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[Isolation and characterization of Azotobacter sp. for the production of poly-beta-hydroxyalkanoates].

A microorganism (Azotobacter sp.) was isolated from soil samples from the Agronomy Faculty campus and its ability to accumulate poly-beta-hydroxyalkanoate (PHAs) polymers was analyzed. The isolated strain (named No 8) accumulated 8 micrograms PHA/m of culture media as intracytoplasmic granules. The following properties of the strain were analyzed: utilization of different carbon sources, antibiotic resistance, optimal pH and temperature, pigment production, nitrogen fixation, proteolytic activity, acid and hydrogen sulphide production, optimal growth temperature, cyst formation, growth on phenol and sodium fluoride, catalase, pleomorfism and mobility. The synthesized polymer showed valuable properties respect to organic solvents resistance.

Argentina↗

PubMed1994

Identification of a nitrogenase protein-protein interaction site defined by residues 59 through 67 within the Azotobacter vinelandii Fe protein.

During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking. In the present study, evidence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum. Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein. First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl. Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction. Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation. Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.

Adenosine Triphosphate↗

PubMed1994

Purification and in vitro activity of a truncated form of ANFA. Transcriptional activator protein of alternative nitrogenase from Azotobacter vinelandii.

The ANFA protein is the transcriptional activator of the sigma 54-dependent anfHDGK operon, which codes for the structural genes of the third nitrogenase system in Azotobacter vinelandii. We have purified, in soluble active form, an N-terminally truncated form of the protein, delta ANFA, which activates transcription from the anfH promoter and other sigma 54-dependent promoters in a purified transcription system. Sequences upstream of the anfH promoter and the presence of the integration host factor protein stimulate transcription, and we have shown that delta ANFA binds to sites situated between 200 and 300 base pairs upstream of the anfH promoter. In common with other sigma 54-dependent activators, ANFA has a highly conserved ATP binding motif in its central domain, and we have demonstrated that ATP or GTP is required for productive complex formation and that the purified truncated protein has a constitutive ATPase activity, which is presumably required to drive open complex formation.

Adenosine Triphosphatases↗

PubMed1994

Purification and characterization of a NADP+/NADPH-specific flavoprotein that is overexpressed in FdI- strains of Azotobacter vinelandii.

Earlier studies have established that mutant strains of Azotobacter vinelandii that do not synthesize ferredoxin I (AvFdI) overexpress another protein designated Protein X (Morgan, T. V., Lundell, P. J., and Burgess, B. K. (1988) J. Biol. Chem. 263, 1370-1375). This protein has now been purified using two-dimensional gel electrophoresis as an assay. The purified protein is a monomer with M(r) approximately 29,000 which degrades slowly to a specific M(r) approximately 22,000 form when stored in solution. The native protein is bright yellow and contains noncovalently attached FAD that is reduced by either dithionite or NADPH without formation of a stable semiquinone. Titration with NADP+/NADPH gives an E0' value of approximately -327 mV versus SHE. Because this E0' is so close to that of the NADP+/NADPH couple it is not clear if Protein X is an NADPH oxidase or an NADP+ reductase in vivo. Comparison of the NH2-terminal sequence and other properties of Protein X with those of other proteins, suggests that it is likely to be related to the Escherichia coli ferredoxin NADP+ reductase (the fpr gene product), and affinity chromatography shows that Protein X binds specifically to AvFdI.

Amino Acid Sequence↗

PubMed1994

Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein.

The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.

Azotobacter vinelandii↗

PubMed1994

MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering.

Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.

Adenosine Diphosphate↗

PubMed1994

Azotobacter vinelandii ferredoxin I. Alteration of individual surface charges and the [4FE-4S]2+/+ cluster reduction potential.

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Peptococcus aerogenes ferredoxin (PaFd), near their analogous [4e-4S]2+/+ clusters, are highly conserved (Backes, G., Mino, Y., Loehr, T.M., Meyer, T.E., Cusanovich, M.A., Sweeney, W.V., Adman, E.T., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 11, 2055-2064). Despite these similarities, the reduction potential (E0') of the AvFdI [4Fe-4S]2+/+ cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to the control of E0' by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitutions of negatively charged surface residues with neutral residues and two involved substitution of buried hydrophobic residues. All AvFdI variants were characterized by x-ray crystallography, absorption, CD, EPR, and 1H NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and 1H NMR spectroscopic properties of AvFdI and had a minor influence on E0'. For all other mutations there were no changes in reduction potential. Thus we conclude, that variations in charged surface residues do not account for the observed differences in E0' between the analogous [4Fe-4S]2+/+ cluster of PaFd and AvFdI. These differences are therefore most likely to be due to differences in solvent accessibility.

Amino Acid Sequence↗

PubMed1993

Dinitrogenase reductase- and MgATP-dependent maturation of apodinitrogenase from Azotobacter vinelandii.

The requirements for iron-molybdenum cofactor (FeMo-co) activation of apodinitrogenase from Azotobacter vinelandii strain UW97, which lacks dinitrogenase reductase activity as assayed by substrate reduction, have been examined. Activation of apodinitrogenase from strain UW97 by FeMo-co requires the addition of both dinitrogenase reductase and MgATP. When the same apodinitrogenase is pretreated with dinitrogenase reductase and MgATP and then partially purified, however, it does not require these components for activation by FeMo-co. This suggests that dinitrogenase reductase and MgATP are involved in processing apodinitrogenase to a FeMo-co activatable form. This processing step coincides with a change in the subunit composition of apodinitrogenase from alpha 2 beta 2 to a form with an additional subunit (gamma) attached. The apodinitrogenase with the associated gamma subunit is apparently the form of the protein that is competent for activation by FeMo-co.

Adenosine Triphosphate↗

PubMed1993

Crystal structures of oxidized and reduced Azotobacter vinelandii ferredoxin at pH 8 and 6.

Crystal structures of Azotobacter vinelandii ferredoxin I (FdI) have been solved and refined at 2.2 to 1.9-A resolution at pH 8 and 6 for both the oxidized and dithionite-reduced proteins. Only the [3Fe-4S] cluster is reduced by dithionite. The four structures (denoted FdI8ox, FdI8red, FdI6ox, and FdI6red) have been compared to address three questions: the effect of reduction at pH 8, the effect of pH change on the structure, and the effect of reduction at pH 6. Comparison of the FdI8ox and FdI8red structures shows that Asp-15 changes conformation in a manner consistent with increased anionic repulsion between this residue and the reduced [3Fe-4S]0 cluster. By revealing an electrostatic interaction between Asp-15 and the [3Fe-4S] cluster, this result supports the conclusion in the accompanying paper (Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, G. M., Stephens, P. J., LaMar, G. N., Gorst, C. M., and Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939) that Asp-15 participates in protonation of the reduced [3Fe-4S]0 cluster at acid pH. The [3Fe-4S]0 cluster in the FdI8red structure also displays a distinct shift within the protein as well as internal distortions when compared to the [3Fe-4S]+ cluster in the FdI8ox structure. Comparison of the FdI8ox and FdI6ox structures shows that pH change does not have any significant effect on the [3Fe-4S]+ cluster or surrounding residues. Comparison of the FdI6ox and FdI6red structures shows that reduction at pH 6 also does not have any significant effect on the [3Fe-4S] cluster or Asp-15. The absence of structural change supports the conclusion that at acid pH, the reduced [3Fe-4S] cluster is protonated, i.e. [3Fe-4S]0-H+ (Shen et al., 1993). The cluster is not shifted or distorted as in the FdI8red structure. Instead, the [3Fe-4S]o-H+ cluster FdI8red is structurally similar to the [3Fe-4S]+ cluster (FdI8ox, FdI6ox), which has the same net charge. An Asp-15-Lys-84 salt bridge is observed in all four structures, indicating that Asp-15 is ionized at pH 8 and 6. An ionized state for Asp-15 is also implied by a lack of conformational change in Lys-84; the side chain of this residue rearranges when Asp-15 is substituted with a neutral amino acid (Shen et al., 1993).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Azotobacter vinelandii ferredoxin I. Aspartate 15 facilitates proton transfer to the reduced [3Fe-4S] cluster.

The [3Fe-4S]+/0 cluster of Azotobacter vinelandii ferredoxin I (AvFdI) has an unusually low and strongly pH-dependent reduction potential (E'0). The reduced cluster exists in two forms, depending upon pH, that exhibit substantially different magnetic circular dichroism (MCD) spectra. Recent studies have established that the MCD changes observed on decreasing the pH from 8.3 (alkaline form) to 6.0 (acid form) cannot be explained either by a change in spin state of the cluster (Stephens, P.J., Jensen, G.M., Devlin, F.J., Morgan, T.V., Stout, C. D., Martin, A.E., and Burgess, B.K. (1991) Biochemistry 30, 3200-3209) or by a major structural change (e.g. ligand exchange) (Stout, C.D. (1993) J. Biol. Chem. 268, 25920-25927). Here, we have examined the influence of aspartate 15 on the pH dependence of the spectroscopic and electrochemical properties of AvFdI by construction of a D15N mutant. Aspartate 15, which is salt-bridged to lysine 84 at the protein surface, is the closest ionizable residue to the [3Fe-4S] cluster. The results show that replacement of aspartate by asparagine results in an approximately 20-mV increase in E'0 for the [3Fe-4S]+/0 cluster at high pH concomitant with an approximately 0.8-pH unit decrease in the pK of the reduced form. The major pH dependence of E'0 is preserved as is the effect observed by MCD. These data eliminate the possibility that the MCD change is due to the presence of Asp-15 and support the conclusion that it originates in direct protonation of the [3Fe-4S]0 cluster, probably on a sulfide ion. Voltammetric studies show that interconversion between [3Fe-4S]+ and [3Fe-4S]0 at acidic pH involves rapid electron transfer followed by proton transfer (for reduction) and then proton transfer followed by electron transfer (for oxidation). Ionized aspartate 15 facilitates proton transfer. Thus, protonation and deprotonation are much slower for D15N relative to the native protein at pH > 5.5. Proton transfer reactions necessary for further reduction of the [3Fe-4S]0 cluster to the [3Fe-4S]- and [3Fe-4S]2- states are also retarded in D15N. The results suggest that the carboxylate-ammonium salt bridge afforded by Asp-15-Lys-84 conducts protons between the cluster and solvent H2O molecules. Overproduction of D15N FdI, but not native FdI, in A. vinelandii has a negative effect on the growth rate of the organism, suggesting that the rate of protonation or deprotonation of the [3Fe-4S]0 cluster may be important in vivo.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of nitrogenase from a delta nifW strain of Azotobacter vinelandii.

Deletion of the nifW gene in Azotobacter vinelandii yields a strain (DJ224) that grows poorly under N2 fixing conditions (Jacobson, M. R., Cash, V. L., Weiss, M. C., Laird, N. F., Newton, W. E., and Dean, D. R. (1989) Mol. & Gen. Genet. 219, 49-57). Here we report the purification of nitrogenase from DJ224. The purified Fe protein was indistinguishable from wild-type. The MoFe protein was indistinguishable from the wild-type MoFe protein by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, native gel electrophoresis, two-dimensional gel electrophoresis, metal analysis, UV/visible and EPR spectroscopies. It was different by the criteria of CD spectroscopy and specific activities. At a 5:1 molar ratio of Fe protein to MoFe protein, H2 evolution under argon was identical to wild-type, C2H2 reduction was inhibited by 27%, N2 reduction was inhibited by 38%, and CO inhibited H2 evolution by 17%. The above data show that the nifW gene product is not required for: 1) detectable alteration of the polypeptide; 2) the synthesis of the metal portion of FeMo cofactor; or 3) FeMo cofactor insertion. The MoFe protein synthesized in the absence of NifW appears to have an alteration near the FeMo cofactor site, possibly at homocitrate, which causes differential inhibition of different substrates.

Amino Acid Sequence↗

PubMed1993

Properties of Azotobacter vinelandii rhodanese.

Rhodanese (thiosulfate: cyanide sulfurtransferase, E.C. 2.8.1.1) was purified from the nitrogen fixing organism Azotobacter vinelandii, and its amino acid composition was determined. The enzyme is a single polypeptide chain of M(r) 29,000 which showed an apparent pI of 4.7 and no presence of isoenzymes. Steady-state initial velocity measurements indicate that the enzyme catalyzes the transfer of sulfane sulfur of thiosulfate either to cyanide or to dihydrolipoate by way of a double displacement mechanism.

Amino Acids↗

PubMed1996

Escherichia coli contains a protein that is homologous in function and N-terminal sequence to the protein encoded by the nifS gene of Azotobacter vinelandii and that can participate in the synthesis of the Fe-S cluster of dihydroxy-acid dehydratase.

In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene. This E. coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0. When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2-. This protein has a reactive sulfhydryl group that is essential for activity. As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein. The purified E. coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a [4Fe-4S] cluster on the apoprotein of E. coli dihydroxy-acid dehydratase. A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state.

Alanine↗

PubMed1977

Inhibition of Azotobacter vinelandii RNA polymerase by cibacron blue F3GA.

Cibacron blue F3GA is a potent inhibitor of the Azotobacter vinelandii DNA-directed RNA polymerase. Addition of 8 micrometer Cibacron blue F3GA prior to initiation results in a greater than 90% inhibition of the poly[d(A-T]-directed synthesis of poly[r(A-U)] while addition of the dye during the course of the reaction is without effect on chain elongation. Binding of RNA polymerase to [3H]poly[d(A-T)] is inhibited by only 15% in the presence of 8 micrometer Cibacron blue F3GA. Inhibition by Cibacron blue F3GA is noncompetitive with regard to ATP, UTP, or template. The poly[d(A-T)]-directed pyrophosphate exchange reaction is relatively resistant to inhibition by Cibacron blue F3GA. Rifampicin added to a similar reaction (in the presence of absence of Cibacron blue F3GA) results in 95% inhibition of the exchange reaction. The interaction of the RNA polymerase core enzyme with Cibacron blue F3GA is shown by the formation of a difference spectrum with a positive maximum at 675 nm which is not affected by the presence of a high concentration (4 micrometer) of rafampicin. The data indicate that Cibacron blue F3GA acts by binding to RNA polymerase and inhibits a step between the synthesis of the initial phosphodiester bond and formation of a stable ternary elongation complex.

Anthracenes↗

PubMed1996

Effect of nutrients on alginate synthesis in Azotobacter vinelandii and characterization of the produced alginate.

The role of nutrients on alginate production by Azotobacter vinelandii was studied in batch cultures. The largest amount of bacterial alginate was obtained in presence of: 0.3 g/l MgSO4.7H2O. 0.4 g/l NaCl, 42 mg/l CaCl2.2H2O,.4 mg/l KH2PO4, 16 mg/l K2HPO4, 2.5 mg/l FeSO4.7H2O, 2.9 mg/l H3BO3, 2 mg/l ZnSO4.7H2O, 2 mg/l Na2MoO4.2H2O, 0.3 mg/l CuSO4.5H2O, 0.2 mg/l MnCl2.4H2O. Alginate production was not enhanced by natural additives or inducing agents, except for acetate, which increased alginate yield. The pure alginate contained 0.36% ash and 0.4% protein. It is similar to algal alginate, but it has an extra acetyl group. It contains 69.5% M-M block, 27.5% M-G block and 3% G-G block.

Alginates↗

PubMed1976

ATP-dependent calcium transport in isolated membrane vesicles from Azotobacter vinelandii.

Membrane vesicles from Azotobacter vinelandii O prepared by osmotic lysis of spheroplasts in tris (hydroxymethyl) aminomethane/acetate buffer (pH 7.8) contain a latent adenosine triphosphatase (ATPase). The ATPase can be activated when the vesicles are incubated in the presence of an electron donor (D-lactate) and a mixture of adenosine diphosphate and inorganic phosphate or by controlled treatment with trypsin. After the ATPase is activated, the membrane vesicles in the presence of adenosine triphosphate accumulate calcium but not glucose or rubidium (in the presence of valinomycin). ATP-dependent calcium uptake follows Michaelis-Menten kinetics with a Km of 48 muM and a Vmax of 20 nmol/min/mg of membrane protein and is highly specific for calcium over cations magnesium, barium, lanthanum, sodium, potassium, and lithium. The calcium accumulated in the presence of ATP is freely exchangeable with external calcium and is rapidly released in the presenceof uncouplers or ATPase inhibitors. Calcium uptake in the presenceof ATP is blocked by dicyclohexylcarbodiimide, ADP, p-chloromercuriphenylsulfonate, by the proton-conducting ionophores m-chlorophenylcarbonylcyanide hydrazone, nigericin, monensin, and gramicidin D, but not by potassium cyanide, anoxia, or valinomycin (in the presence of potassium). Measurements of the external pH of vesicle suspensions reveal that protons are actively taken up by the membranes during hydrolysis of ATP. These results suggest that vesicles prepared under these conditions have a topology which is inverted with respect to the intact cell and that calcium is accumulated by means of proton antiport.

Adenosine Diphosphate↗

PubMed1997

Induction of Encystment and Poly-beta-Hydroxybutyric Acid Production by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201, when grown under nitrogen-free conditions with excess glucose, accumulated poly-beta-hydroxybutyric acid amounting to 75% of cell dry weight at the late exponential phase. This led to induction of encystment, which increased steadily with concomitant intracellular degradation of the polymer. Increase in encystment and PHB production were parallel up to 0.5% (wt/vol) glucose. Further increase in glucose reduced cyst formation but enhanced PHB accumulation. Replacement of glucose by n-butyl alcohol and metabolically related compounds identified crotonate as the best encystment inducer followed by beta-hydroxybutyrate and butyrate, but PHB production was inhibited in general. Supplementation of medium with these compounds enhanced the onset of encystment, and only beta-hydroxybutyrate increased PHB accumulation significantly.

Journal Article↗

PubMed2000

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.

Journal Article↗