Bioaccumulation of silver by a multispecies community of bacteria

A stable community of bacteria that had unusually high tolerance of soluble silver was isolated from soil by chemostat enrichment. The community consisted of three bacteria: Pseudomonas maltophilia, Staphylococcus aureus and a coryneform organism. The pseudomonas was primarly responsible for the silver resistance. The tolerance of high silver concentrations, up to 100 mM Ag⁺, was greatly reduced when the community was grown in the absence of silver. Pseudomonas maltophilia comprised approximately 50% by numbers of the community when grown in chemostats in the presence or absence of Ag⁺ but large fluctuations occurred in population sizes of the other two bacteria; the S. aureus population was small (less than 1%) in the presence of Ag⁺ but comparised a third of the total numbers when Ag⁺ was omitted from the medium. Silver-resistant respiration of the silveradapted community was significant even when it was confronted with high concentrations of Ag⁺. In contrast the respiration of the coryneform organism and particularly S. aureus was highly sensitive to silver. The inhibition constants for silver-sensitive respiration were 0.78 mM and 0.04 mM for silver acclimatized and nonacclimatized communities respectively.The community had great capacity for silver bioaccumulation. Maximum concentrations of over 300 mg silver per g dry weight of biomass were recorded at an accumulation rate of 21 mg Ag⁺ h^-1 (g biomass)^-1. The extent of silver removal from solution was a function of initial concentration of silver; at low external concentrations (ca. 1 mM) all the silver was rapidly removed from solution, at high concentrations (ca. 12 mM) 84% removal occurred in 15 h.     

Engineering the homoserine dehydrogenase and threonine dehydratase control points to analyse flux towards L-isoleucine in Corynebacterium glutamicum

 The synthesis of L-isoleucine by Corynebacterium glutamicum involves 11 reaction steps, with at least 5 of them regulated in activity or expression. Using gene replacement we constructed a vector-free C. glutamicum strain having feedback-resistant aspartate kinase and feedback-resistant homoserine dehydrogenase activity. Isogenic strains carrying in addition one or several copies of feedback-resistant threonine dehydratase were made and their product accumulations compared. With strain SM1, with high threonine dehydratase activity, accumulation of 50 mM L-isoleucine was achieved, whereas with the parent strain only 4 mM L-isoleucine was obtained. Applying a closed-loop control fed-batch strategy to strain SM1 a final titre of 138 mM L-isoleucine was achieved with an integral molar yield of 0.11 mol/mol, and a maximal specific productivity of 0.28 mmol (g h)^-1. This shows that high L-isoleucine yields can be obtained in the presence of one copy of feedback-resistant homoserine dehydrogenase by applying the appropriate fermentation strategy. In addition, the specific profiles of 2-oxoglutarate and pyruvate accumulation during fermentation revealed a major transition of the metabolism of C. glutamicum during the fermentation process. Received: 16 October 1995/Received revision: 21 December 1995/Accepted: 8 January 1996     

Considerations on the use of commercially available yeast biomass for the treatment of metal-containing effluents

Summary Three strains ofSaccharomyces cerevisiae and one strain of aCandida sp. obtained from different industrial sources were screened for uptake of silver and copper. Considerable differences in metal uptake capacities were found between the different strains ofS. cerevisiae and betweenS. cerevisiae and theCandida sp. used. Copper uptake capacities ranged from 0.05 mmol g^–1 dry wt to 0.184 mmol g^–1 dry wt while values of 0.034 mmol Ag g^–1 dry wt and 0.193 mmol Ag g^–1 dry wt biomass were observed. Use of ion-selective electrodes (ISEs) enabled the detection of copper complexing agents (possibly proteins and carbohydrates) released by yeasts into the surrounding medium. In contrast, these compounds had no silver complexation abilities. Langmuir and Scatchard transformations of metal adsorption isotherms suggested differences in the mechanisms involved in metal uptake by the various yeasts. The differences between strains ofS. cerevisiae were due possibly to differences in cell wal composition. Different methods of preparation of biomass (fresh, air, oven and freeze-dried) had little effect on metal uptake in comparison with fresh biomass. Storage of fresh waste biomass at 4°C for 20 days had no effect on metal biosorption capacities. It was also observed that individual batches of waste biomass produced from different fermentation runs had consistent metal uptake capacities. The implications of the above results on the use of waste yeast biomass for treatment of metal-containing effluents are discussed.     

Silver tolerance and accumulation in yeasts

Summary Debaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO₃. Although Ag⁺ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag⁺ to Ag⁰ resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO₃-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag⁰ although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag⁺ reduction to Ag⁰.     

Zinc biosorption from aqueous solution by a halotolerant cyanobacterium Aphanothece halophytica

We have investigated the characteristics of zinc biosorption by Aphanothece halophytica. Zinc could be rapidly taken up from aqueous solution by the cells with an equilibrium being reached within 15 min of incubation with 100 mg L⁻¹ ZnCl₂. The adsorbed zinc was desorbed by treatment with 10 mM EDTA. The presence of glucose, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N′-dicyclohexylcarbodiimide (DCCD) did not affect the uptake of zinc. The specific uptake of zinc increased at low cell concentration and decreased when cell concentration exceeded 0.2 g L⁻¹. The binding of zinc followed Langmuir isotherm kinetics with a maximum zinc binding capacity of 133 mg g⁻¹ and an apparent zinc binding constant of 28 mg L⁻¹. The presence of an equimolar concentration of Mn²⁺, Mg²⁺, Co²⁺, K⁺, or Na⁺ had no effect on zinc biosorption, whereas Ca²⁺, Hg²⁺, and Pb²⁺ showed an inhibitory effect. The biosorption of zinc was low at a pH range from 4 to 6, but increased progressively at pH 6.5 and 7. Received: 12 December 2001 / Accepted: 11 January 2002     

Comparison of Rhizopus nigricans in a pelleted growth form with some other types of waste microbial biomass as biosorbents for metal ions

Biosorption of metal ions (Li⁺, Ag⁺, Pb²⁺, Cd²⁺, Ni²⁺, Zn²⁺, Cu²⁺, Sr²⁺, Fe²⁺, Fe³⁺ and Al³⁺) by Rhizopus nigricans biomass was studied. It was shown that metal uptake is a rapid and pH-dependent process, which ameliorates with increasing initial pH and metal concentrations. Different adsorption models: Langmuir, Freundlich, split-Langmuir and combined nonspecific-Langmuir adsorption isotherm were applied to correlate the equilibrium data. The maximum biosorption capacities for the individual metal ions were in the range from 160 to 460 mol/g dry weight. Scatchard transformation of equilibrium data revealed diverse natures of biomass metal-binding sites. The binding of metals was also discussed in terms of the hard and soft acids and bases principle. The maximum biosorption capacities and the binding constant of R. nigricans were positively correlated with the covalent index of metal ions.The following types of waste microbial biomass originating as by-products from industrial bioprocesses were tested for biosorption of metal ions: Aspergillus terreus, Saccharomyces cerevisiae, Phanerochaete chrysosporium, Micromonospora purpurea, M. inyoensis and Streptomyces clavuligerus. The determined maximum biosorption capacities were in the range from 100 to 500 mol/g dry weight. The biosorption equilibrium was also represented with Langmuir and Freundlich sorption isotherms.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

Copper biosorption in an aerobic bioreactor

Abstract

This study evaluates the biosorption of copper by aerobic biomass that was selected from surface waters of the San Pedro River in Sonora, Mexico. Using a batch system, 73% biosorption of copper was obtained in 75 minutes. Continuous biosorption assays were carried out for 133 days in an ascending flow aerobic reactor packed with zeolite (AFAR-PZ) that was inoculated with a bacterial consortium. Strains were grown until 1g L^?1 of biomass was obtained. Tests using continuous biosorption were performed as follows: (i) the addition of 50 mg Cu²⁺ L^?1 without recirculation of biomass; (ii) the addition of 20 mg Cu²⁺ L^?1without recirculation of biomass; and (iii) the biomass were recirculated with the addition of 20 mg Cu²⁺ L^?1 to pH 3 to 4. The fourth and fifth assays varied pH between 4 and 5, with 20 mg Cu²⁺ L^?1and the biomass recirculated. Biosorption capacity of the first and second assays was 96% on the first day of experimentation. During the third trial 97% of biosorption was obtained during 6 days and the process was improved by varying the pH. Copper biosorption equilibrium was investigated under the same operating conditions. Langmuir adsorption isotherms were used to fit experimental data. The biosorption capacity of aerobic biomass was 3.08 mmol g^?1. It was demonstrated that this biomass is capable of biosorbing copper and this method has potential for the treatment of industrial effluents contaminated with heavy metals.     

Silver ion (Ag+)-Induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: Reversal by thiol reagents

Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag⁺) on K⁺ transport and respiration with extracellular K⁺ and O₂ electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO₃) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K⁺ efflux (K _m =10^–4 m,V _max=379 nmol K⁺/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO₂) (13.9±1.1 to 25.7±4.4 nmol O₂/min/mg,P<0.001), was observed 19 sec after the K⁺ efflux induced by AgNO₃ (10^–4 m), suggesting a delayed increase in Na⁺ entry into the cell. Ouabain-insensitive QO₂, nystatin-stimulated QO₂, and CCCP-uncoupled QO₂ were not significantly affected, indicating preserved function of the Na⁺, K⁺-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K⁺ transport and QO₂. We conclude that Ag⁺ causes early changes in the permeability of the cell membrane to K⁺ and then to Na⁺ at concentrations that do not limit Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag⁺ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

The change in the electro-rotation of yeast cells effected by silver ions

Pre-treatment of brewer's yeast (Saccharomyces cerevisiae) cells with silver acetate or nitrate at concentrations of 20 nmol/l or higher caused a dramatic increase in the number of cells which rotated in the same direction as the field (‘Co-field rotation’). The change in rotation of single cells correlated very well with the chemically observed loss of potassium induced by Ag⁺. The sensitivity to Ag⁺ was lowered by increasing the cell concentration, and the extent of this change can be used to estimate the binding of Ag⁺ per cell and the limiting sensitivity of the method. The Ag⁺ concentration required to induce a response was found to be increased significantly in the presence of alkali ions (especially K⁺) during the Ag⁺ incubation. The Ag⁺ sensitivity was, therefore, observed to be a function of the type and strength of buffer used in the incubation. Under certain conditions, 1 mM Ca²⁺ increased the Ag⁺ sensitivity. These observations show that the presence or absence of ions that are so common that they are often overlooked may have interesting consequences for the bio-assay of heavy metals.     

BspA (CyuC) in Lactobacillus fermentum BR11 Is a Highly Expressed High-Affinity L-Cystine-Binding Protein

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 M. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1–2 × 10⁵ BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Control of in vitro rooting and plant development in Corymbia maculata by silver nitrate, silver thiosulfate and thiosulfate ion

Plant regeneration and transformation in vitro is often improved by adding silver ion (Ag⁺) to the culture media as AgNO₃ or silver thiosulfate (STS). Ag⁺ reacts with substances to form insoluble precipitates, while thiosulfate (S₂O₃ ²⁻) interferes with these reactions. We studied the implications of silver precipitation and S₂O₃ ²⁻ in the medium for culture development by (1) examining formation of Ag⁺ precipitates from AgNO₃ versus STS in agar gels and their possible dependence on agar type; (2) comparing Corymbia maculata culture responses to AgNO₃ and STS and determining which better suits control of culture development; (3) clarifying whether STS-dependent alterations in culture development are due to Ag⁺ alone or also to a separate influence of S₂O₃ ²⁻. Silver precipitates appeared in aqueous gels of four agar brands supplemented with AgNO₃, but not in Phytagel^™, which remained transparent. No precipitation was observed in gels with STS. Indole-3-butyric acid (IBA)-mediated adventitious root induction and shoot growth were higher in C. maculata shoot tips cultured on gels with STS versus AgNO₃ (6–25 μM Ag⁺). IBA-treated shoot tips exhibited enhanced adventitious root regeneration, accelerated root elongation, increased frequency of lateral root formation, and stimulated shoot growth mediated by 100–250 μM sodium thiosulfate (Na₂S₂O₃) in medium without Ag⁺. The potency of S₂O₃ ²⁻ in facilitating culture development has never been recognized. It is inferred that superiority of STS in stimulating multiple responses of C. maculata culture results from sustained biological activity of Ag⁺ through prevention of its precipitation, and from impact of S₂O₃ ²⁻ on cell differentiation and growth.     

Silver tolerance and silver accumulation of microorganisms from soil materials of a silver mine

Summary Silver-tolerant microoganisms were isolated from soil materials of a silver mine. The bacterial count decreased approximately linearly with increasing silver concentration. The fungal count, however, remained almost constant in all flasks, up to a concentration of 1 mM silver. At 10 mM Ag⁺ (about 1 g/l) and more, neither bacterial nor fungal growth could be observed.All silver-tolerant isolates were tested for silver accumulation capacity. Bacteria accumulated a mean 23 mg Ag⁺/g dry weight, hyphomycetes 6.7 mg/g dry wt. and yeasts 0.46 mg/g dry wt. The accumulation process of the hyphomycete with the highest accumulation capacity (20 mg/g dry wt.) was shown to be completed after about 30 min. Between 4°C and 80°C the process was nearly independent of temperature; as to the optimum pH, a slight preference for the neutral range was observed. Mycelium destroyed by formaldehyde solution showed the same accumulation pattern. These results would indicate a binding of silver to the surface of the cell.     

Production of L-arabitol from L-arabinose by Candida entomaea and Pichia guilliermondii

 Lignocellulosic biomass, particularly corn fiber, represents a renewable resource that is available in sufficient quantities from the corn wet milling industry to serve as a low cost feedstock for production of fuel alcohol and valuable coproducts. Several enzymatic and chemical processes have potential for the conversion of cellulose and hemicellulose to fermentable sugars. The hydrolyzates are generally rich in pentoses (D-xylose and L-arabinose) and D-glucose. Yeasts produce a variety of polyalcohols from pentose and hexose sugars. Many of these sugar alcohols have food applications as low-calorie bulking agents. During the screening of 49 yeast strains capable of growing on L-arabinose, we observed that two strains were superior secretors of L-arabitol as a major extracellular product of L-arabinose. Candida entomaea NRRL Y-7785 and Pichia guilliermondii NRRL Y-2075 produced L-arabitol (0.70 g/g) from L-arabinose (50 g/l) at 34°C and pH 5.0 and 4.0, respectively. Both yeasts produced ethanol (0.32–0.33 g/g) from D-glucose (50 g/l) and only xylitol (0.43–0.51 g/g) from D-xylose (50 g/l). Both strains preferentially utilized D-glucose>D-xylose>L-arabinose from mixed substrate (D-glucose, D-xylose and L-arabinose, 1:1:1, 50 g/l, total) and produced ethanol (0.36–0.38 g/g D-glucose), xylitol (0.02–0.08 g/g D-xylose) and L-arabitol (0.70–0.81 g/g L-arabinose). The yeasts co-utilized D-xylose (6.2–6.5 g/l) and L-arabinose (4.9–5.0 g/l) from corn fiber acid hydrolyzate simultaneously and produced xylitol (0.10 g/g D-xylose) and L-arabitol (0.53–0.54 g/g L-arabinose). Received: 24 April 1995/Received revision: 9 August 1995/Accepted: 7 September 1995     

Lead biosorption by different morphologies of fungus Mucor indicus

Biosorption characteristics of Pb⁺² ions from aqueous solution were investigated using fungus Mucor indicus biomass treated with NaOH. Biosorption was measured as a function of biomass morphology, pH, biomass concentration, contact time, and metal concentration. The morphology of M. indicus biomass was manipulated towards filamentous or yeast-like forms. The highest and lowest biosorption capacities were observed for purely filamentous and yeast-like forms, respectively. Models of Langmuir, Freundlich, Temkin, and Scachard were applied to describe adsorption isotherm and fitted appropriately. Biosorption kinetics was successfully described using Ho’s pseudo-second-order model. Maximum and minimum values of biosorption capacity of Pb²⁺ were 22.1 and 12.1 mg g⁻¹ for purely filamentous and yeast-like morphologies, respectively. Increasing pH resulted in higher biosorption of Pb⁺² ions up to pH 5.5. Biosorption capacity of individual Pb⁺² ions was reduced in the presence of other metal ions in bi- or multi-metal ion experiments. Metal ions adsorption by the biomass could be eluted effectively with HNO₃.     

Removal and Recovery of Toxic Silver Ion Using Deep-Sea Bacterial Generated Biogenic Manganese Oxides

Products containing silver ion (Ag⁺) are widely used, leading to a large amount of Ag⁺-containing waste. The deep-sea manganese-oxidizing bacterium Marinobacter sp. MnI7-9 efficiently oxidizes Mn²⁺ to generate biogenic Mn oxide (BMO). The potential of BMO for recovering metal ions by adsorption has been investigated for some ions but not for Ag⁺. The main aim of this study was to develop effective methods for adsorbing and recovering Ag using BMO produced by Marinobacter sp. MnI7-9. In addition, the adsorption mechanism was determined using X-ray photoelectron spectroscopy analysis, specific surface area analysis, adsorption kinetics and thermodynamics. The results showed that BMO had a higher adsorption capacity for Ag⁺ compared to the chemical synthesized MnO₂ (CMO). The isothermal absorption curves of BMO and CMO both fit the Langmuir model well and the maximum adsorption capacities at 28°C were 8.097 mmol/g and 0.787 mmol/g, for BMO and CMO, respectively. The change in enthalpy (ΔH^θ) for BMO was 59.69 kJ/mol indicating that it acts primarily by chemical adsorption. The change in free energy (ΔG^θ) for BMO was negative, which suggests that the adsorption occurs spontaneously. Ag⁺ adsorption by BMO was driven by entropy based on the positive ΔS^θ values. The Ag⁺ adsorption kinetics by BMO fit the pseudo-second order model and the apparent activation energy of E_a is 21.72 kJ/mol. X-ray photoelectron spectroscopy analysis showed that 15.29% Ag⁺ adsorbed by BMO was transferred to Ag(0) and meant that redox reaction had happened during the adsorption. Desorption using nitric acid and Na₂S completely recovered the Ag. The results show that BMO produced by strain MnI7-9 has potential for bioremediation and reutilization of Ag⁺-containing waste.     

Enzymatic preparation of optically active 3-trimethylsilylalanine

 We constructed an efficient system for preparing optically active 3-trimethylsilylalanine (TMS-Ala) by kinetic resolution with acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC 3.5.1.14). Racemic TMS-Ala was chemically synthesized and acetylated. Enantioselective deacetylation of N-acetyl-DL-TMS-Ala with acylase I from porcine kidney or from Aspergillus melleus was then attempted. Both enzymes could catalyze the deacetylation of N-acetyl-DL-TMS-Ala, and the porcine enzyme was found to have much higher activity than the enzyme from A. melleus. The optimum pH of the porcine-acylase-catalyzed reaction was 7.5, and the addition of 0.5 mM Co²⁺ accelerated the reaction. Optically pure L-TMS-Ala (>99% enantiomeric excess, ee) was obtained in 72% yield under the optimized conditions. Furthermore, highly optically pure D-TMS-Ala (96% ee) could also be obtained in 76% yield by chemically hydrolyzing the residual substrate. Received: 6 June 1995/Received revision: 3 July 1995/Accepted: 19 July 1995     

Production, purification and characterization of a constitutive intracellular α-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular α-galactosidase (1.33 U mg⁻¹ protein) within 48 h at 45°C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65°C and an optimum pH of 5.0. Humicola α-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The K _m S for p-nitrophenyl-α-D-galactopyranoside, O-nitrophenyl-α-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag⁺ and Hg²⁺. D-Galactose inhibited α-galactosidase competitively and the inhibition constant (K _i) for galactose was 11 mM. Received 28 January 1999/ Accepted in revised form 07 April 1999