Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

Enzymatic synthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera.

The enzyme activity synthesizing poly-beta-hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction of Zoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction when the bacterium grew dispersedly in a glucose-starved medium. The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose. PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated with D(-)-beta-hydroxybutyryl CoA in vitro. Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity for D(-)-beta-hydroxybutyryl CoA, and showed the same pH optimum at 7.0.     

Accumulation of heavy-metal ions by Zoogloea ramigera

Biomass has been produced from glucose using the organism Zoogloea ramigera 115. This biomass has been used to remove copper, cadmium, and uranyl ions from water solutions. The metal uptake was studied with two different methods: either by spectrophotometric measurements on the solutions after flocculation or by potentiometric measurements with amalgam electrodes in order to follow the entire complex formation. The metal-biopolymer interaction in 0.1M NaClO(4) is practically the same as when no neutral salt is added. The metal uptake is dependent on pH and is selective. A reversible process suitable for metal complexation is described.     

Production of Extracellular Polysaccharide by Zoogloea ramigera

In batch cultures of Zoogloea ramigera the maximum rate of exopolysaccharide synthesis occurred in a partly growth-linked process. The exopolysaccharide was attached to the cells as a capsule. The capsules were released from the cell walls after 150 h of cultivation, which caused the fermentation broth to be highly viscous. Ultrasonication could be used to release capsular polysaccharide from the microbial cell walls. Treatment performed after 48 to 66 h of cultivation revealed exopolysaccharide concentration and apparent viscosity values in accordance with values of untreated samples withdrawn after 161 h of cultivation. The yield coefficient of exopolysaccharide on the basis of consumed glucose was in the range of 55 to 60% for batch cultivations with an initial glucose concentration of 25 g liter⁻¹. An exopolysaccharide concentration of up to 38 g liter⁻¹ could be attained if glucose, nitrogen, and growth factors were fed into the batch culture. The oxygen consumption rate in batch fermentations reached 25 mmol of O₂ liter⁻¹ h⁻¹ during the exopolysaccharide synthesis phase and then decreased to values below 5 mmol of O₂ liter⁻¹ h⁻¹ during the release phase. The fermentation broth showed pseudoplastic flow behavior, and the polysaccharide was not degraded when growth had ceased.     

Production of Extracellular Polysaccharide Matrix by Zoogloea ramigera

Zoogloea ramigera 115 synthesized large amounts of matrix polymer from fructose, galactose, glucose, lactose, mannose, soluble starch, and sucrose when these carbohydrates were used as supplements to a chemically defined medium. All of them supported polymer synthesis to the extent that cultures thickened to a gel. Concentration of carbohydrate nutrients in the range 0.5 to 2.0% was not a critical factor in determining eventual total thickening to a gel, except in relation to the incubation time required. Glucose disappeared from the growth medium rapidly and correlated with increasing cell growth and poly-beta-hydroxybutyrate (PHB) accumulation. PHB concentration decreased as extracellular polymer was synthesized, suggesting a link between PHB and extracellular polymer production.     

Fractal dimensions and porosities of Zoogloea ramigera and Saccharomyces cerevisae aggregates

The fractal nature microbial aggregates is a function of the type of microorganism and mixing conditions used to develop aggregates. We determined fractal dimensions from length-projected area (D(2)) and length-number scaling (D(3)) relationships. Aggregates of Zoogloea ramigera developed in rotating test tubes were both surface and mass fractals, with fractal dimensions of D(2) = 1.69 +/- 0.11 and D(3)= 1.79 +/- 0.28 (+/-standard deviation), respectively. When we grew this bacteria in a bench-top fermentor, aggregates maintained their surface fractal characteristics (D(2) = 1.78 +/- 0.11) but lost their mass fractal characteristics (D(3) = 2.99 +/- 0.36). Yeast aggregates (Saccharomyces cerevisae) grown in rotating tests tubes had higher average fractal dimensions than bacterial aggregates grown under physically identical conditions, and were also considered fractal (D(2) = 1.92 +/- 0.08 and D(3) = 2.66 +/- 0.34). Aggregates porosity can be expressed in term of a fractal dimensions, but average porosities are higher than expected. The porosities of yeast aggregates (0.9250-0.9966) were similar to porosities of bacterial aggregates (0.9250-0.9966) cultured under the same physical conditions, although bacterial aggregates developed in the reactor had higher average porosities (0.9857-0.9980). These results suggest that that scaling relationships based on fractal geometry may be more useful than equations derived from Euclidean geometry for quantifying the effects of different fluid mechanical environments on aggregates morphology and characteristics such as density, porosity, and projected surface area.     

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.     

Isolation of Zoogloea ramigera I-16-M exopolysaccharide biosynthetic genes and evidence for instability within this region.

The genetics of the biosynthesis of an exocellular polysaccharide (EPS) from Zoogloea ramigera I-16-M is being investigated. Tn5 insertion mutants deficient in EPS production were isolated by screening for the absence of fluorescence on plates containing the dye Cellufluor (Polysciences Chemicals). Complementation of these mutations was achieved with a Z. ramigera I-16-M gene library constructed in a broad-host-range cosmid vector and introduced into the I-16-M mutants by conjugation. Four recombinant plasmids able to restore EPS production to all of these mutants were found to contain at least 14 kilobases of common insert DNA. Subcloning of the common region and restriction mapping the locations of Tn5 insertions have identified two complementation groups contained within a chromosomal segment of DNA that is between 4.6 and 6.5 kilobases in size. We have clearly demonstrated genetic instability in this region which leads to spontaneous deletions and possibly rearrangements resulting in the loss of EPS production.     

Ester Synthesis by Zoogloea ramigera 115 Grown in the Presence of Ethanol

The ester ethyl butyrate is produced by Zoogloea ramigera 115, a bacterium isolated from an aerobic waste treatment plant, when ethanol is present in culture media. The cells appear to produce butyric acid which is then esterified with residual ethanol in the culture medium.     

Oxygen and nitrate reduction kinetics of a nonflocculating strain of Zoogloea ramigera

The oxygen and nitrate reduction kinetics of a nonflocculating strain of Zoogloea ramigera were determined. Axenic, nitrate-reducing bacterial suspensions were acclimated to various oxygen levels in a chemostat while measuring nitrate reduction in the presence of high ammonium nitrogen concentrations. Significant nitrate reduction was observed at oxygen concentrations up to 8 mg L^–1. Oxygen consumption was inhibited by oxygen concentrations in excess of 2 mg L^–1.     

Identification and characterization of poly-3-hydroxybutyrate granule-associated protein, PGA12 and PGA16 in Zoogloea ramigera I-16-M

Poly-3-hydroxybutyrate (PHB) granules of Zoogloea ramigera I-16-M contained two major PHB granule-associated proteins (PGA12 and PGA16) as revealed by sodium dodecyl sulfate-polyacrylamide gel elecrophoresis. N-terminal amino acid sequences of these proteins were determined. The genes encoding these proteins were cloned and sequenced. The structural genes of PGA12 and PGA16 were 351 and 447 bp long, which encode polypeptides with deduced molecular masses of 12.3 and 16.0 kDa, respectively. PGA12 and PGA16 were expressed in Escherichia coli. PHB granules were isolated from cells of recombinant strains of E. coli JM109, which harbored and expressed the PHB-synthetic genes of Ralstonia eutropha H16 and PGA12 or PGA16. These PHB granules contained PGA12 or PGA16 as a major protein. The presence of pga12 or pga16 did not affect the amount of PHB synthesized in E. coli. PGA12 and PGA16 bound to crystalline and amorphous PHB granules.     

Development of a continuous process for metal accumulation by Zoogloea ramigera

Biomass, mainly consisting of an acidic polysaccharide produced by Zoogloea ramigera, has been used as an adsorbing agent in a continuous process for the recovery of metal. The adsorption of copper has been studied with respect to retention time, biomass concentration, and reaction pH, in order to determine the optimal conditions for copper recovery. The results indicate that the uptake of copper is rapid and efficient. About 0.17 g Cu is adsorbed per gram of biomass within 10 min. At high biomass concentrations, the total amount of copper removed from solution is high, but the specific amount of copper adsorbed to biomass is low. The biomass exhibits a higher adsorptive uptake at low concentrations.     

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.     

Effects of yeast extract on the production and the quality of the exopolysaccharide, zooglan, produced by Zoogloea ramigera 115SLR

Although many studies have examined the influence of culture conditions on the production and composition of polysaccharides, little is known about the factors influencing the quality of exopolysaccharides (EPS). In this work we studied the effect of yeast extract on the production, composition and molecular weight of the EPS zooglan produced by Zoogloea ramigera 115SLR. This bacterium was grown on a new completely defined synthetic medium and on a medium containing yeast extract. Growth and polysaccharide production performances were comparable on the two media with a glucose to exopolysaccharide conversion yield of 35% (g/g). The polysaccharides produced on these two media have an identical composition but a different molecular weight and molecular weight distribution. The yeast extract medium leads to a more homogeneous polysaccharide solution. Received: 12 June 1998 / Received revision: 19 September 1998 / Accepted: 11 October 1998     

Determination of the biosorption heats of heavy metal ions on Zoogloea ramigera and Rhizopus arrhizus

The biosorption of Fe(III), Cr(VI), Pb(II), Cu(II) and Ni(II) ions on Zoogloea ramigera (activated sludge bacterium) and Rhizopus arrhizus (filamentous fungus) has been studied as a function of initial metal ion concentration and temperature. The applicability of the Langmuir model for each metal-microorganism system has been tested at different temperatures. The enthalpy change for the biosorption process has been evaluated by using the Langmuir constant b, related to the energy of adsorption. Thermodynamic parameters indicate the exothermic nature of Cu(II) and Ni(II) biosorption on both microorganisms. Fe(III), Cr(VI) and Pb(II) biosorption is determined to be an endothermic process since increased binding occurs as the temperature is increased in the range 15-45 degrees C.     

The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera

Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.