Mixing,mass transfer,and scale-up of polysaccharide fermentations

Microbial polysaccharides are rapidly emerging as a new and important source of polymeric materials. These biopolymers have novel and unique properties and already have found a wide range of applications in the food, pharmaceutical, and other industries. In view of the impending importance of polysaccharides as an industrial commodity, there is renewed interest in the area of product and process development. This paper summarizes the state-of-the art in polysaccharide fermentations. An attempt is being made to review the following areas: rheological characteristics of polysaccharide solutions, mixing and power requirements of polysaccharides and other highly viscous non-Newtonian systems, oxygen mass transfer, and scale-up problems encountered in polysaccharide fermentations.     

Structural and rheological properties of polysaccharides from mango (Mangifera indica L.) pulp

The structure and rheological properties of water-soluble polysaccharides from industrialized mango pulp were investigated. Soluble fraction (SF) 2 was heterogeneous on high performance size exclusion chromatography, giving two peaks as determined by multi-angle laser light scattering and refractive index detectors. The presence of starch in SF2 was demonstrated by a positive iodine reaction and by 13C nuclear magnetic resonance (NMR) spectroscopy. The presence of pectic polysaccharides was shown by a calorimetric method, 13C-NMR spectroscopy and carboxyl reduction. The main pectic polysaccharide was polygalacturonic acid; type I rhamnogalacturonan was also detected. Analysis of the rheological properties of SF2 showed a pseudoplastic behavior up to 3 g x l(-1). 'Creep and recovery' tests and analysis performed under a dynamic state revealed a weak gel character for solutions at concentrations of 15, 20 and 30 g x l(-1).     

Water dynamics in charged and uncharged polysaccharide gels by quasi-elastic neutron scattering

Using a microeV neutron spectrometer we have studied the mobility of water in gels formed by two polysaccharides: agarose and hyaluronic acid. Agarose is a nearly uncharged polysaccharide; its gels are fairly stiff, quasi-random networks of fibre bundles. Hyaluronic acid is a highly charged polysaccharide capable of retaining large amounts of water in entangled meshworks with unusual rheological properties. We have analysed sets of quasi-elastic lineshapes broadened by two proton populations with different degrees of freedom. The resulting microscopic mobility parameters and their temperature dependence reveal a complex behaviour. The overall effect of the biopolymer network is to increase translational as well as rotational relaxation times, but the changes observed are not dramatic and cannot fully account for the strikingly different macroscopic properties of these gels. Local electrostatic interactions (over 3 to 20 A) do not appear to influence significantly the rheological behaviour.     

Absorption of oxygen in highly viscous newtonian and non-Newtonian fermentation model media in bubble column bioreactors

Summary Mean relative gas holdup, slip velocity, bubble size distribution, mean specific interfacial area, and volumetric mass transfer coefficient of oxygen were estimated in sparged columns 14 cm in diameter and 380 and/or 390 cm high with two different aerator types (porous plate and injector nozzle) in highly viscous Newtonian (glycerol solutions) and non-Newtonian (CMC solutions) fluids.For the Newtonian liquids the above properties were estimated as function of the viscosity of the liquid. For the non-Newtonian liquids they were determined as function of the fluid consistency index and flow behavior index. Significant differences between Newtonian and non-Newtonian systems appear. In Newtonian medium k_L a drops with increasing viscosity and already approaches a constant value at =40 cP. In pseudoplastic medium k_L a varies with the fluid consistency and flow behavior indexes in the entire investigated range.In both of these systems the primary bubble population changes into two or three populations along the reactor: the medium bubbles gradually disappear and small and large bubbles are formed.     

Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.¹ Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.     

Structure of the extracellular gelling polysaccharide produced by Enterobacter (NCIB 11870) species

The gelling polysaccharide produced by a species of Enterobacter (NCIB 11870) contains L-fucose, D-glucose, and D-glucuronic acid in the ratios 1:2:1. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide revealed terminal non-reducing glucose, (1----3)-linked fucose, (1----3,1----4)-linked glucose, and (1----4)-linked glucuronic acid in the ratios 1:1:1.2:0.8. From the results of Smith degradation of the polysaccharide and spectroscopic studies of the acidic tetra- and octa-saccharides produced by bacteriophage-induced enzymic depolymerization of the polysaccharide, the following tetrasaccharide repeating-unit is proposed. (Formula: see text). This repeating-unit is identical to that of the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 except for the absence of O-acetyl groups. The effects of the O-acetyl groups on the secondary structure and rheological properties of these polysaccharides are discussed.     

Pulsatile blood flow and oxygen transport past a circular cylinder

The fundamental study of blood flow past a circular cylinder filled with an oxygen source is investigated as a building block for an artificial lung. The Casson constitutive equation is used to describe the shear-thinning and yield stress properties of blood. The presence of hemoglobin is also considered. Far from the cylinder, a pulsatile blood flow in the x direction is prescribed, represented by a time periodic (sinusoidal) component superimposed on a steady velocity. The dimensionless parameters of interest for the characterization of the flow and transport are the steady Reynolds number (Re), Womersley parameter (alpha), pulsation amplitude (A), and the Schmidt number (Sc). The Hill equation is used to describe the saturation curve of hemoglobin with oxygen. Two different feed-gas mixtures were considered: pure O(2) and air. The flow and concentration fields were computed for Re=5, 10, and 40, 0< or =A< or =0.75, alpha=0.25, 0.4, and Schmidt number, Sc=1000. The Casson fluid properties result in reduced recirculations (when present) downstream of the cylinder as compared to a Newtonian fluid. These vortices oscillate in size and strength as A and alpha are varied. Hemoglobin enhances mass transport and is especially important for an air feed which is dominated by oxyhemoglobin dispersion near the cylinder. For a pure O(2) feed, oxygen transport in the plasma dominates near the cylinder. Maximum oxygen transport is achieved by operating near steady flow (small A) for both feed-gas mixtures. The time averaged Sherwood number, Sh, is found to be largely influenced by the steady Reynolds number, increasing as Re increases and decreasing with A. Little change is observed with varying alpha for the ranges investigated. The effect of pulsatility on Sh is greater at larger Re. Increasing Re aids transport, but yields a higher cylinder drag force and shear stresses on the cylinder surface which are potentially undesirable.     

Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan

Ozone is known to add across and cleave carbon–carbon double bonds. Ozonolysis is widely used for the preparation of pharmaceuticals, for bleaching substances and for killing microorganisms in air and water sources. Some polysaccharides and oligosaccharides, such as those prepared using chemical or enzymatic β-elimination, contain a site of unsaturation. We examined ozonolysis of low-molecular-weight heparins (LMWHs), enoxaparin and logiparin, and heparosan oligo- and polysaccharides for the removal of the nonreducing terminal unsaturated uronate residue. 1D ¹H NMR showed that these ozone-treated polysaccharides retained the same structure as the starting polysaccharide, except that the C4–C5 double bond in the nonreducing end unsaturated uronate had been removed. The anticoagulant activity of the resulting product from enoxaparin and logiparin was comparable to that of the starting material. These results demonstrate that ozonolysis is an important tool for the removal of unsaturated uronate residues from LMWHs and heparosan without modification of the core polysaccharide structure or diminution of anticoagulant activity. This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan.     

Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate

The oscillatory and steady shear rheological properties of concentrated solutions of proteoglycan subunit (PGS) and aggregate (PGA) from bovine articular cartilage have been studied using a Rheometrics fluids spectrometer. At comparable concentrations in the physiological range tan delta increases from 0.5 to 1.0 for PGA as the oscillation frequency (omega) increases from 10(-1) to 10(2) rads/s, compared to a decrease from 40 to 5 for PGS. Thus PGA solutions exhibit predominantly elastic response whereas those of PGS exhibit primarily viscous behavior. PGA solutions show pronounced shear-thinning behavior at all shear rates (gamma) in the range 10(-2) less than gamma (s-1) less than 10(2), whereas PGS solutions exhibit predominantly Newtonian flow. For PGA, the small-strain complex viscosity eta* (omega) is substantially smaller than the steady-flow viscosity eta(gamma) at comparable values of omega and gamma. These observations indicate that the presence of proteoglycan aggregates leads to formation of a transient or weak-gel network. Since aggregation leads to a large increase in molecular hydrodynamic volume and hence in the relaxation times for macromolecular rotation, it appears that role of aggregate formation is to shift the linear viscoelastic response from the terminal viscous flow into the plateau elastomeric regime of relaxational behavior. Normal or pathological changes that produce a decrease in aggregation will result in a loss of elastomeric behavior of the proteoglycan matrix.     

Novel bacterial exopolysaccharides from deep-sea hydrothermal vents

Five bacterial strains recovered from deep-sea hydrothermal vents were studied for their ability to secrete extracellular polymers. A preliminary characterization displayed four different polysaccharides in terms of both chemical composition and rheological properties. One of them was secreted by Alteromonas macleodii subsp. fijiensis and exhibited similarities with xanthan, a commercial polysaccharide. Two of the three Pseudoalteromonas species were shown to produce the same polymer. The last polymer was secreted by a bacterium belonging to the Vibrio genus. They all contained glucose, galactose, mannose, glucuronic and galacturonic acids as the main sugars with the exception of the last one which was only constituted by uronic acids and hexosamines, in that similar to the structure of heparin, a glycosaminoglycan useful in pharmaceutical area. Applications for these polysaccharides could be expected in various biotechnological fields including the food industry, the wastewater treatment and pharmaceutical areas.     

Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitations of nutrients

This research studied the production of polysaccharides from Ganoderma lucidum under the various limitations of nutrients, including carbon-source, nitrogen-source, phosphate-source, magnesium-source, and dissolved oxygen. The different responses of polysaccharide production were observed under different limitations of nutrients. The concentration of polysaccharides was from 1.79 g/L decreasing to 0.91 g/L when the concentration of glucose was from 60 g/L decreasing to 20 g/L. The highest specific polysaccharide production was found at 60 g/L glucose media with 0.299 g/g-cell, and the lowest molecular weight was found in carbon-source limitation. Under nitrogen-source limitation the concentration of cells was low, but both polysaccharide production (1.61 g/L) and specific polysaccharide production (0.492 g/g-cell) were the highest. The lowest molecular weight of polysaccharides was found under nitrogen-source limitation. Both the phosphate-source and magnesium-source limitations showed low cell growth. With the phosphate-source limitation both low polysaccharide production and a lower molecular weight of polysaccharides was found. In the magnesium-source limitation low polysaccharide production, but a higher molecular weight of polysaccharides, was found. For the factor of oxygen supply the best polysaccharide production was found with sufficient oxygen for the first 5 days’ cultivation and then, after changing to oxygen limitation for another 5 days’ cultivation. On the other hand, the highest molecular weight of polysaccharides was found from the beginning with oxygen limitation throughout the process of fermentation.     

The extracellular polysaccharides of the red microalgae: Chemistry and rheology

Cells of red microalgae encapsulated within sulphated polysaccharides, are thought to have a wide range of potential industrial applications. Our group is thus carrying out a comprehensive research program aimed at bringing these biopolymers into industrial use. The program includes physiological studies on polysaccharide production, outdoor cultivation of the microalgae, and characterisation of the polysaccharides. Chemical composition and structure and physicochemical properties were investigated for the polysaccharides of three red microalgae, Porphyridium sp., P. aerugineum and Rhodella reticulata. Differences were found among the three species in the composition of the monosugars, half ester sulphate groups and glucuronic acid content, but a disaccharide isolated was identical in all the species examined. This disaccharide is thought to be the basic building block of these polysaccharides. In addition, monosugar sulphates were isolated and characterised. Fractionation by charge showed the polysaccharides to be heterogenous and composed of at least two fractions that differed in their composition. Although the polysaccharides differed in composition, their rheological characteristics were found to be similar. Aqueous solutions of the biopolymers were stable over a wide range of pH values and temperatures and were compatible with monovalent cations. Mixtures of the algal polysaccharides with locust bean gum exhibited synergism and syneresis. When the gel strength was compared with that of agar gel at the same concentration the polysaccharide gels were found to be weaker.     

Rheological properties of guar gum and hydroxyethyl guar gum in aqueous solution

The rheological properties of aqueous solutions of guar gum (GG) and hydroxyethyl guar gum (HEG) have been investigated. The flow properties of these polysaccharide solutions were studied at the shear rate in the range 1.5–1310s⁻¹ using a Rheotest-2 viscometer. The flow of these polysaccharide solutions was described by equation of state based on Cross model. The basic rheological parameters, like zero shear rate viscosity (η_o), elasticity modulus (G_o) and relaxation time (gl_o) were calculated using simple and established relations. Master viscosity curves indicated that the molecular weight distribution of native guar gum has been changed by hydroxyethylation under specified reaction conditions. The effect of concentration and temperature on η_o and λ_o has been studied, and the relations among these were established by simple equations.     

Effect of additives on mass transfer in turbine aeration

Mass transfer coefficients and interfacial areas were determined for the aeration of aqueous solutions in a turbine agitated vessel. The mass transfer coefficients measured for water without additive and for sodium chloride solutions matched very well to measurements in the literature for air bubbles of the same diameter in free rise. Thus the only effect of agitation was to determine the bubble size which then in turn set the coefficient. Two surface active agents were studied: sodium dodecyl sulfate and Dow Corning Antifoam C. The rate of mass transfer increased with the former additive but decreased with the latter; however, the mass transfer coefficient was the exact same function of bubble diameter in both cases and the different rates are attributed to the quite different effects on interfacial area.     

Extracellular polysaccharides of a bacterium associated with a fungal canker disease of Eucalyptus sp

Extracellular polysaccharides (EPSs) produced by an Erwinia sp associated with a fungal canker disease of Eucalyptus were fractionated into one polysaccharide that was identified with that produced by Erwinia chrysanthemi strains SR260, Ech1, and Ech9, and the other distinctively different from any other EPS produced by E. chrysanthemi strains so far studied. Their structures were determined using a combination of chemical and physical techniques including methylation analysis, low pressure gel-filtration, and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy. The new polysaccharide, identified as EPS Teranera, has the following structure: [structure: see text] The molecular weights of the polysaccharides range from 3.2-6.2 x 10(5) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.     

Two phase aqueous extraction

Rheological properties have been measured for aqueous solutions of dextran, polyethylene glycol and bovine serum albumin. Mixtures of these materials have also been studied. A rotating concentric cylinder viscometer was used to study the rheological properties of these materials over the temperature range 10 to 40°C. Over the range of concentrations, molecular weights, temperature and shear rates covered in this work, all aqueous solutions exhibited Newtonian behaviour. Correlations have been reported for viscosities of dextran, polyethylene glycol, and bovine serum albumin. The viscosity of mixtures of these materials is not linear with respect to concentration.     

Rheological behaviour of the culture medium during growth of the microalga Botryococcus braunii

A non-axenic strain of the microalga Botryococcus braunii Kützing, isolated from a small lake in Portugal, when cultured at 25°C in mineral medium and under continuous illumination, showed a poor production of hydrocarbons (5% of the dry biomass) but excreted remarkably high quantities of an exopolysaccharide (4–4·5 g litre⁻¹) into the medium. The production of the soluble polysaccharide, which contains galactose, fucose and uronic acid residues, occurs mainly after the exponential phase of growth.The rheological properties of broth during growth were studied. The increase of polysaccharide concentration as a consequence of its continuous biosynthesis, changes the medium behaviour from Newtonian to non-Newtonian with a flow characterized by a power-law equation. This behaviour becomes Newtonian again, when the culture is maintained for a longer period of time.     

九种蕨类植物多糖提取物抗动植物病原菌活性

从9种蕨类植物中提取多糖,以苯酚-硫酸比色法测定其含量,并采用纸片法进行抗动植物病原菌实验。结果表明,9种蕨类多糖含量差别较大,其多糖提取物表现出不同程度的抑制动植物病原菌活性。     

Investigation of bacterial resistance to the immune system response: cepacian depolymerisation by reactive oxygen species

Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.     

NMR investigations of the 4-ethyl guaicol self-diffusion in iota (ι)-carrageenan gels

Self-diffusion coefficient of an aroma molecule (4-ethyl guaicol) was measured using the pulsed field gradient spin echo NMR (PGSE-NMR) method in order to investigate the influence of a macromolecular matrix on its diffusion and release processes. Iota (ι)-carrageenan was used for its ability to form thermoreversible gels in aqueous salt solutions. Variations of the ι-carrageenan and the salt concentrations permitted various gels with different thermal and rheological properties to be obtained. These latter were modified by an isotope effect obtained by preparing gels in D₂O. The NMR self-diffusion measurements realised for water and the aroma molecules indicated neither chemical interactions with ι-carrageenan, nor obstruction effects from the polysaccharide chains. In ι-carrageenan gels, the diffusional phenomenon was highly dependent on the heterogeneous gel structure and controlled by hydrodynamic interactions due to frictional drag between each molecule of the system and water microviscosity changes.