Volatilization of Mercury by Immobilized Bacteria (Klebsiella pneumoniae) in Different Support by Using Fluidized Bed Bioreactor

Klebsiella pneumoniae, a mercury-resistant bacterial strain able to reduce ionic mercury to metallic mercury, was isolated from wastewater of Casablanca. This strain exhibits high minimal inhibition concentrations for heavy metals such as mercury 2400 μM, lead 8000 μM, silver 2400 μM, and cadmium 1000 μM. This bacterium was immobilized in alginate, polyacrylamide, vermiculite, and cooper beech and was used for removing mercury from a synthetic water polluted by mercury by using a fluidized bead bioreactor. Immobilized bacterial cells of Klebsiella pneumoniae could effectively volatilize mercury and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells. The highest cleanup and volatilization rates were obtained when Klebsiella pneumoniae was entrapped in alginate beads, with a cleanup rate of 100% and a volatilization rate of 89%. Immobilized cells in alginate continuously volatilized mercury even after 10 days without loss of activity. Received: 21 February 2001 / Accepted: 13 March 2001     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Mercury accumulation and volatilization in immobilized algal cell systems

Summary Rapid removal of mercury from growth medium and its uptake by free and alginate-entrappedChlorella has been observed. Immobilized cell systems accumulated more mercury than free cell systems. In addition, both volatilized significant quantities of mercury. Studies show, however, that mercury lost in this way may re-enter the aqueous phase and subsequently be accumulated by immobilized cells.     

New form of lignolytically active mycelium generated by immobilization of protoplasts isolated from the white rot fungi Heterobasidion annosum and Polyporus pinsitus

Summary Immobilized mycelia regenerated from immobilized protoplasts isolated from lignin-degrading Basiodiomycetes have been shown to be able to decompose specifically ¹⁴C-labelled dehydropolymers of coniferylalcohol (DHP-lignin) and monomeric lignin-related compounds more intensively than native mycelium, by decarboxylation, demethylation, ring and side chain cleavage. Protoplasts of two white rot fungi were immobilized by entrapment in Na- alginate gel and remained intact after the immobilization procedure. Within the first 3 days of incubation in culture medium, regeneration of hyphal cells occurred. Since hyphal cells regenerated from protoplasts within gel beads were hindered from stretching by the matrix, the microbial immobilized cells differed from native mycelium in terms of their morphology. The time course and extent of lignin degradation by native mycelium and regenerated mycelium of the examined white rot fungi also differed, a sign that there may also be differences between them in terms of the physiology of lignin degradation.     

Removal of inorganic and organic mercurials by immobilized bacteria having mer-ppk fusion plasmids

Feasibility of biological mercury removal from wastewater was examined by using alginate-immobilized cells of Escherichia coli carrying mer-ppk fusion plasmid pMKB18. Immobilized cells engineered to express mercury-transport system, organomercurial lyase and polyphosphate efficiently removed organic and inorganic mercury from contaminated wastewater over a wide concentration range of mercurials, probably via intracellular accumulation mediated by ppk-specified polyphosphate. Bioaccumulation of mercury was selective compared to other metals such as Cd(2+), Pb(2+) and Cr(6+). The immobilized cells could be used repeatedly (at least three times) without large loss of mercury removal activity. From these results, it is concluded that the mer-ppk fusion plasmid and the immobilized cells are useful for simultaneous removal of organic and inorganic mercury from contaminated wastewater.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Continuous hydrolysis of tallow with immobilized lipase in a microporous membrane

Thermostable lipase from Thermomyces lanuginosus was immobilized in untreated microporous membranes. Melted tallow pumped through the membrane did not wash the enzyme out. From 0.4 to 0.9% of the soluble activity remained after immobilization with half-lives of 1-2 months or more at 50 degrees C. Membranes can be acid/base washed and reloaded with enzyme with no adverse effects. Buffer was required for a long half-life, and recycling the buffer improved the mass transfer of glycerol out of the immobilized lipase reactor. Immobilized activity was unaffected when the pH of the aqueous product changed from 5.5 to 6.5.     

Mercury bioremediation by mercury accumulating Enterobacter sp. cells and its alginate immobilized application

The effective microbial remediation of the mercury necessitates the mercury to be trapped within the cells without being recycled back to the environment. The study describes a mercury bioaccumulating strain of Enterobacter sp., which remediated mercury from the medium simultaneous to its growth. The transmission electron micrographs and electron dispersive X-ray analysis revealed the accumulation of remediated mercury as nano-size particles in the cytoplasm as well as on the cell wall. The Enterobacter sp. in the present work was able to accumulate mercury, without being engineered in its native form. The possibility of recovering the accumulated mercury from the cells is also indicated. The applicability of the alginate immobilized cells in removing mercury from synthetic and complex industrial effluent in a batch mode was amply demonstrated. The initial load of 7.3 mg l⁻¹ mercury in the industrial effluent was completely removed in 72 h. The cells immobilized in calcium alginate were similarly effective in the complete removal of 5 mg l⁻¹ HgCl₂ of mercury from the synthetic effluent in less than 72 h. The immobilized cells could be reused for multiple cycles.     

Tris–sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61

The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris–sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.     

Genotoxicity of mercury compounds. A review

This article reviews literature data concerning the genotoxicity of 29 mercury-containing agents, including laboratory compounds as well as ingredients of preparations used as fungicides, dyes, disinfectants and drugs. A variety of genetic end-points were investigated in bacteria, yeasts, moulds, plants, insects, cultured cells from fishes, rodents or humans, aquatic organisms, amphibians, mammalia and exposed humans. The overall evaluation is quite complex. Mercury compounds failed to induce point mutations in bacteria but often exerted clastogenic effects in eukaryotes, especially by binding SH groups and acting as spindle inhibitors, thereby causing c-mitosis and consequently aneuploidy and/or polyploidy. Inorganic mercury compounds were also found to induce the generation of reactive oxygen species and glutathione depletion in cultured mammalian cells. Although different mercury compounds tended to produce qualitatively comparable genetic effects, which suggests the involvement of a common toxic entity, methylmercury derivatives and other ionizable organomercury compounds were more active in short-term tests than either non-ionizable mercury compounds (e.g., dimethylmercury) or inorganic mercury salts (e.g., mercuric chloride). The results of cytogenetic monitoring in peripheral blood lymphocytes of individuals exposed to elemental mercury or mercury compounds from accidental, occupational or alimentary sources were either negative or borderline or uncertain as to the actual role played by mercury in some positive findings. Both genotoxic and non-genotoxic mechanisms may contribute to the renal carcinogenicity of mercury, which so far has been convincingly demonstrated only in male rodents treated with methylmercury chloride.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.     

Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein

Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.     

Isolation of an extracellular Mn-dependent enzyme mineralizing melanoidins from the white rot fungus Trametes versicolor

Abstract Morphological and physiological properties of Tetrahymena thermophila immobilized by encapsulation in calcium-alginate hollow spheres were found to be substantially different from those of suspended cells. Immobilized T. thermophila reached lengths of 70–100 μm, whereas the average cell of suspension cultures was about 40 μm long. Suspended cells appeared typically pear-shaped while immobilized cells developed a proboscis-like anterior end. Contrary to suspended T. thermophila , encapsulated cells were functionally deficient in phagocytosis although developing an oral apparatus. The diameter of the macronucleus of immobilized cells was about two times larger than the macronucleus of suspended cells and contained twice as much DNA, while the DNA content of the micronucleus remained unchanged. High cell density fermentations of suspended cells indicated that the alterations observed in immobilized cells were not due to close physical contacts between the cells.     

Production of cellulolytic enzymes by immobilized Sporotrichum thermophile.

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was considerably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.     

Studies on the immobilization of trypsin on sand

Trypsin was immobilized on sand using five different methods. Attempts were made to attach amino-functional groups onto sand using 3-aminopropyltriethoxysilane, hexamethylenetetramine, hexamethylenediamine, and melamine. Glutaraldehyde was used as a bifunctional agent in all the methods. Methods for the estimation of the proteolytic 1activity and protein content of immobilized trypsin were standardized. The maximum retained activity was observed for trypsin immobilized on sand via 3-aminopropytriethoxysilane and glutaraldehyde. Immobilized trypsin showed a shift in the pH optimum toward the acidic side over that of soluble trypsin in all five cases. The optimum temperature for both native and immobilized trypsin prepared by the silane-glutaraldehyde method was found to be 45°C. However, the pH and thermal stabilities of immobilized trypsin were observed to be better than that of the native enzyme.     

Changes in monoclonal antibody productivity of recombinant BHK cells immobilized in collagen gel particles

Animal cell perfusion high density culture is often adopted for the production of biologicals in industry. In high density culture sometimes the productivity of biologicals has been found to be enhanced. Especially in immobilized animal cell culture, significant increase in the productivity has been reported. We have found that the specific monoclonal antibody (MAb) productivity of an immobilized hybridoma cell is enhanced more than double. Several examples of enhancing productivities have been also shown by collagen immobilized cells. Immobilized cells involve some different points from non-immobilized cells in high density culture: In immobilized culture, some cells are contacted together, resulting in locally much higher cell concentration more than 10⁸ cells/ml. Information originating from a cell can be easily transduced to the others in immobilized culture because the distance between cells is much nearer. Here we have performed collagen gel immobilized culture of recombinant BHK cells which produce a human IgG monoclonal antibody in a protein-free medium for more than three months. In this high density culture a stabilized monoclonal antibody production was found with around 8 times higher specific monoclonal antibody productivity compared with that in a batch serum containing culture. No higher MAb productivity was observed using a conditioned medium which was obtained from the high density culture, indicating that no components secreted from the immobilized cells work for enhancing monoclonal antibody production. The MAb productivity by the non-immobilized cells obtained by dissolving collagen using a collagenase gradually decreased and returned to the original level in the batch culture using a fresh medium. This suggests that the direct contact of the cells or a very close distance between the cells has something to do with the enhancement of the MAb productivity, and the higher productivity is kept for a while in each cell after they are drawn apart.     

Characteristics of yeast β-galactosidase immobilized on calcium alginate gels

Summary Saccharomyces anamensis having -galactosidase activity, has been immobilized in calcium alginate gel matrix that retained 78.6% enzyme activity to that of native cells. Optimum pH(7.0) was negligibly affected by immobilization. K_m values for immobilized and native cells were 119 mM and 102 mM respectively. Protective agents like dithioerythritol, bovine serum albumin, enhance the enzyme activity when added prior to immobilization. Immobilized cells can be stored in refrigeration(4°C) for 42 days without a significant loss of enzyme activity.     

Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles

As part of a research program aimed at producing biodiesel fuel from plant oils enzymatically cells of Rhizopus oryzae (R. oryzae) IFO4697 (with a 1,3-positional specificity lipase) immobilized within biomass support particles (BSPs) were investigated for the methanolysis of soybean oil. The R. oryzae cells easily became immobilized within the BSPs during batch operation. To enhance the methanolysis activity of the immobilized cells under the culture conditions used, various substrate-related compounds were added to the culture medium. Among the compounds tested, olive oil or oleic acid was significantly effective. In contrast, no glucose was necessary. Immobilized cells were treated with several organic solvents, but none gave higher activity than untreated cells. When methanolysis was carried out with stepwise additions of methanol using BSP-immobilized cells, in the presence of 15% water the methyl esters (MEs) content in the reaction mixture reached 90% - the same level as that using the extracellular lipase. The process presented here, using a whole cell biocatalyst, is considered to be promising for biodiesel fuel production in industrial applications.     

固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

Characteristics and applications of adsorbents for pyrogen removal

Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated. Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range. The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent. The apparent dissociation constant was 1.57 X 10(-9) M. The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed. The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length. Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.