Alginate production by Azotobacter vinelandii DSM576 in batch fermentation

The kinetics of growth and alginate production from glucose in a nitrogen and phosphate-rich medium by Azotobacter vinelandii DSM576 were studied in a laboratory fermenter at pH 7 and 35°C. Batch fermentations were carried out both without control of dissolved oxygen concentration (DO) and at 1, 2, 5 and 10% DO. Although growth was faster at higher DO, maximum biomass concentration was lower. No alginate was produced at 10% DO. Alginate production was faster at 5 and 2% DO but higher alginate concentrations and yields were obtained without DO control. Alginate production was growth-associated at 5% DO, but significant amounts of alginate were produced after growth had stopped at lower DO values. In fermentations without DO control the molecular weight of the polymer reached a maximum (11–17.6 × 10⁴) when specific growth rate was between 0.02 and 0.04 h⁻¹ and residual concentration of ammoniacal nitrogen was between 0.01 and 0.02 g L⁻¹ and then sharply decreased. Received 15 August 1997/ Accepted in revised form 08 January 1998     

Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-β-hydroxybutyrate (PHB) synthesis

Aims:  A two-stage fermentation strategy, based on batch cultures conducted first under non-oxygen-limited conditions, and later under oxygen-limited conditions, was used to improve alginate production by Azotobacter vinelandii (AT6), a strain impaired in poly-β-hydroxybutyrate (PHB) production.
Methods and Results:  The use of sucrose as carbon source, as well as a high oxygen concentration (10%), allowed to obtain a maximum biomass concentration of 7·5 g l⁻¹ in the first stage of cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9·5 g l⁻¹, after 50 h of cultivation.
Conclusion:  Alginate concentration obtained from the AT6 strain was two times higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature. However, the mean molecular mass of the alginate produced in the second stage of the process by the mutant AT6 was lower (400 kDa) than the polymer molecular mass obtained from the cultures developed with the parental strain (950 kDa).
Significance and Impact of the Study:  The use of a mutant of A. vinelandii impaired in the PHB production in combination with a two-stage fermentation process could be a feasible strategy for the production of alginate at industrial level.     

Continuous monitoring of nitrogenase activity in Azotobacter vinelandii fermentation using off-gas mass spectrometry

This study evaluated the feasibility of monitoring nitro-genase activity in situ through measurement of N(2) uptake rate (NUR) using off-gas mass spectrometry. Four 50-L cultures of Azotobacter vinelandii were grown aer-obically in nitrogen-free medium to cell densities of 1.0-1.3gL(-1) magnetic-sector mass spectrometer was used to monitor NUR along with other gas exchange rates. The small specific uptake rate (1.2 mmol g(-1) h(-1)) and low cell density were found to lead to a NUR below the measurement accuracy limits under normal conditions. An operating strategy and feed gas mixture (40% O(2), 45% N(2) 15% Ar) were designed to improve the signal-to-noise ratio while maintaining dissolved O(2) and N(2) levels in desired ranges. The fraction of N(2) removed from the air stream was increased approximately 5-fold from 0.2% to 1.0% and the measurement noise was reduced 25-fold from a baseline of +/-5to +/-0.2 mmol L(-1) h(-1). The NUR measurements were compared against in vivo and in vitro acetylene reduction assays as well as on-line cell growth rate measurements. While electron transfer requirements predict an NUR-to-acetylene reduction rate ratio of 0.33, measured ratios for the in vivo and in vitro assays were 0.8 and 0.44, respectively. This suggests that other rate-limiting steps were present in the case of the in vivo assay. In accordance with reports in the literature, no concomitant hydrogen evolution was detected. This is the first reported continuous and direct measurement of NUR in fermentation and demonstrates a novel approach for improving measurement accuracy through rational adjustment of operating conditions. The technique has potential to provide useful insight for development and control of microbial nitrogen fixation processes.(c) John Wiley & Sons, Inc.     

Components in the inoculum determine the kinetics of Azotobacter vinelandii cultures and the molecular weight of its alginate

In cultures of Azotobacter vinelandii inoculated using washed cells (avoiding exhausted broth components) alginates of a higher molecular weight (1200 kDa) than those obtained in cultures conventionally inoculated (350 kDa), were produced. Also, when comparing conventionally inoculated cultures with those inoculated with washed-cells, the alginate lyase activity was delayed and the final polymer concentration decreased from 4.8 to 3.5 g l^–1. This suggests that components in the exhausted inoculum broth play important regulatory roles in alginate biosynthesis and needs to be taken into account when describing polymer biosynthesis.     

Physiology of exopolysaccharide production by Azotobacter vinelandii from 4-hydroxybenzoic acid

The relationship between exopolysaccharide (EPS) production by Azotobacter vinelandii ATCC 12837 from 4-hydroxybenzoic acid as sole carbon source and other physiological parameters was investigated. In relation to growth, Azotobacter needed more time in 4-hydroxybenzoic acid to reach levels of biomass similar to those obtained when sugars were used, although the phenolic compound led to a more extensive exponential phase. The encystment process was initiated after cells had grown for 24 h, in which small amounts of EPS were synthesized and poly-β-hydroxybutyrate (PHB) accumulation began. Both polymers, EPS and PHB, showed a similar evolution with time, as well as the formation of cysts, which points out the existence of a relation between these parameters. This was corroborated by a statistical study, in which significant correlations (P<0.05) were observed when each parameter was compared to the two others. Journal of Industrial Microbiology & Biotechnology (2002) 29, 129–133 doi:10.1038/sj.jim.7000288 Received 01 February 2002/ Accepted in revised form 13 June 2002     

Characterization of Azotobacter vinelandii aggregation in submerged culture by digital image analysis

A simple and accurate method for determining the distribution of sizes of single cells and aggregates of Azotobacter vinelandii by image analysis has been developed. A staining procedure using methylene blue helps to enhance the contrast between aggregates and background without altering aggregate size distribution. Sample dilution affected the distribution of the population and therefore should be avoided. Mixing and aeration conditions during culture play an important role in the aggregation of A. vinelandii. Cells grown under mild mixing conditions (unbaffled flasks) presented a thick slime layer and formed aggregates of up to 35 microm of average equivalent diameter. In contrast, under strong agitation conditions (baffled flasks) practically no aggregates were formed throughout cultivation. The method described can be used for the characterization of aggregation of other microbial cultures.     

Utilization of the metal-cyano complex tetracyanonickelate (II) by Azotobacter vinelandii

AIMS: The ability of Azotobacter vinelandii, a N(2)-fixing bacterium, to biodegrade tetracyanonickelate (TCN) was evaluated. METHODS AND RESULTS: The amounts of TCN were measured spectrophotometrically. Ammonia was determined colorimetrically by the indophenol method. The produced methane from TCN conversion by A. vinelandii was detected by gas chromatography. Results showed that A. vinelandii was able to biodegrade 1 mmol l(-1) of TCN. Ammonia and methane were detected during the process of TCN degradation. Effects of exogenous nitrogen sources on TCN degradation were addressed in this study. Results revealed that the addition of ammonia (1, 5 and 10 mmol l(-1)) into the reaction mixtures caused decrease of TCN degradation rate during a 24-h incubation period. This inhibition was also observed when nitrite (5 and 10 mmol l(-1)) was added, whereas TCN degradation still proceeded after the addition of nitrate at the same concentrations. Furthermore, the rate of TCN utilization was strikingly enhanced when 0.8% of glucose was added. CONCLUSIONS: Azotobacter vinelandii can degrade 1 mmol l(-1) of TCN into ammonia and methane. However, the inhibitory effects of exogenous ammonia and nitrite on TCN degradation by this bacterium were found in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report defining the capability of A. vinelandii to degrade TCN. This bacterium might have potential value in applied strategies for removing metal-cyano wastes. Furthermore, these findings would be helpful in designing a practical system inoculated with A. vinelandii for the treatment of TCN.     

The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii

 Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and molybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were determined by a simultaneous spectrophotometric and potentiometric titration as log K ₅=3.65(5), log K ₄=7.41(3) and log K ₃=8.54(4). The metal-ligand equilibrium constant for [MoO₂(L)]^3– was obtained from analysis of the absorbance concentration data: at 20  °C and pH 6.6, log K _eq=4(1). Based on an average log K _a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K _f(MoL) = [MoO₂L^{3 –}]/[MoO₂ ²⁺][L^{5 –}] = 10³⁵ M^–1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on siderophore biosynthesis was followed by UV-vis spectroscopy and HPLC. It could be shown that the formation of molybdenum siderophore complexes clearly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentrations up to 100 μM, the molybdenum azotochelin complex is the predominant molybdenum species, suggesting that azotochelin might also possess sequestering activity towards molybdenum. Even higher molybdate levels result in a complete repression of the synthesis of the tetradentate siderophore azotochelin, while they initiate the alternative release of the more efficient iron chelator, the hexadentate siderophore protochelin. Received: 20 April 1998 / Accepted: 29 June 1998     

Effect of PEG and Salt on Crystallization of Bacterioferritin and Nitrogenase MoFe Protein from Azotobacter vinelandii

MgCl2 was added to the supernatant of the first crystallization of MoFe protein to give a final concentration of 14.6 mmol/L, followed by centrifugation. The treated supematant solution and MoFe protein could be crystallized by using method of siting drop with PEG 6000 and MgC12 as a precipitant and salt, respectively. The larger crystal from the supermatant was observed when the final concentration of PEG and MgCl2 was 4.5% and 15.6 mmol/L, respectively; but small crystal was observed when the concentration was 0 and 23.8 mmol/L, respectively. The larger crystal in brown rectangular prism of MoFe protein was also obtained using the same crystallization method when the final concentration of PEG and MgCI2 was 7.44% and 338.0 mmol/L, respectively. It suggests that the two protein crystals seem to be different, the former being bacterioferritin and the later as nitrogenase MoFe protein.     

Properties of polysaccharide produced by Azotobacter vinelandii cultured on 4-hydroxybenzoic acid

AIMS: Characterization of the exopolysaccharide produced by Azotobacter vinelandii grown on 4-hydroxybenzoic acid (EPS I), and the comparison between this exopolysaccharide and commercial alginate, constituted the main objective of this work. METHODS AND RESULTS: Total carbohydrates, uronic acids, acetyl and pyruvyl groups and proteins were determined by colorimetric methods and composition was confirmed by Nuclear Magnetic Resonance studies. Rheological properties were analysed under different physical and chemical conditions. Results showed differences between EPS I and commercial alginate, in relation to both composition and viscosity. Higher amount of guluronnosyl residues were found in EPS I, whereas commercial alginate contained the same proportion of mannuronosyl and guluronnosyl residues. In accordance with this result, EPS I gave rise to solutions of higher viscosity than commercial alginate, although solutions of this polysaccharide showed greater stability when conditions were altered. CONCLUSIONS: The exopolysaccharide produced by A. vinelandii grown on 4-hydroxybenzoic acid showed a different composition in comparison with commercial alginate, which leads to higher viscosity values for the aqueous solutions of EPS I. SIGNIFICANCE AND IMPACT OF STUDY: This work describes for the first time the characteristics of an exopolysaccharide produced by A. vinelandii from 4-hydroxybenzoic acid, a substrate rarely used as sole carbon source.     

Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture

Abstract Beet molasses (BM) has proven to be an excellent feedstock for polyhydroxyalkanoate (PHA) production by Azotobacter vinelandii UWD. The substrate-cost for PHA production from BM in fed-batch culture was one-third of that using glucose. Copolymers containing β-hydroxyvalerate are readily formed in BM medium when valerate is used as a precursor. The origin of the hydroxyvalerate monomer was most likely a β-ketoacyl-CoA intermediate in the β-oxidation of the odd-length n -alkanoates. BM also contained unidentified factors that stimulated PHA production to a greater extent than cell growth. Analysis of BM fractions has suggested that amino-N compounds may be required for PHA-yield-promotion. Thus the addition of a small amount of commercial peptone to mineral salts medium containing pure or other impure sugar sources has led to significantly increased PHA yields.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Adenine nucleotide content and energy charge in dry cells and cysts of Azotobacter vinelandii

Abstract The levels of intracellular adenine nucleotides, energy charge, oxygen consumption and poly-β-hydroxybutyric acid stored, have been investigated in dry vegetative cells and cysts of Azotobacter vinelandii . The data show that under desiccation conditions the cysts retain viability at energy charge values of 0.20 and an ATP/ADP ratio of 0.24, whereas under the same desiccation conditions, vegetative cells die at energy charge values <0.5 and an ATP/ADP ratio of <0.6.     

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii

A membrane-bound cytochrome oxidase from Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using an ascorbate-TMPD oxidation assay. The oxidase was ‘solubilized’ from a sonic-type electron-transport particle (R₃ fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27–70% (NH₄)₂SO₄. The highly purified cytochrome oxidase has a V of 60–78 μgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN₃ and NH₂OH; NaNO₂ (but not NaNO₃) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c₄ and cytochrome o; cytochromes a₁ and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c₄?o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a₃ oxidase of mammalian mitochondria.     

Influence of nutritional and environmental factors on polysaccharide production by Azotobacter vinelandii cultured on 4-hydroxybenzoic acid

The capacity of 4-hydroxybenzoic acid to support exopolysaccharide (EPS) biosynthesis was investigated. Carbon source concentration, nitrogen supplementation, and other nutritional and environmental factors were optimized to obtain maximal EPS recovery. Higher EPS yields were obtained in nitrogen-free media amended with 20–30 mM 4-hydroxybenzoic acid. In general, modifications in inorganic salt concentration did not alter EPS production, except in the case of magnesium ions. Increased levels of this cation were correlated to greater EPS yields. Production was strongly influenced by certain environmental factors. Optimal values of 34°C, 80 rpm and neutral or slightly basic conditions were selected. Under these conditions, more than 25% of the carbon source supplied was converted to EPS and the production was improved about 42% in comparison to that observed in the initial media. Journal of Industrial Microbiology & Biotechnology (2001) 27, 5–10. Received 05 November 2000/ Accepted in revised form 30 April 2001     

The Relationship Between the Metal Clusters and the Conformation of Molybdenum-Iron Protein from Azotobacter vinelandii

The ultraviolet CD spectrum of nitrogenase MoFe protein from Azotobacter vinelandii had a negative trough with double peaks at 208 nm and 222 nm, respectively, and the shape of the trough was similar to those of other proteins with a-helix structure. After treatment with o-phenanthroline under an aerobic or anaerobic condition, the height of the peak at 222 nm (h222 nm) decreased with the decrease of the C2H2-reduction activity, Fe content and CD spectra at both 450 nm and 660 nm, or at 450 nm of the treated proteins. However, after reconstituting with a reconstituent solution containing Na2MoO4, Na2S, dithiothreitol and either ferric homocitrate or ferric citrate, the h222 nm Of the reconstituted proteins could be restored as well as the activity, Fe content and CD spectra at both of 450 nm and 660 nm. The results show that there is a significant relationship between the metal clusters (FeMoco and P-cluster) and the conformation of MoFe protein.     

Purification and Properties of Nitrogenase MoFe Protein from a nif Z Deletion Strain of Azotobacter vinelandii

The MoFe protein of the nif Z deletion strain (△nif Z MoFe protein) of Azotobac ter vinelandii designated DJ 194 was purified and some properties were studied. The cell free extract of DJ 194 was more sensitive to O2 and heat than the wild-type extract. The specific activity of the purified DJ 194 protein was 283 nmol C2H2 reduced/(min · mg protein), which was much lower than that of purified wild-type A. vinelandii MoFe protein. The △nif Z MoFe protein exhibited a visible similar absorption spectra as the wild type MoFe protein, yet showed significant difference in CD and MCD spectra at the region about 450 mm com paring with the spectral property of the wild-type MoFe protein. This seems to indicate that the P-cluster of the △nif Z MoFe protein was modified, which might be the cause of the low activity of the DJ 194 MoFe protein.     

Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii

Abstract To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii , plasmids carrying vnfE-, vnfH- , or vnfD-lacZ fusions were transferred to Escherichia coli . These genes were expressed only if VnfA was present. Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA. This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters. Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression. In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs. The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA.