Characterization of a fungal strain isolated from a polyphenol polluted site

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.     

Archaeal and bacterial communities of heavy metal contaminated acidic waters from zinc mine residues in Sepetiba Bay

Mining of metallic sulfide ore produces acidic water with high metal concentrations that have harmful consequences for aquatic life. To understand the composition and structure of microbial communities in acid mine drainage (AMD) waters associated with Zn mine tailings, molecular diversity of 16S genes was examined using a PCR, cloning, and sequencing approach. A total of 78 operational taxonomic units (OTUs) were obtained from samples collected at five different sites in and around mining residues in Sepetiba Bay, Brazil. We analyzed metal concentration, physical, chemical, and microbiological parameters related to prokaryotic diversity in low metal impacted compared to highly polluted environments with Zn at level of gram per liter and Cd–Pb at level of microgram per liter. Application of molecular methods for community structure analyses showed that Archaea and Bacteria groups present a phylogenetic relationship with uncultured environmental organisms. Phylogenetic analysis revealed that bacteria present at the five sites fell into seven known divisions, α-Proteobacteria (13.4%), β-Proteobacteria (16.3%), γ-Proteobacteria (4.3%), Sphingobacteriales (4.3%), Actinobacteria (3.2%) Acidobacteria (2.1%), Cyanobacteria (11.9%), and unclassified bacteria (44.5%). Almost all archaeal clones were related to uncultivated Crenarchaeota species, which were shared between high impacted and low impacted waters. Rarefaction curves showed that bacterial groups are more diverse than archaeal groups while the overall prokaryotic biodiversity is lower in high metal impacted environments than in less polluted habitats. Knowledge of this microbial community structure will help in understanding prokaryotic diversity, biogeography, and the role of microorganisms in zinc smelting AMD generation and perhaps it may be exploited for environmental remediation procedures in this area.     

Characterization of Desmodesmus pleiomorphus isolated from a heavy metal-contaminated site: biosorption of zinc

Microalgae have been proven efficient biological vectors for heavy metal uptake. In order to further study their biosorption potential, a strain of Desmodesmus pleiomorphus (L) was isolated from a strongly contaminated industrial site in Portugal. Under different initial Zn²⁺ concentrations, metal removal by that strain reached a maximum of 360 mg Zn/g biomass after 7 days, at 30 mg Zn/l, after an initial rapid phase of uptake. Comparative studies were carried out using a strain of the same microalgal species that is commercially available (ACOI 561): when exposed to 30 mg Zn/l, it could remove only 81.8 mg Zn/g biomass. Biosorption experiments using inactivated biomass of the isolated strain reached a maximum Zn²⁺ uptake of 103.7 mg/g. Metal removal at various initial pH values was studied as well; higher removal was obtained at pH 5.0. The microalga strain L, isolated from the contaminated site, exhibited a much higher removal capacity than the commercial strain, and the living biomass yielded higher levels of metal removal than its inactivated form.     

Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterial consortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia). Microorganisms identified in the consortium were Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp. In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l⁻¹ of methyl parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l⁻¹ of both pesticides was used the percentage decreased to 72% for methyl parathion and 39% for chlorpyrifos. With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l⁻¹ of the pesticides in the mixture. 4 treatments were carried out in soil that included the addition of glucose with microorganisms, the addition of sugar cane with microorganisms, microorganisms without nutrient addition and without the addition of any item. In the treatment in which glucose was used, degradation percentages of methyl parathion and chlorpyrifos of 98% and 97% respectively were obtained in 120 h. This treatment also achieved the highest percentage of reduction in toxicity, monitored with Vibrio fischeri.     

Comparative proteomic approach to identify proteins involved in flooding combined with salinity stress in soybean

Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean seedlings at the emergence (VE) stage were treated with 100 mM NaCl and flooded with water or 100 mM sodium chloride solution for 2 days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome level that are both specific and part of a common or shared response. The identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to a combination of flooding and salinity.     

New developments in treatment of heavy metal contaminated soils

Contamination of soil and groundwater by heavymetals is a widespread problem due to theformer activities of metal processing, sufracetreatment and mining industry and theuncontrolled dumping of waste in landfills.Several methods do exist to treat soil andgroundwater. This paper will pay attention tosome new developed methods based on removal ofthe metals from the soil (simultaneousextraction and binding to biomass) or thegorundwater (aboveground treatment by sulphatereducing bacteria). Due to the fact that veryoften large areas are affected by heavy metalcontamination a removal is difficult. Thereforesome methods are developed to keep the metalsin the soil but reduce the risks related tothis presence. This risk reduction is based ona decrease in bioavailability by in situimmobilisation processes. These in situimmobilisation processes allow the treatment oflarge diffusely contaminated areas. Twoapproaches are presented. The first approach isbased on the addition of soil additives toimmobilize the metals. In this case specialattention is paid to the the biologicalevaluation methods of the reduction in metalbioavailability. The second approach uses againSRBs for the in situ precipitation ofmetal sulphides.     

Characterization of polychlorinated biphenyl-degrading bacteria isolated from contaminated sites in Czechia

Biphenyl-utilizing polychlorinated biphenyls (PCB)-degrading bacteria were isolated from sites highly contamined by PCBs, and their degradation abilities were determined using GC for typical commercial PCB mixtures (Delor 103 and Delor 106). Out of twelve strains which utilized biphenyl as a sole source of carbon and energy, strainsPseudomonas alcaligenes KP2 andP. fluorescens KP12, characterized by the BIOLOG identification system and the NEFERM test, were shown to significantly co-metabolize the PCB mixture Delor 103. DNA-DNA hybridization was used to compare both strains with well-known PCB-degradersBurkholderia cepacia strain LB400 andRalstonia eutropha strain H850. The strain KP12 employs the samemeta-fission route for degradation of chlorobenzoates as a chlorobiphenyl degraderPseudomonas cepacia P166. Both isolates KP2 and KP12 belong to different phylogenetic groups, which indicates that the same geographical location does not ensure the same ancestor of degradative enzymes. We confirmed that also highly chlorinated and the most toxic congeners, which are contained in commercial PCB mixtures, can be biotransformed by members of indigenous bacterial-soil community under aerobic conditions.     

Glycogen-lead relationship in the earthworm Dendrobaena rubida from a heavy metal site

Summary Control individuals contained no lead in the chloragocytes but high -glycogen rosette reserves. Starvation of contaminated earthworms for 4d caused a lead loss and the chloragocytes possessed fewer debris vesicles than those of unstarved worms, suggesting that the debris vesicles may be the route for at least some of the lead loss. No glycogen deposits were observed in the chloragocytes of starved or unstarved earthworms from contaminated soil. Maintenance of contaminated earthworms in potting compost caused lead losses similar to those sustained by starvation, but the chloragocyte cytoplasm possessed -glycogen reserves. Specimens maintained in lead-spiked potting compost showed lead levels similar to those of earthworms taken directly from contaminated soil. No -glycogen accumulations were observed under this enriched regime.Although the possible interference of lead in carbohydrate metabolism is discussed, the results do not wholly support metabolic inhibition by lead. It is hypothesised that lead sequestration is energy-demanding and that in the absence of an energy-rich diet glycogen stores fail to accumulate. In the presence of an organic-rich medium, elevated lead levels preclude glycogen formation, because of the high sequestration-demand, but at lower lead levels -glycogen deposits occur if a high organic diet is available.     

Glycogen-lead relationship in the earthworm Dendrobaena rubida from a heavy metal site

Control individuals contained no lead in the chloragocytes but high alpha-glycogen rosette reserves. Starvation of contaminated earthworms for 4d caused a lead loss and the chlorgocytes possessed fewer debris vesicles than those of unstarved worms, suggesting that the debris vesicles may be the route for at least some of the lead loss. No glycogen deposits were observed in the chloragocytes of starved or unstarved earthworms from contaminated soil. Maintenance of contaminated earthworms in potting compost caused lead losses similar to those sustained by starvation, but the chloragocyte cytoplasm possessed beta-glycogen reserves. Specimens maintained in lead-spiked potting compost showed lead levels similar to those of earthworms taken directly from contaminated soil. No beta-glycogen accumulations were observed under this enriched regime. Although the possible interference of lead in carbohydrate metabolism is discussed, the results do not wholly support metabolic inhibition by lead. It is hypothesised that lead sequestration is energy-demanding and that in the absence of an energy-rich diet glycogen stores fail to accumulate. In the presence of an organic-rich medium, elevated lead levels preclude glycogen formation, because of the high sequestration-demand, but at lower lead levels beta-glycogen deposits occur if a high organic diet is available.     

A proteomic approach to apoplastic proteins involved in cell wall regeneration in protoplasts of Arabidopsis suspension-cultured cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines

Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

Composition of the saprophytic bacterial communities in freshwater systems contaminated by heavy metals

The bacterial communities of three aquatic systems were analyzed in order to compare the influence of heavy metals. The first system was a sedimentation pond in a zinc-copper factory. The second was the bank of the Belgian river Meuse covered by the mossPlatyhypnidium riparioides (Hedw.) Dix. contaminated with heavy metals. The third was the bank of the same river covered by the same uncontaminated moss.The study was focused mainly on cadmium.The reciprocal averaging method showed that some bacterial strains could develop in very high concentrations of cadmium, but their physiological characteristics were not the same as those of the sensitive strains. In addition, the characteristics of the resistant strains depended on the environment. Correlation between resistance to heavy metals and to antibiotics was observed but was not the same in all communities. The density of resistant strains was roughly related to the level of toxicity in the environment.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Effect of heavy metal-solubilizing microorganisms on zinc and cadmium extractions from heavy metal contaminated soil with Tricholoma lobynsis

The macrofungus, Tricholoma lobynsis, was chosen to remedy Zn–Cd–Pb contaminated soil. To enhance its metal-extracting efficiency, two heavy metal resistant microbes M6 and K1 were applied owing to their excellent abilities to solubilize heavy metal salts. The two isolated microbial strains could also produce indole acetic acid (IAA), siderophore and solubilize inorganic phosphate, but neither of them showed 1-aminocyclopropane-1-carboxylate deaminase activity. The strains M6 and K1 were identified as Serratia marcescens and Rhodotorula mucilaginosa based on 16S rDNA and ITS sequence analysis respectively. Pot experiment showed that spraying to T. lobynsis-inoculated soil with M6 and K1 respectively could increase total Cd accumulations of this mushroom by 216 and 61%, and Zn by 153 and 49% compared to the uninoculated control. Pb accumulation however, was too low (<1 mg kg⁻¹) to be determined. The results illustrated that special microbes and macrofungi can work together to remedy polluted soil as plant and plant growth promoting microbes do, probably because of excellent metal-accumulating abilities of macrofungi and IAA-siderophore production, phosphate solubilization abilities of the assisted-microbes. This kind of macrofungi-microbe interaction can be developed into a novel bioremediation strategy.     

Characterization of microtubule-associated proteins isolated from bovine adrenal gland

Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e.     

Identification of disease-associated proteins by proteomic approach in ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic systemic inflammatory disorder of the axial skeleton and shows significant inherited susceptibility. Auto-immune responses have been traditionally considered as a putative pathogenetic event in AS. However, no consistent self-antigen has been identified to responsible for the disorders in AS to this day. In this study, serum protein profiles of AS patients and healthy controls from a large Chinese AS family were investigated by two dimensional electrophoresis analysis. A group of four highly expressed protein spots was observed in all AS patients' profiles and subsequently identified as isoforms of haptoglobin precursor (pre-Hp) by ESI-Q-TOF MS/MS. Increased expression of haptoglobin precursor were also observed in sera of sporadic AS patients. Moreover, bioinformatics analysis revealed epitopes derived from haptoglobin precursor with high affinity binding to HLA-B( *)2705, a primary subtype associated with AS. These results indicate that pre-Hp may be involved in the pathogenesis of AS.