Cadmium(II) Removal by a Hyperaccumulator Fungus <Emphasis Type="Italic">Phoma</Emphasis> sp. F2 Isolated from Blende Soil

A cadmium(II)-resistant fungus, strain F2, isolated from blende soil was identified as Phoma sp. by morphological study and internal transcribed spacer sequencing. This strain could accumulate 280 mg of Cd(II)/g dry weight mycelium. In liquid medium containing 163.8 mg Cd(II)/L, 96% of Cd(II) was removed by the actively growing mycelium. In addition, both oven-dried and lyophilized mycelium could effectively adsorb Cd(II). There were removed 91% and 46.2% of Cd(II) from 51.6 mg Cd(II)/L solution by lyophilized biomass and oven-dried biomass respectively. Transmission electron microscopy and energy-dispersive X-ray analysis showed the accumulation of Cd(II) in the mycelium cell walls. Our results demonstrated that Phoma sp. F2 was a hyperaccumulator for the removal of Cd(II) from contaminated soil and water.     

Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Kinetic and Equilibrium Studies of Cr(VI) Biosorption by Dead <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Biomass

Many studies have been carried out on the biosorption capacity of different kinds of biomass. However, reports on the kinetic and equilibrium study of the biosorption process are limited. In our experiments, the removal of Cr(VI) from aqueous solution was investigated in a batch system by sorption on the dead cells of Bacillus licheniformis isolated from metal-polluted soils. Equilibrium and kinetic experiments were performed at various initial metal concentrations, pH, contact time, and temperatures. The biomass exhibited the highest Cr(VI) uptake capacity at 50°C, pH 2.5 and with the initial Cr(VI) concentration of 300 mg/g. The Langmuir and Freundlich models were considered to identify the isotherm that could better describe the equilibrium adsorption of Cr(VI) onto biomass. The Langmuir model fitted our experimental data better than the Freundlich model. The suitability of the pseudo first-order and pseudo second-order kinetic models for the sorption of Cr(VI) onto Bacillus licheniformis was also discussed. It is better to apply the pseudo second-kinetic model to describe the sorption system.     

Biosorption of heavy metals by the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis>

The biosorption of several toxic heavy metals (Pb, Cd, Co, Ni, Zn and Cu) by the exopolysaccharide (EPS) produced by Paenibacillus jamilae, a potential biosorbent for metal remediation and recovery was studied. Firstly, the biochemical composition of this bacterial polymer was determined. Glucose was the most abundant neutral sugar, followed by galactose, rhamnose, fucose and mannose. The polymer presented a high content of uronic acids (28.29%), which may serve as binding sites for divalent cations. The presence of carboxylic groups was also detected by infrared spectroscopy. The EPS presented an interesting affinity for Pb in comparison with the other five metals. Lead biosorption (303.03 mg g⁻¹) was tenfold higher (in terms of mg of metal adsorbed per gram of EPS) than the biosorption of the rest of metals. Biosorption kinetics, the effect of pH and the effect of competitive biosorption were determined. Finally, we found that the EPS was able to precipitate Fe(III), but the EPS-metal precipitate did not form with Fe(II), Pb(II), Cd(II), Co(II), Ni(II), Cu(II) and Zn(II).     

Biosorption of Co<Superscript>2+</Superscript> ions by lichen <Emphasis Type="Italic">Hypogymnia physodes</Emphasis> from aqueous solutions

Cobalt is one of the possible contaminants originating from radioactive wastes or from metal mines and refineries. This paper describes sorption of cobalt by the foliose lichen Hypogymnia physodes from CoCl₂ solutions spiked with ⁶⁰Co²⁺ in laboratory experiments. Maximum uptake was reached within 1 hour; the biosorption after 24 hours is not pH-dependent within the range of pH 4–7, negligible at pH 2 and is not dependent on metabolic activity. The process can be described by the Freundlich adsorption isotherm with ln k = 2.77, 1/n = 0.22 and R ² = 0.94. Bivalent metal ions showed a concentration-dependent competitive effect on cobalt biosorption, decreasing in the order: Cu > Ni > Ca > Mg. Monovalent ions, such as K⁺ and Na⁺, showed only very weak competitive effect. Up to 98% of Co taken up by lichen can be removed by washing with 0.1 M NiCl₂ at 20°C. This means that only a small fraction of the cobalt is localized intracellularly. These results can be used for elucidating the behaviour of lichens as bioindicators of cobalt pollution in water systems, including the risk of cobalt leakage from lichen probes under the influence of rain, snow and atmospheric humidity.     

Kinetics and thermodynamics of a novel endoglucanase (CMCase) from <Emphasis Type="Italic">Gymnoascella </Emphasis><Emphasis Type="Italic">citrina</Emphasis> produced under solid-state condition

Gymnoascella citrina produced two isoforms of endoglucanases (CMCase-I and -capital I, Ukrainiancapital I, Ukrainian) under solid-state condition. Purified CMCase-I was novel because it was apparently holoenzyme in nature. The enzyme was monomeric as its native and subunit mass were almost the same, i.e., 43 and 42 kDa, respectively. Ea for carboxymethylcellulose (CMC) hydrolysis was 36.2 kJ mol(-1). The enzyme was stable over a pH range of 3.5-6.5, while temperature optimum was 55 degrees C. Vmax, Km and k (cat )for CMC hydrolysis were 39 U mg(-1) protein, 6.25 mg CMC mL(-1) and 27.5 s(-1), respectively. The pKa1 and pKa2 of ionizable groups of active site were 2.8 and 7.4, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: DeltaH*=33.5 kJ mol(-1), DeltaG*=70.42 kJ mol(-1) and DeltaS*=-114.37 J mol(-1) K(-1). The removal of metals resulted into complete loss of enzymatic activity and was completely recovered in the presence of 1 mM Mn2+, whereas inhibition initiated at 5 mM. The other metals like Ca2+, Zn2+ and K1+ showed no inhibition up to 7 mM, Co2+ completely inhibited the activity, while Mg2+ could not recover the initial activity up to 7 mM. So we are reporting for the first time, kinetics and thermodynamics of CMCase-Iota from G. citrina.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

<Emphasis Type="Italic">Elliptochloris bilobata</Emphasis> var. <Emphasis Type="Italic">corticola</Emphasis> var. nov. (Trebouxiophyceae,Chlorophyta), a novel subaerial coccal green alga

We investigated a previously unidentified subaerial corticolous strain of the genus Elliptochloris Tschermak-Woess. The alga shares the generic morphological characters with Elliptochloris bilobata, the type species of the genus, but it has a thicker cell wall of adult globular cells, different chloroplast structure and it also differs in shape of elliptical autospores. The differences of the autospore shape between both species were evaluated using landmark-based geometric morphometrics. The 18S rDNA gene sequence of the new alga forms a monophyletic clade with the authentic strain of E. bilobata within the green algal class Trebouxiophyceae close to representatives of the genus Coccomyxa. We describe the new alga as Elliptochloris bilobata var. corticola var. nov. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Alanine racemase from the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Summary. Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 μmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.     

<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> infections in captive macaques (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>) in the Philippines

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.     

Cultivation of the edible green seaweed <Emphasis Type="Italic">Gayralia</Emphasis> (Chlorophyta) in southern Brazil

During the last two decades, the monostromatic green seaweed Gayralia sp. has been harvested sporadically by local fishermen on the Paraná coast of southern Brazil and sold to Japanese restaurants. However, the production is erratic and its economic impact very small. This paper provides basic information about a technique to cultivate this seaweed on suspended nets in Paranaguá Bay, southern Brazil, aiming to develop a more reliable and sustainable source of income for impoverished coastal dwellers. Gayralia sp. occurs year round in the region, usually growing on mangrove stems and roots. Polypropylene nets (10 m long × 1 m wide with 16 cm mesh) were placed close to the mangrove fringe. Recruitment occurred year round reaching a peak of 500 recruits m⁻² during early spring. Higher recruitment occurred at periods of low temperature (21–23°C) and high salinity (30–33 psu). Growth rates of Gayralia sp. ranged from 5.75 ± 0.56% to 6.50 ± 0.43% day⁻¹ during the winter and from 1.43 ± 1.65% to 4.65 ± 2.17% day⁻¹, during the summer. Production ranged from 22 ± 6 g m⁻² DW in June to 58 ± 21 g m⁻² DW in September 2004 in 45 days after zooid settlement. The simplicity of the cultivation method, reasonable growth rates and extensive favorable area for cultivation suggest that mariculture of Gayralia sp. may become a good alternative of income for the local inhabitants.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Biosorption and Bioreduction of Trivalent Aurum by Photosynthetic Bacteria <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

Biosorption has been shown to be an eco-friendly approach to remove heavy metal ions. In this study, the photosynthetic bacteria Rhodobacter capsulatus was screened and found to have strong ability to adsorb Au(III). The maximum specific uptake of living cells was over 92.43 mg HAuCl₄/g dry weight of cell in the logarithmic phase. Biosorpion ability would be enhanced by an acidic environment. As the main cations, during biosorption the quantity of Mg²⁺ exchanged was more than Na⁺. Biosorbed Au(III) could be reduced by carotenoid and enzymes embedded and/or excreted by R. capsulatus, which might be the mechanism of photosynthtic bacteria metal tolerance.     

Flutolanil and carboxin resistance in<Emphasis Type="Italic"> Coprinus cinereus</Emphasis> conferred by a mutation in the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>560</Subscript> subunit of succinate dehydrogenase complex (Complex II)

A gene that confers resistance to the systemic fungicide flutolanil was isolated from a mutant strain of the basidiomycete Coprinus cinereus. The flutolanil resistance gene was mapped to a chromosome of approximately 3.2 Mb, and a chromosome-specific cosmid library was constructed. Two cosmid clones that were able to transform a wild-type, flutolanil-sensitive, strain of C. cinereus to resistance were isolated from the library. Analysis of a subclone containing the resistance gene revealed the presence of the sdhC gene, which encodes the cytochrome b ₅₆₀ subunit of the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial membrane. Comparison between the sdhC gene of a wild-type strain and that of a mutant strain revealed a single point mutation, which results in the replacement of Asn by Lys at position 80. Measurements of succinate-cytochrome c reductase activity in the transformants with mutant sdhC gene(s) suggest that flutolanil resistance of the fungus is caused by a decrease in the affinity of the SDH complex for flutolanil. This sdhC mutation also conferred cross-resistance against another systemic fungicide, carboxin, an anilide that is structurally related to flutolanil. In other organisms carboxin resistance mutations have been found in the genes sdhB and sdhD, but this is the first demonstration that a mutation in sdhC can also confer resistance. The mutant gene cloned in this work can be utilized as a dominant selectable marker in gene manipulation experiments in C. cinereus.Communicated by E. Cerdá-Olmedo