Synergism within polyhexamethylene biguanide biocide formulations

G ilbert , P., P emberton , D. & W ilkinson , D.E. 1990. Synergism within polyhexamethylene biguanide biocide formulations. Journal of Applied Bacteriology 69 , 593–598.
Polyhexamethylene biguanides (PHMB) are mixtures of polymeric biguanides with an average polymer length ( n ) of 5, but containing high ( n > 15, mol. wt 3300) and low molecular weight material ( n = 2, mol. wt 400). Studies involving discrete molecular weight fractions of PHMB have shown that antimicrobial activity of PHMB increases with increasing polymer length. Cell suspensions which had not been subjected to centrifugation and/or washing during their preparation were employed. Whilst activity was still observed to increase with n , the trend was much reduced as n exceeded six. Centrifugation and washing of cells markedly increased the activity of high but not low molecular weight materials and corresponded to losses upon centrifugation of envelope lipopolysaccharide (LPS). Such envelope LPS represented high affinity binding sites on the surfaces of the cells. Combinations of various molecular weight fractions of PHMB were evaluated against filter-washed cells and revealed a profound synergy between extremes of polymer length.     

Binding of some polyhexamethylene biguanides to the cell envelope of Escherichia coli ATCC 8739

Absorption and antibacterial action of some polyhexamethylene biguanides upon Escherichia coli has been investigated. An amine-ended-dimer (AED) (n = 2), a polydisperse mixture sold by ICI Limited as the active ingredient of vantocil IB (PHMB) (n = 5.5), and a high molecular weight fraction of PHMB (HMW, n greater than or equal to 10) were used. Bactericidal activity was determined over a range of pH (5-9). Similarly, adsorption of drug to the cell surface, indicated by changes in electrophoretic mobility, and overall drug absorption by the cells were determined. Maximal activity occurred at pH 6 for PHMB and HMW and at pH 5 for AED. This corresponded to minimal surface adsorption and maximal distribution of drug to the underlying cytoplasm and cytoplasmic membrane. Uptake of drug corresponded to high affinity isotherms and indicated a rapid transfer of biocide into the cells following their initial interaction at the surface.     

Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli

The interaction between some polyhexamethylene biguanides and the cell envelope of Escherichia coli has been investigated. An amine-ended dimer, (AED, n = 2), a polydisperse mixture (ICI plc) available as the active ingredient of Vantocil IB, (PHMB, n = 5.5), and a high molecular weight fraction, (HMW, n = greater than or equal to 10) of PHMB were used. The sensitivity of batch cultures depleted of magnesium (M-dep), phosphorus (P-dep) or glycerol (C-dep) towards the biocides was assessed by monitoring the rate and extent of potassium ion leakage. P-dep suspensions were particularly resistant to all these agents and possessed less than half the quantity of phospholipid of other cell types. This was compensated for by a proportionate increase in fatty acid and neutral lipid content of the cells. The reduction in phospholipid content was accounted for by decreases in phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) and phosphatidylserine (PS) content of the cultures remained unaffected by the depleting nutrient. Fourier-transform n.m.r. spectroscopy was used to study proton nuclei during the interaction of HMW, AED and PHMB with various phospholipid-vesicle preparations. The results strongly suggest that the biocides acted preferentially on the acidic phospholipids PG and DPG, rather than towards PE or PS. Resistance of P-dep cultures therefore reflected reductions in PG content. A molecular basis for the interaction of these compounds and membranes is proposed.     

Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli

The interaction between some polyhexamethylene biguanides and the cell envelope of Escherichia coli has been investigated. An amine-ended dimer, (AED, n = 2), a polydisperse mixture (ICI plc) available as the active ingredient of Vantocil IB, (PHMB, n = 5.5), and a high molecular weight fraction, (HMW, n =≧ 10) of PHMB were used. The sensitivity of batch cultures depleted of magnesium (M-dep), phosphorus (P-dep) or glycerol (C-dep) towards the biocides was assessed by monitoring the rate and extent of potassium ion leakage. P-dep suspensions were particularly resistant to all these agents and possessed less than half the quantity of phospholipid of other cell types. This was compensated for by a proportionate increase in fatty acid and neutral lipid content of the cells. The reduction in phospholipid content was accounted for by decreases in phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) and phosphatidylserine (PS) content of the cultures remained unaffected by the depleting nutrient. Fourier-transform n.m.r. spectroscopy was used to study proton nuclei during the interaction of HMW, AED and PHMB with various phospholipid-vesicle preparations. The results strongly suggest that the biocides acted preferentially on the acidic phospholipids PG and DPG, rather than towards PE or PS. Resistance of P-dep cultures therefore reflected reductions in PG content. A molecular basis for the interaction of these compounds and membranes is proposed.     

Barrier properties of the Gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides

G ilbert , P., P emberton , D. & W ilkinson , D.E. 1990. Barrier properties of the Gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides. Journal of Applied Bacteriology 69 , 585–592.
The antimicrobial activities of four discrete molecular weight fractions of polyhexamethylene biguanides towards a number of Escherichia coli strains have been investigated. Whilst activity of the polymers was observed to increase in proportion to polymerization number, the dependence of activity upon molecular weight was five times greater towards sphaeroplasts than towards whole cells. This suggested that the cell envelope, whilst not conferrring complete resistance to the agents, did provide a significant exclusion barrier. Comparison of the activities towards rough and deep-rough lipopolysaccharide strains showed growth inhibitory activity, but not bactericidal activity nor respiratory inhibition, to be enhanced in the rough strains. Uptake studies showed mixed H- and C-type adsorption with significantly greater numbers of high-affinity binding sites being associated with rough than deep-rough lipopolysaccharide. The binding affinity of polyhexamethylene biguanides towards cells was also enhanced in the rough strains. Binding affinity was, in all cases, significantly reduced in the presence of magnesium and suggested a mechanism of self-promoted uptake for these biocides, facilitated through core lipopolysaccharide.     

Barrier properties of the gram-negative cell envelope towards high molecular weight polyhexamethylene biguanides

The antimicrobial activities of four discrete molecular weight fractions of polyhexamethylene biguanides towards a number of Escherichia coli strains have been investigated. Whilst activity of the polymers was observed to increase in proportion to polymerization number, the dependence of activity upon molecular weight was five times greater towards sphaeroplasts than towards whole cells. This suggested that the cell envelope, whilst not conferring complete resistance to the agents, did provide a significant exclusion barrier. Comparison of the activities towards rough and deep-rough lipopolysaccharide strains showed growth inhibitory activity, but not bactericidal activity nor respiratory inhibition, to be enhanced in the rough strains. Uptake studies showed mixed H- and C-type adsorption with significantly greater numbers of high-affinity binding sites being associated with rough than deep-rough lipopolysaccharide. The binding affinity of polyhexamethylene biguanides towards cells was also enhanced in the rough strains. Binding affinity was, in all cases, significantly reduced in the presence of magnesium and suggested a mechanism of self-promoted uptake for these biocides, facilitated through core lipopolysaccharide.     

High and low molecular weight fractions of humic and fulvic acids

Summary The present investigation was initiated to study molecular weight fractionation of humic compounds isolated from surface samples of an Ultisol (Red Yellow Pc lzolic soil) and a Spodosol (Podzol), using fine (mol. wt. cutoff 3500) and coarse (mol. wt. cutoff 12000) dialysis membranes and sephadex gel filtration. Characterization of the humic fractions was conducted by elemental analysis and infrared spectroscopy. The results confirmed that fulvic acid (FA) was higher in ash and elemental content than humic acid (HA). With careful purification the amount of ash was found to be reduced to a minimum, but not to zero. Sephadex gel filtration revealed that fine HA (obtained with fine membranes) was composed of smaller amounts of HA-I (high mol. wt.) and large amounts of HA-II (low mol. wt. fraction). Coarse HA (obtained with coarse membranes) had almost equal amounts of HA-I and HA-II. Fine or coarse FA yielded only low molecular weight components after elution through sephadex. Infrared spectra of the humic fractions were indicative for the presence of phosphoglyceric acid as a possible constituent of the low molecular weight fraction of humic compounds.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.Contribution of the Univ. of Georgia, Agric. Expt. Sta., College Sta., Athens, Ga. Permission for the publication herein of Sadtler Standard spectrum has been granted, and all rights are reserved by Sadtler Res., Inc.     

Polyhexamethylene biguanide exposure leads to viral aggregation

Aims: This study reports the activity of two biguanides against MS2 bacteriophage used as a surrogate virus for nonenveloped mammalian viruses and provides an explanation as to their apparent limited efficacy. Methods and Results: When tested in a standard suspension test, two polyhexamethylene biguanides (PHMB), VANTOCIL? TG and COSMOCIL? CQ, reduced the viability of MS2 by only 1–2 log₁₀ PFU ml^?1. Exposure time up to 30 min did not affect the activity of the biguanides, although both PHMB were shown to strongly interact with MS2 proteins. Conclusions: Inactivation kinetics and change in virus hydrophobicity suggested that PHMB induces the formation of viral aggregates. This hypothesis was supported using dynamic light scattering that showed an increase in viral aggregates sizes (up to 500 nm) in a concentration‐dependent manner. Significance and Impact of the Study: It has been reported that viral aggregation is responsible for virus survival to the biocide exposure. Here, this might be the case, because the virucidal activity of the biguanides was modest and viral aggregation important. The formation of viral aggregates during virus exposure to PHMB was unlikely to overestimate the virucidal potential of the biguanides.     

Human urine deoxyribonuclease increases endogenous thymidine in the mouse thymocyte interleukin 1 assay: an artificial interleukin-1 inhibitor

Molecular size chromatography of urine from normal individuals showed two peaks of apparent IL-1 suppressive activities when tested by the murine thymocyte comitogenic assay (mol wt greater than 600 kD and 20 to 60 kD). However, the high molecular weight inhibitory activity disappeared if the concentration of PHA was increased during assay, and the low molecular weight inhibitory activity subsided in the presence of a high concentration of [3H]thymidine. The 20 to 60 kD fractions contained DNase activity which acted on DNA liberated from the considerable number of dying thymocytes during the course of the assay. Thus, incubating the urine fractions with freeze-killed murine T cells, whose DNA was prelabeled with [3H]thymidine, showed the appearance of supernatant [3H]thymidine correlating quantitatively with the DNase activity in the fractions. This indicates that urine DNase together with phosphatase(s) in the thymocyte cultures increase the level of extracellular, unlabeled thymidine, thereby diluting the specific activity of the tracer. These artificial IL-1-inhibitors may explain why urine from both normal and febrile individuals 'inhibits' IL-1 only when tested for thymocyte-activating activity but not when tested for other biological activities.     

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.     

Two separate receptors for prolactin in the rabbit mammary gland

Rabbit mammary gland PRL receptors in the microsome fraction were solubilized with the zwitterionic detergent Chaps, and were separated into two fractions (Fr. A and B) by ion-exchange chromatography. The number of receptors in Fr. B was about 2.2 times greater than in Fr. A. In sucrose gradient centrifugation analysis, PRL receptors in Fr. A and Fr. B sedimented at different positions. After binding 125I-PRL, the apparent molecular weight (mol wt) of the PRL receptor in Fr. A changed from 42,400 to 65,500 and that in Fr. B changed from 89,400 to 108,000, suggesting that each binding subunit interacts with one PRL molecule. Cross-linking 125I-PRL to receptors revealed little change following SDS-PAGE, in the autoradiogram patterns of the microsome PRL receptors, either in the presence or absence of dithiothreitol. Both the microsome and the Chaps extract contained two major binding subunits (mol wt, 83,200 and 36,800) and one minor subunit (mol wt, 20,800). The mol wt of the dominant PRL receptors in Fr. A and Fr. B were 36,800 and 83,200, respectively. The latter form did not dissociate into a 36,800 mol wt form, suggesting that the rabbit mammary gland contains two independent binding subunits with mol wt of 36,800 and 83,200. Data showed that PRL receptors in the rabbit mammary gland are mostly the high Kd type receptor with a mol wt of 83,200.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

Differential Expressions of Two Types of Alkaline Phosphatase during Developmental Growth of Embryoid Bodies of Mouse Teratocarcinoma OTT-6050 In Vitro

Ascites teratocarcinoma OTT-6050 is a totipotent tumor line producing indefinitely the simple type of embry-oid bodies (EBs). In culture with fetal calf serum (FCS) in Eagle's minimal essential medium (MEM), these EBs show developmental growth, only in which some differentiative events result. EBs also show this developmental growth in MEM supplemented with two fractions of FCS separated with a Amicon PM 10 membrane, i.e. a low molecular weight Fraction L (mol. wt. less than 10,000) and a high molecular weight Fraction H (mol. wt. more than 10,000). Fraction H is necessary for the survival of EBs in vitro. Fraction L enhances the uptake of ³H-thymidine into EB cells with increase in the Vmax , but no change in the K m. On culture of EBs with both Fractions, a marked bimodal increase in alkaline phosphatase (ALPase) activity is seen on day 1–2 and 4, resulting from the differential expressions of two electrophoretically distinct ALPases (Bands I and II). The differential expressions of ALPase are also observed cytochemically, one activity being on the inner cells and the other on the surrounding cells of EBs. From the cytochemical similarity of ALPase activity to that of normal mouse embryos, Band I ALPase is inferred to be the epiblast (developmentally totipotent stem cell)-type and Band II ALPase to be the distal (parietal) endoderm-type.     

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.     

Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.     

Purification of the enzyme NADPH: protochlorophyllide oxidoreductase.

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.     

Experimental allergic encephalomyelitis. Isolation of basic proteins and polypeptides from central nervous tissue

1. Basic protein (mol.wt. 16500) and polypeptides (mol.wt. 3500) were isolated from bovine spinal cord by a procedure involving defatting, acid extraction of the defatted material and repeated chromatography on Sephadex G-50. Similar fractions were isolated from guinea-pig brain. 2. These fractions produced experimental allergic encephalomyelitis in guinea pigs. 3. The polypeptides appeared to be derived from a basic protein of myelin as a result of the action of an acid proteinase during extraction with acid. Similar proteolysis might also occur in the isolation of other biologically active polypeptides from acetone-dried powders of nervous tissue. The activity of the acid proteinase was lowered by defatting with chloroform-methanol. 4. Peptides from tryptic digests of encephalitogenic polypeptides and protein were also encephalitogenic, which suggests that the encephalitogenic determinant may be quite a short sequence of amino acids. 5. These encephalitogenic polypeptides are further examples of antigens of low molecular weight.     

Liproprotein inhibitor of bone marrow cells in tumor-bearing rats.

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. 59Fe-heme synthesis, [3H]thymidine DNA synthesis, and 14C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (greater than 400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipoprotein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

Some permeability properties of angiosperm pollen grains,pollen tubes and generative cells

Summary The permeability of pollen grains, pollen tubes and generative cells of Helleborus foetidus and Galanthus nivalis has been investigated using four probes spanning a wide range of molecular weights: 4,6-diamidino-2-phenyl indole (DAPI; mol.wt. 350). Evans blue (mol.wt. 960), FITC-dextran (average mol.wt. 19400) and FITC-albumin (average mol.wt. 67000). DAPI penetrated into the vegetative cells of desiccated and hydrated pollen, and also entered growing pollen tubes. In contrast, the generative cells of hydrated pollen and of pollen tubes were highly resistant to penetration, as they were when isolated in osmotically balancing medium. Evans blue failed to enter intact generative cells under any of the conditions tested. The dye ultimately entered the vegetative cells of some pollen grains, but these were non-germinable. Growing pollen tubes invariably resisted penetration. Neither of the high molecular weight conjugates entered germinable pollen grains or intact pollen tubes. The results suggest that it is highly unlikely that DNA fragments of high molecular weight can enter viable pollen, pollen tubes or generative cells under any normal conditions.     

Influence of dissolved oxygen tension and agitation speed on alginate production and its molecular weight in cultures of Azotobacter vinelandii*

The alginate production by Azotobacter vinelandii, as well as the molecular weight of the polymer, are strongly influenced by the dissolved oxygen tension (DOT) and stirring speed of the culture. Under high DOT (5% of air saturation), the bacteria produced more alginate (4.5 g/l) than that obtained at low (0.5%) oxygen tension (1.0 g/l) in cultures conducted at 300 rpm. On the other hand, under constant DOT (3%), the higher the stirring speed (from 300 to 700 rev./min), the higher the specific growth rate and the alginate production rate. However, low agitation speed (300 rev./min) lead the culture to produce a polymer of high molecular weight (680 000 g/g mol) whereas a low molecular weight (352 000 g/g mol) alginate was isolated from cultures conducted at high (700 rev./min) stirring speed. At 700 rev./min, the MMW increased to a plateau between 1 and 3% DOT and then decreased to a minimum of 0.11 x 10(6) g/g mol at 7%. Microscopic observations revealed the presence of cell aggregates (one order of magnitude larger than individual cells) when the culture was conducted at 300 rev./min. Oxygen gradients occurring within the aggregates could be responsible of this phenomenon. At high agitation rate, the MMW of the alginate dropped towards the end of the culture in all conditions evaluated. Alginase activity was detected, which would be responsible for this phenomenon.