分析,采用单因素法对培养温度、转速、培养基成分等进行优化,并考察优化条件下的靛蓝合成曲线。【结果】构建了一株重组大肠杆菌E.coli IND_AB,该菌株能够在LB培养基生长的过程中合成蓝色色素,产物分析表明该色素为靛蓝;菌株IND_AB在30°C和150 r/min条件下能在LB培养基中合成22.9 mg/L靛蓝,优化培养条件后产量达到25.4 mg/L;优化LB培养基各组分浓度后产量可提高到35.1 mg/L;外加50.0 mg/L吲哚或0.1 g/L色氨酸后靛蓝产量可分别提高到57.7 mg/L和64.4 mg/L,相比初始产量提高了152.0%和181.2%;靛蓝合成曲线表明在添加吲哚或色氨酸的培养基中,菌株IND_AB前6 h没有靛蓝生成,6-15 h为靛蓝合成加速期,18 h达到产量平衡。【结论】重组大肠杆菌IND_AB可用于生物合成高纯度靛蓝,为靛蓝的微生物合成提供了有效的基因资源。     

Long-Term Fertilization Modifies the Structures of Soil Fulvic Acids and Their Binding Capability with Al

The binding characteristics of organic ligands and minerals in fulvic acids (FAs) with Al are essential for understanding soil C sequestration, remain poorly understood. In this study, Fourier transform infrared (FTIR) spectroscopy combined with two-dimensional correlation spectroscopy (2DCOS) analysis was applied for the first time to explore the binding of Al with organic ligands and minerals in soil FAs. For these analyses, two contrasting treatments were selected from a long-term (i.e., 22-year) fertilization experiment: chemical (NPK) fertilization and swine manure (SM) fertilization. The results showed that the long-term application of organic and inorganic fertilizers to soils had little effect on the compositions of the fluorescent substances and organic ligands in the soil FAs. However, long-term SM fertilization increased the weathered Al and Si concentrations in the soil FAs compared with long-term chemical fertilization. Furthermore, organic ligands in the soil FAs were mainly bound with Al in the NPK treatment, whereas both organic ligands and minerals (Al-O-Si, Si-O) were bound with Al under the M fertilization conditions. Both transmission electron microscopy (TEM) images and X-ray diffraction spectra demonstrated that amorphous and short-range-ordered nanominerals were abundant in the soil FAs from the SM plot in contrast to the soil FAs from the NPK plot. This result illustrates the role nanominerals play in the preservation of soil FAs by during long-term organic fertilization. In summary, the combination of FTIR and 2D correlation spectroscopy is a promising approach for the characterization of the binding capability between soil FAs and Al, and a better understanding FA-Al binding capability will greatly contribute to global C cycling.     

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.

     

Adhesion of Pseudomonas putida onto Palygorskite and Sepiolite Clay Minerals

Adhesion of bacteria to clay minerals is of great importance in both natural soil environments and technological applications. In the present study, equilibrium experiments along with Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and energy-dispersive X-ray analysis (EDX) were used to investigate the adhesion of Pseudomonas putida to the palygorskite and sepiolite clay minerals. The results showed that bacterial adhesion was rapid and reached equilibrium within 30 min. Equilibrium data showed that sepiolite has higher capacity and affinity than palygorskite for P. putida retention. Mixed FT-IR peak features of the clay minerals and P. putida were observed in the clay–bacteria complex spectra, indicating significant adhesion of P. putida to the minerals. However, some differences in the position of the individual bands were observed between infrared spectra obtained for pure bacteria or clay minerals and their corresponding clay–bacteria complexes, which are believed to be related to clay–bacteria interactions via different mechanisms. SEM/EDX analysis demonstrated fine fibrous clay particles adhered to the surface of individual P. putida cells. The results revealed rapid and close interactions of palygorskite and sepiolite with P. putida cells, which is important for better understanding the fate of bacteria in soil systems dominated by fibrous clay minerals and their practical applications in bioengineering and biotechnology.     

Synthesis and characterization of a novel polyamidoamine-cyclodextrin crosslinked copolymer

A novel insoluble crosslinked copolymer containing β-cyclodextrin (β-CD) structural units has been synthesized with polyamidoamine (PAMAM, generation 2) as comonomer. The polymer was characterized using Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), elemental analysis, scanning electron microscopy (SEM), pores and surface area analysis, X-ray diffraction analysis (XRD), and thermal analysis (thermogravimetric and differential scanning calorimetric measurement, TG/DSC). The results reveal that PAMAM-CD copolymer has been synthesized successfully and two β-CD molecules were cross-linked by one PAMAM (G2.0) molecule (on average). The copolymer has a reef-like surface with many irregular nanocavities, and its thermal stability is > 180°C in an argon atmosphere. The synthesis strategy presented in this work provides an innovative route for the synthesis of a PAMAM-CD-based copolymer. In preliminary sorption experiments, the PAMAM-CD copolymer exhibits high sorption capacities and high removal efficiencies toward both the heavy-metal ions (Cu(2+) and Pb(2+)) and organic compounds (2,4-dichlorophenol, 2,4,6-trichlorophenol, and ponceau 4R (C.I. 16255)). The polymer may provide many possibilities for applications in biomedical sensing, flocculation, sorption, and therapeutics.     

Crystal Structure of the Complex of Concanavalin A and Hexapeptide

The X-ray structure analysis of a cross-linked crystal of concanavalin A soaked with a hexapeptide molecule as a probe molecule showed an electron density corresponding to full occupation in the binding pocket. The site lies on the surface of concanavalin A and is surrounded by three symmetry-related molecules. The crystal structure of the hexapeptide complex was refined at 1.93- resolution, to an R-factor of 19% (R _free factor of 25%), with an RMS deviation in bond distances of 0.01 . The model includes all 237 residues of concanavalin A, one manganese ion, one calcium ion, 95 water molecules, one glutaraldehyde molecule, one isopropanol molecule, and one hexapeptide molecule. This X-ray structure analysis also provides an approach to mapping the binding surface of crystalline protein with a probe molecule that is dissolved in the mixture of organic solvent with water or in neat organic solvent but is hardly dissolved in aqueous solution.     

大肠杆菌异源表达的indigoidine与靛蓝的稳定性比较

【背景】Indigoidine是一种来源于微生物的无毒天然蓝色素。【目的】比较Indigoidine和靛蓝的色素稳定性,进而评价Indigoidine的色素稳定性。【方法】构建重组菌株Escherichia coli DH5α/p28s异源表达Indigoidine,考察可见光、紫外线、pH、温度、氧化还原剂、食品添加剂和金属离子对其与商品级靛蓝色素稳定性的影响。【结果】以N,N-二甲基甲酰胺为溶剂,Indigoidine和靛蓝都对可见光、紫外线敏感;2种色素在pH1.0-11.0时稳定,强碱性pH对色素破坏作用很大;Indigoidine抗Vc还原能力强于靛蓝,氧化剂可不同程度地降低2种色素的保存率;2种色素热稳定性不佳,在75℃以下时Indigoidine的色素稳定性优于靛蓝;食品添加剂中的柠檬酸和苯甲酸分别对Indigoidine和靛蓝均具有显著的护色效果;对这2种色素,Ca²⁺、Mg²⁺均具有护色效果,Na⁺、K⁺、Li⁺总体上没有明显的破坏作用,而Zn²⁺、Al³⁺、Cu²⁺、Fe²⁺、Fe³⁺则具有显著的破坏作用。【结论】Indigoidine色素稳定性明显优于靛蓝,具有广阔的开发应用前景。     

Crystal structure of the complex of concanavalin A and tripeptide

The X-ray structure analysis of a cross-linked crystal of concanavalin A soaked with the tripeptide molecule as the probe molecule showed electron density corresponding to full occupation in the binding pocket. The site lies on the surface of concanavalin A and is surrounded by three symmetry-related molecules. The crystal structure of the tripeptide complex was refined at 2.4-Å resolution to an R-factor of 17.5%, (R_free factor of 23.7%), with an RMS deviation in bond distances of 0.01 Å. The model includes all 237 residue of concanavalin A, 1 manganese ion, 1 calcium ion, 161 water molecules, 1 glutaraldehyde molecule, and 1 tripeptide molecule. This X-ray structure analysis also provides an approach to mapping the binding surface of crystalline protein with a probe molecule that is dissolved in a mixture of organic solvent with water or in neat organic solvent but is hardly dissolved in aqueous solution.     

Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo.     

Identification of indigo-related pigments produced by Escherichia coli containing a cloned Rhodococcus gene.

Pigments produced by Escherichia coli containing a cloned piece of DNA from Rhodococcus sp. ATCC 21145 were extracted in chloroform and separated into blue and pink components. Evidence from TLC, NMR spectroscopy, absorption spectrum analysis and solubility behaviour suggested that the blue pigment was indigo and the pink pigment was indirubin, a structural isomer of indigo. The proposed pathway for pigment production on LB agar involves the conversion of tryptophan to indole by tryptophanase of E. coli and the oxidation of indole to indigo by the product of the cloned Rhodococcus DNA insert.     

Microscale Characterization and Trace Element Distribution in Bacteriogenic Ferromanganese Coatings on Sand Grains from an Intertidal Zone of the East China Sea

An ancient wood layer dated at about 5600 yr BP by accelerator mass spectrometry (AMS) ¹⁴C was discovered in an intertidal zone of the East China Sea. Extensive and horizontally stratified sediments with black color on the top and yellowish-red at the bottom, and some nodule-cemented concretions with brown surface and black inclusions occurred in this intertidal zone. Microscale analysis methods were employed to study the microscale characterization and trace element distribution in the stratified sediments and concretions. Light microscopy, scanning electron microscopy (SEM) and backscattered electron imaging (BSE) revealed the presence of different coatings on the sand grains. The main mineral compositions of the coatings were ferrihydrite and goethite in the yellowish-red parts, and birnessite in the black parts using X-ray powder diffraction (XRD). SEM observations showed that bacteriogenic products and bacterial remnants extensively occurred in the coatings, indicating that bacteria likely played an important role in the formation of ferromanganese coatings. Post-Archean Australian Shale (PAAS)-normalized middle rare earth element (MREE) enrichment patterns of the coatings indicated that they were caused by two sub-sequential processes: (1) preferentially release of Fe-Mn from the beach rocks by fermentation of ancient woods and colloidal flocculation in the mixing water zone and (2) preferential adsorption of MREE by Fe-Mn oxyhydroxides from the seawater. The chemical results indicated that the coatings were enriched with Sc, V, Cr, Co, Ni, Cu, Zn, Ba, especially with respect to Co, Ni. The findings of the present study provide an insight in the microscale features of ferromanganese coatings and the Fe-Mn biogeochemical cycling during the degradation of buried organic matter in intertidal zones or shallow coasts.     

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora,Prostomatea)

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.     

Aluminium reactions with polygalacturonate and related organic ligands

Aluminium (Al), in inorganic monomeric forms, has been recognised as a limiting factor for root growth in many acid soils. Plant tolerance to Al may be achieved by the detoxification (complexation) of Al by organic ligands present in the rhizosphere. The Al-complexing ability of seven organic ligands, citric, oxalic, gluconic, glucuronic, mucic, galacturonic and polygalacturonic (pectin) acids, was investigated. The proportion of organically-complexed Al was determined using colorimetric methods based on differences in reaction rate with pyrocatechol violet or aluminon. The colorimetric methods confirmed that citric acid forms a strong complex with Al at pH 4.2. In contrast, pectin and related organic ligands weakly complexed Al in acidic conditions. In an additional study, the Al-binding ability of pectin and Ca-pectate was compared at a biologically significant concentration of 32 µM Al. Only 29% of free Al remained in solution in the presence of Ca-pectate, while 54% remained when pectin was present. This suggests that Ca-pectate, rather than pectin, is responsible for binding Al in root cell walls and consequently plays an important role in Al toxicity to plants. Root growth of mungbean (Vigna radiata (L.) Wilczek) confirmed differences in the ability of citrate, oxalate and galacturonate to complex Al.