Bacterial degradation of emulsan

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Enzymatic depolymerization of emulsan

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.     

Bacterial degradation of emulsan.

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Properties of hydrocarbon-in-water emulsions stabilized by Acinetobacter RAG-1 emulsan

Emulsan is a polymeric extracellular emulsifying agent produced by Acinetobacter RAG-1. Hydrocarbon-in-water emulsions (V(f) of hydrocarbon of 0.01-0.10) were stabilized by small quantities of emulsan (0.02-0.2 mg/mL). Although both aliphatic and aromatic hydrocarbon emulsions were stabilized by emulsan, mixtures containing both aliphatics and aromatics were better substrates for emulsan than the individual hydrocarbon by themselves. The emulsan remained tightly bound to the hydrocarbon even after centrifugation as determined by (a) residual emulsan in the aqueous phase and (b) the fact that the resulting "cream" readily dispersed in water to reform stable emulsions. With hexadecane-to-emulsan weight ratio of 39 and 155, the noncoalescing oil droplets had average droplet diameters of 2.0 and 4.0 mum, respectively. Dialysis studies showed that the water-soluble dye Rhodamine B adsorbed tightly to the interface of hexadecane-emulsan droplets although the dye did not bind to either hexadecane or emulsan alone. At saturating concentrations of dye, 2.2 mumol of dye were bound per mg emulsan.     

Biosynthesis of emulsan biopolymers from agro-based feedstocks

AIMS: The need for biocompatible, biodegradable, and versatile biopolymers permeates many fields including environmental and food technology. The goal of the study presented here is to establish the utility of agricultural oils as an inexpensive carbon source to produce materials useful for biomedical materials and offer positive attributes in terms of green chemistry. METHODS AND RESULTS: Structural variants of the complex acylated polysaccharide, emulsan, secreted from Acinetobacter venetianus RAG-1, were biosynthesized in cultures supplemented with agricultural feedstocks to examine the feasibility of conversion of these substrates into value-added biopolymers. Acinetobacter venetianus produced chemically and biologically distinct emulsan variants in culture on soy molasses and tallow oil. These variants possess significant biological function, including macrophage activation and adjuvant activity, in similar range to that observed for the standard emulsan formed on ethanol-fed A. venetianus. CONCLUSIONS: The results indicate that this novel family of biopolymers can be produced in significant quantities from the readily available renewable agricultural feedstocks and the resulting structures and functions can be correlated to the chemistry of these feedstocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant quantities of agricultural oils produced annually represent an untapped source for bioconversion to valuable products. The results of this study confirm that the important polymer emulsan can be synthesized from this inexpensive carbon source.     

Protein engineering of wzc to generate new emulsan analogs

Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Uranium Biosorption by the Lichen Trapelia involuta at a Uranium Mine

Metal localisation was investigated in the lichenised ascomycete Trapelia involuta growing on a range of uraniferous minerals including metazeunerite [Cu(UO₂)₂(AsO₄)₂·8H₂O], metatorbernite [Cu(UO₂)₂(PO₄)₂·8H₂O], autunite [Ca(UO₂)₂(PO₄)₂·10H₂O] and uranium-enriched iron oxide and hydroxide minerals at the abandoned South Terras mine site, Cornwall, UK. Apothecia from samples collected from waste dumps at the mine have an unusually dark colour that decolorized with NaOCl, an observation which together with Fourier Transform Infrared Spectroscopy of apothecial extracts, suggested the presence of melanin-like pigments. X-ray element mapping and probe traverses across the lichen-rock interface identify the highest U, Fe, and Cu concentrations in the outer parts of melanised apothecia. Accumulation of mineral particulates and complexing with lichen acids are not considered responsible for this since element ratios in the traverses do not correspond with those of likely mineral phases and lichen metabolites are localised in different tissues. Metal biosorption by melanin-like pigments are likely to be responsible for the observed metal fixation. No detectable U or Cu was observed in control samples although Fe showed a similar localisation in some specimens. The high concentrations of mucopolysaccharides and P recorded inside apothecia (within asci containing reproductive spores and hypothecium) suggests that the formation of melanised tissues may help protect vital reproductive tissues from the toxic effects of U and other metals, since the uranyl ion complexes strongly with phosphate species.     

Uranium phosphate biomineralization by fungi

Geoactive soil fungi were investigated for phosphatase‐mediated uranium precipitation during growth on an organic phosphorus source. Aspergillus niger and Paecilomyces javanicus were grown on modified Czapek–Dox medium amended with glycerol 2‐phosphate (G2P) as sole P source and uranium nitrate. Both organisms showed reduced growth on uranium‐containing media but were able to extensively precipitate uranium and phosphorus‐containing minerals on hyphal surfaces, and these were identified by X‐ray powder diffraction as uranyl phosphate species, including potassium uranyl phosphate hydrate (KPUO₆.3H₂O), meta‐ankoleite [(K_1.7Ba_0.2)(UO₂)₂(PO₄)₂.6H₂O], uranyl phosphate hydrate [(UO₂)₃(PO₄)₂.4H₂O], meta‐ankoleite (K(UO₂)(PO₄).3H₂O), uramphite (NH₄UO₂PO₄.3H₂O) and chernikovite [(H₃O)₂(UO₂)₂(PO₄)₂.6H₂O]. Some minerals with a morphology similar to bacterial hydrogen uranyl phosphate were detected on A. niger biomass. Geochemical modelling confirmed the complexity of uranium speciation, and the presence of meta‐ankoleite, uramphite and uranyl phosphate hydrate between pH 3 and 8 closely matched the experimental data, with potassium as the dominant cation. We have therefore demonstrated that fungi can precipitate U‐containing phosphate biominerals when grown with an organic source of P, with the hyphal matrix serving to localize the resultant uranium minerals. The findings throw further light on potential fungal roles in U and P biogeochemistry as well as the application of these mechanisms for element recovery or bioremediation.     

Reconstitution of emulsifying activity of Acinetobacter calcoaceticus BD4 emulsan by using pure polysaccharide and protein.

Acinetobacter calcoaceticus BD4 and BD413 produce extracellular emulsifying agents when grown on 2% ethanol medium. For emulsifying activity, both polysaccharide and protein fractions were required, as demonstrated by selective digestion of the polysaccharide with a specific bacteriophage-borne polysaccharide depolymerase, deproteinization of the extracellular emulsifying complex with hot phenol, and reconstitution of emulsifier activity with pure polysaccharide and a polysaccharide-free protein fraction. Chemical modification of the carboxyl groups in the polysaccharide resulted in a loss of activity. The protein required for reconstitution of emulsifying activity was purified sevenfold. The BD4 emulsan apparently derives its amphipathic properties from the association of an anionic hydrophilic polysaccharide with proteins.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus

An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.     

Enhanced Uranium Immobilization and Reduction by Geobacter sulfurreducens Biofilms

Biofilms formed by dissimilatory metal reducers are of interest to develop permeable biobarriers for the immobilization of soluble contaminants such as uranium. Here we show that biofilms of the model uranium-reducing bacterium Geobacter sulfurreducens immobilized substantially more U(VI) than planktonic cells and did so for longer periods of time, reductively precipitating it to a mononuclear U(IV) phase involving carbon ligands. The biofilms also tolerated high and otherwise toxic concentrations (up to 5 mM) of uranium, consistent with a respiratory strategy that also protected the cells from uranium toxicity. The enhanced ability of the biofilms to immobilize uranium correlated only partially with the biofilm biomass and thickness and depended greatly on the area of the biofilm exposed to the soluble contaminant. In contrast, uranium reduction depended on the expression of Geobacter conductive pili and, to a lesser extent, on the presence of the c cytochrome OmcZ in the biofilm matrix. The results support a model in which the electroactive biofilm matrix immobilizes and reduces the uranium in the top stratum. This mechanism prevents the permeation and mineralization of uranium in the cell envelope, thereby preserving essential cellular functions and enhancing the catalytic capacity of Geobacter cells to reduce uranium. Hence, the biofilms provide cells with a physically and chemically protected environment for the sustained immobilization and reduction of uranium that is of interest for the development of improved strategies for the in situ bioremediation of environments impacted by uranium contamination.     

Uranium mobilization from low-grade ore by cyanobacteria

Summary Three cyanobacterial isolates (two LPP-B forms and one Anabaena or Nostoc species) from different environments could mobilize uranium from low-grade ores. After 80 days, up to 18% uranium had been extracted from coal and 51% from carbonate rock by the filamentous cyanobacterium OL3, a LPP-B form. Low growth requirements with regard to light and temperature optima make this strain a possible candidate for leaching neutral and alkaline low-grade uranium ores.     

Relationship between emulsifying activity and carbohydrate backbone structure of emulsan from Acinetobacter calcoaceticus RAG-1

Various emulsan samples with the different degrees of branching of the carbohydrate backbone were obtained from Acinetobacter calcoaceticus under different culture conditions. The emulsifying activity of emulsan had a linear correlation to the branching degrees of the carbohydrate backbone (r²= 0.930) suggesting that the structure of carbohydrate backbone was an important factor influencing emulsifying activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Uranium accumulation by Pseudomonas sp. EPS-5028

Summary Pseudomonas sp. EPS-5028 was examined for the ability to accumulate uranium from solutions. The uptake of uranium by this microorganism is very rapid and is affected by pH but not by temperature, metabolic inhibitors, culture time and the presence of various cations and anions. The amount of uranium absorbed by the cells increased as the uranium concentration of the solution increased up to 55 mg uranium/g cell dry weight. Electron microscopy indicated that uranium accumulated intracellularly as needle-like fibrils. Uranium could be removed chemically from the cells, which could then be reused as a biosorbent. Offprint requests to: A. M. Marqués     

Uranium (VI) Reduction by Denitrifying Biomass

Groundwater near the S3 ponds at the US Department of Energy's Y-12 site in Oak Ridge, Tennessee, is contaminated by high levels of nitrate (up to 160 mM) and U(VI) (∼0.3 mM). To minimize nitrate inhibition, the authors proposed extraction of contaminated groundwater, nitrate removal in a denitrifying fluidized bed bioreactor (FBR), and return of nitrate-free effluent to the aquifer to stimulate in situ microbial reduction of U(VI). In the presence of carbonate, U(VI) sorption to biomass was negligible, but in its absence, sorption was significant. Biomass reduced U(VI) to U(IV), exhibiting slow first-order removal with respect to U(VI). Addition of electron donor increased rates. Addition of an inhibitor of sulfate reduction (molybdate) slowed the rate and inhibited sulfate reduction. Denitrifying β-Proteobacteria dominated clone libraries of SSU rRNA and dsrA gene sequences. Approximately 10% were low-G+C microorganisms that had 90% to 92% sequence identity with Sporomusa, Acetonema, and Propionispora. The dsrA sequences were dominated by a single clone with ∼80% nucleotide identity to dsrA of Desulfovibrio vulgaris sub sp. oxamicus. The authors conclude that some members of this denitrifyng community reduce uranium, and that sulfate-reducing bacteria likely contribute to this capability.     

Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.     

Uranium accumulation by a bacterium isolated from electroplating effluent

Pseudomonas MGF-48, a gram-negative, motile, oxidase-negative, catalase-positive, yellow-pigmented bacterium isolated from electroplating effluent, was found to accumulate uranium with high efficiency. Uptake of uranium was rapid and the amount increased in direct proportion to concentration, e.g., from 50 to 200 mg uranium per liter. The largest amount of uranium uptake was 174 mg per gram dry weight bacterial biomass and was observed to occur in stationary phase during incubation at 30 °C. Uptake was determined by flow injection analysis. Maximum uranium accumulation occurred at pH 6.5, with 86% of the uranium being taken up within 5 min of incubation. Release of uranium bound to the cells was accomplished by addition of sodium carbonate and EDTA solution (0.1 M), the cells were reusable, and served as a biosorbent. Cells immobilized in polyacrylamide gel took up 90% of the uranium. Pseudomonas MGF-48 showed excellent efficiency in biosorbing uranium, by both immobilized and free cells. The results of this study, compared with those of other reports of uranium accumulation by microorganisms, leads us to conclude that Pseudomonas MGF-48 shows excellent potential for bioremediating uranium-polluted aqueous effluents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Uranium accumulation by immobilized cells of aCitrobacter sp.

Uranium was removed from challenge flows presented to immobilized cells of aCitrobacter sp. In excess of 90% of the presented metal was recovered, giving high yields of accumulated metal which could be subsequently released from the immobilized cellsin situ.