Cyst formation and poly-beta-hydroxybutyric acid accumulation in Azotobacter

Stevenson, L. H. (Louisiana State University, Baton Rouge), and M. D. Socolofsky. Cyst formation and poly-beta-hydroxybutyric acid accumulation in Azotobacter. J. Bacteriol. 91:304-310. 1966.-The relationship between cyst formation and the accumulation of poly-beta-hydroxybutyric acid (PHB) in Azotobacter vinelandii (A. agilis) was investigated. After various periods of growth, the cells were harvested, and the amount of PHB and the extent of encystment were determined. The polymer content of the cells increased sharply and reached a maximum on the 2nd day of growth followed by a gradual decline as the culture aged. At maximal accumulation, the PHB content was 35% of the dry weight, and the PHB-nitrogen ratio was 11:1. Those substrates promoting the highest polymer content (glucose, butanol) also promoted 95 to 100% encystment. Manipulation of the carbon and nitrogen supply in the medium indicated that both the maximal PHB content and the extent of cyst formation could be controlled. A direct correlation was noted between the amount of polymer accumulated and the percentage of cysts formed, indicating a possible role of PHB as a carbon or energy source, or both, for the encystment process.     

Acetate assimilation by Nitrobacter agilis in relation to its "obligate autotrophy"

Acetate (1 to 10 mm) had no effect on the rate of nitrite oxidation or exponential growth by Nitrobacter agilis. However, acetate-1-(14)C and -2-(14)C were both assimilated by growing cultures, and acetate carbon contributed 33 to 39% of newly synthesized cell carbon. Carbon from acetate was incorporated into all of the major cell constituents, including most of the amino acids of cell protein and poly-beta-hydroxybutyrate (PHB). Cultures grown in the presence of acetate showed a significant increase in turbidity, attributable in part to protein synthesis and the accumulation of PHB in the "post-exponential phase," when the supply of nitrite was completely exhausted. Cell suspensons of N. agilis assimilated acetate in the absence of bicarbonate and even in the absence of nitrite. However, the addition of nitrite increased the rate of acetate assimilation by cell suspensions. The distribution of (14)C-acetate incorporated by cell suspensions was qualitatively similar to that found with growing cultures. Cell suspensions of N. agilis slowly oxidized acetate to CO(2). Addition of nitrite suppressed CO(2) production from acetate but increased the assimilation of acetate carbon into cell material. N. agilis contained all the enzymes of the tricarboxylic acid cycle. Growth of N. agilis in the presence of acetate did not significantly affect the levels of the enzymes of the tricarboxylic acid cycle, but did result in a 100-fold increase in the specific activity of isocitratase. In contrast, carboxydismutase was partially repressed. N. agilis was grown heterotrophically through seven transfers on a medium containing acetate and casein hydrolysate. The addition of nitrite increased the rate of heterotrophic growth. Heterotrophically grown organisms still retained their ability to grow autotrophically with nitrite. However, these organisms oxidized nitrite at a slower rate. Organisms from autotrophic and heterotrophic cultures were analyzed to determine the mean guanine plus cytosine content of their deoxyribonucleic acid; in both cases this mean was 61.2 +/- 1%. We concluded that N. agilis is not an obligate autotroph; it appears to be a facultative autotroph which resembles the novel facultative autotroph, Thiobacillus intermedius, very closely.     

Production of poly-β-hydroxybutyrate by Azotobacter chroococcum H23 in chemically defined medium and alpechin medium

M.V. MARTINEZ-TOLEDO, J. GONZALEZ-LOPEZ, B. RODELAS, C. POZO AND V. SALMERON. 1995. Azotobacter chroococcum H23 is able to produce large amounts of poly-β-hydroxybutyrate (PHB) during growth in chemically-defined medium (N-free or with NH⁺₄) and alpechin (wastewater from olive oil mills) medium. Polymer production was not dependent of the nutrient limitation. Strain H23 was capable of accumulating PHB up to 70% of the cell dry weight after 24 h incubation in chemically-defined media containing 1% glucose, fructose, mannitol, saccharose or starch. Azotobacter chroococcum grown on NH⁺₄-medium supplemented with alpechin formed PHB up to 50% of the cell dry weight after 24 h, suggesting that these wastes could be utilized by Azotobacter as a cheap substrate for producing PHB.     

New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products

A recombinant E. coli strain (K24K) was constructed and evaluated for poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20 degrees C higher than those of PHBs from the natural producer strains.     

Isolation, Identification, and Metabolic Role of the Sudanophilic Granules of Zoogloea ramigera

Organisms isolated from activated sludge and identified as Zoogloea ramigera accumulated large amounts of sudanophilic granules as the cultures flocculated. The granules were extracted by chloroform and precipitated with ether from acid-hydrolyzed cells. Identification of the sudanophilic granules as poly-β-hydroxybutyric acid (PHB) was confirmed by physical, chemical, and infrared spectral analyses. The isolated polymer accounted for 12.0 to 50.5% of the dry weight of the cells. The polymer was not synthesized when the culture was grown in a growth-limiting concentration of organic substrate; it did accumulate when the culture was grown in medium enriched with carbon and energy sources. An increase in concentration of intracellular PHB was directly proportional to optical density and uptake of glucose. Aside from intracellular storage of PHB as endogenous metabolite, the accumulation of PHB is noted as a possible mechanism of flocculation.     

EFFECT OF TUNGSTATE ON THE UPTAKE AND FUNCTION OF MOLYBDATE IN AZOTOBACTER AGILIS.

Bulen, William A. (Charles F. Kettering Foundation, Yellow Springs, Ohio). Effect of tungstate on the uptake and function of molybdate in Azotobacter agilis. J. Bacteriol. 82:130-134. 1961.-The reported competitive inhibition of molybdate by tungstate was investigated in an effort to elucidate molybdenum functions associated with nitrogen fixation by Azotobacter agilis (A. vinelandii). Growth, respiration, and N(2) (15)-incorporation experiments with normal and molybdenum-deficient cells indicated that tungstate inhibits the uptake of molybdate but does not compete with the metabolically functional molybdenum of cells metabolizing N(2). Neither a molybdenum requirement nor a tungstate inhibition was observed with cells metabolizing urea.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Role of poly-β-hydroxybutyric acid in cyst formation byAzotobacter

Several parameters associated with the growth ofAzotobacter vinelandii in liquid culture were examined in order to investigate the relationship between the accumulation and degradation of poly-β-hydroxybutyric acid (PHB), the development of viscous capsular components, and cyst formation. The amount of intracellular PHB, which increased markedly during the log phase of growth, reached a maximum during the early stationary phase and subsequently declined. During polymer degradation there was a concurrent increase in the extent of encystment in the cultures supplemented with CaCO₃. An increase was noted in the viscosity of culture supernatants during polymer degradation when CaCO₃ was deleted from the medium and the culture pH was controlled by the periodic addition of 0.1m KOH. The extent of encystment and the amount of PHB accumulated were directly proportional to the substrate concentration. The PHB was selectively labeled by the addition of sodium acetate-2-¹⁴C to late log-phase cells. During polymer utilization in either encysting or nonencysting cultures 20% of the label was evolved as CO₂. In the nonencysting cultures, 45% of the radioactivity was distributed between residual PHB and other cellular components, and 35% was in the supernatant polysaccharide-like material. Intact cysts retained 80% of the label. Experiments with ruptured cysts indicated that about 35% of the radioactivity was present in the intine material.     

Lipid metabolism during encystment of Azotobacter vinelandii.

The formation of cysts by Azotobacter vinelandii involves the synthesis of lipids as major metabolic products. Cells which encyst at low levels in aging glucose cultures undergo the same pattern of lipid synthesis as cells which undergo reasonably synchronous encystment in beta-hydroxybutyrate or n-butanol. The accumulation of poly-beta-hydroxybutyrate (PHB) precedes the synthesis of 5-n-heneicosylresorcinol and 5-n-tricosylresorcinol (AR1), which is then followed in about 6 h by the synthesis of the 5-n-alkylresorcinol galactosides (AR2). In the mature cyst, PHB, AR1, and AR2 account for 8, 5.6, and 4.5%, respectively, of the dry weight. Phospholipid formation levels off 4 h postinduction, which coincides with the final cell division, but fatty acids synthesis continues at a very low level throughout encystment, suggesting some turnover of fatty acid. Distribution studies show that AR1 and AR2 are found in roughly equal amounts in the exine and central body of the cysts, with only trace amounts recovered from the intine. Studies of cysts labeled during encystment with [14C]beta-hydroxybutyrate or during vegetative growth with [14C]glucose suggest that the exine structure is synthesized during encystment, but that the intine is composed largely of vegetative cell components.     

Poly-3-hydroxybutyrate metabolism in the type II methanotroph Methylocystis parvus OBBP

Differences in carbon assimilation pathways and reducing power requirements among organisms are likely to affect the role of the storage polymer poly-3-hydroxybutyrate (PHB). Previous researchers have demonstrated that PHB functions as a sole growth substrate in aerobic cultures enriched on acetate during periods of carbon deficiency, but it is uncertain how C(1) metabolism affects the role of PHB. In the present study, the type II methanotroph Methylocystis parvus OBBP did not replicate using stored PHB in the absence of methane, even when all other nutrients were provided in excess. When PHB-rich cultures of M. parvus OBBP were deprived of carbon and nitrogen for 48 h, they did not utilize significant amounts of stored PHB, and neither cell concentrations nor concentrations of total suspended solids changed significantly. When methane and nitrogen both were present, PHB and methane were consumed simultaneously. Cells with PHB had significantly higher specific growth rates than cells lacking PHB. The addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption, but the addition of glyoxylate (a source of C(2) units) did not. This and results from other researchers suggest that methanotrophic PHB metabolism is linked to the supply of reducing power as opposed to the supply of C(2) units for synthesis.     

Influence of glutamic acid on the endogenous respiration of Bacillus subtilis

Clifton, C. E. (Stanford University, Stanford, Calif.), and John Cherry. Influence of glutamic acid on the endogenous respiration of Bacillus subtilis. J. Bacteriol. 91:546-550. 1966.-Amino acids serve as the major initial endogenous substrate for Bacillus subtilis. The endogenous activity of freshly harvested washed cells is high and falls off rapidly with time of shaking at 30 C to lower but still significant levels. The rate of O(2) consumption after the addition of glutamic acid also decreases as the cells age, but more slowly than noted for endogenous respiration. When cells were fed glutamate as soon as possible after harvesting, an apparent stimulation of endogenous respiration was noted. However, endogenous activity was inhibited if the cell suspensions were shaken for at least 1 hr before addition of the glutamate. Similar results were obtained with glycerol or glucose as exogenous substrates. Variation in rates of respiration with age of the cells, inherent instability of B. subtilis, and possible utilization of substances initially excreted by the cells appear to account for the variations noted regarding the influence of an exogenous substrate on endogenous respiration.     

Poly(3-hydroxybutyrate) as an endogeneous substrate for H 2 evolution in Rhodovulum sulfidophilum

In the absence of an external substrate, H 2 was evolved in Rhodovulum sulfidophilum under light-anaerobic conditions, along with degradation of poly(3-hydroxybutyrate) (PHB). Cells grown with succinate as a sole carbon source accumulated only a small amount of PHB compared with that in cells grown with a multiple substrate consisting of a mixture of four organic acids. Unlike PHB-containing cells, PHB-deficient cells did not evolve H in the absence of an external substrate. Nitrogenase activity was expressed while no hydrogenase activity was detected during the incubation of PHB-containing cells. These results suggest that intracellular PHB serves as a substrate for the H evolution catalyzed by nitrogenase when an external substrate is lacking.     

Production of poly (3-hydroxybutyrate) from starch by Azotobacter chroococcum

Production of poly (3-hydroxybutyrate) (PHB) from starch was investigated in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask culture, PHB content increased up to 74% of dry cell wt with increasing culture volume. In batch culture, PHB content increased to 44% with O2 limitation. In fed-batch culture, cell concentration of 71 g/l with 20% PHB was obtained without O2 limitation, whereas cell concentration of 54 g/l with 46% PHB was obtained with O2 limitation.     

Effects of Granule-Associated Protein PhaP on Glycerol-Dependent Growth and Polymer Production in Poly(3-Hydroxybutyrate)-Producing Escherichia coli

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.     

Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

Phosphate-limited culture of Azotobacter vinelandii.

Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly-beta-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.     

Internal Membrane Control in Azotobacter vinelandii

Azotobacter vinelandii was grown on N(2), NH(4) (+), or NO(3) (-), and an internal membrane network was observed by electron microscopy of thin sections of cells. Cells obtained in early exponential growth contained less internal membrane than did cells from cultures in late exponential growth. It seems likely that O(2) has a role in regulating the amount of internal membrane structure.     

Suitability of commercial beet molasses fractions as substrates fro polyhydroxyalkanoate production byAzotobacter vinelandii UWD

Summary Beet molasses that had been fractionated commercially by ion exclusion resulted in two waste-streams: extract molasses (EM) and concentrated separator by-product (CSB). Only EM at 4–5% w/v contained sufficient sugar to promote polyhydroxybutyrate (PHB) formation byAzotobacter vinelandii UWD, but the yield of PHB/protein was less than that obtained in unfractionated beet molasses. EM and especially CSB added at 0.5–2.0% w/v to media containing a variety of sugar sources promoted an increased yield of PHB/protein. The best use of these beet molasses fractions was, therefore, as a minor addition to media containing sugars to increase PHB yield, but not as a primary substrate for PHB production.     

Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803

Poly-beta-hydroxybutyrate (PHB) accumulation in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, was studied under various cultural and nutritional conditions. Under controlled condition, cells harvested at the stationary phase of growth depicted maximum accumulation of PHB, i.e., 4.5% (w/w of dry cells) as compared to lag (1.8%) or logarithmic (2.9%) phases of cultures. A temperature range of 28-32 degrees C and pH between 7.5 and 8.5 were preferred for PHB accumulation. Cells cultivated under regular light-dark cycles accumulated more PHB (4.5%) than those grown under continuous illumination (2.4%). Nitrogen and phosphorus starvation stimulated PHB accumulation up to the tune of 9.5 and 11% (w/w of dry cells), respectively. Synechocystis cells pre-grown in glucose (0.1%)-supplemented BG-11 medium when subjected to P-deficiency in presence of acetate (0.4%), PHB accumulation was boosted up to 29% (w/w of dry cells), the value almost 6-fold higher with respect to photoautotrophic condition. Fishpond discharges were found as suitable media for PHB accumulation in the test cyanobacterium.