Ultrastructural Study of Poly-β-Hydroxybutyrate Granules from Bacillus cereus

The freeze-etching technique was used to examine the effects of fracturing and etching on the appearance of poly-beta-hydroxybutyrate granules from Bacillus cereus. These granules were examined in extracts isolated by hypochlorite or by sonic treatment, and also in fixed and unfixed intact cells; in the latter case they were compared with granules in thin sections of intact cells. After freeze-fracturing, the diameter of the granules in intact cells was between 240 and 720 nm. The granules consisted of a central core, of diameter between 140 and 370 nm, which occupied less than 50% of the volume of the granule and which was either stretched or removed on fracturing; the core was surrounded by an outer coat which may be bounded by a membrane.     

Poly-β-Hydroxybutyrate Metabolism During Growth and Sporulation of Clostridium botulinum

The granules observed in the cytoplasm of cells of sporogenic and asporogenic strains of Clostridium botulinum type E were isolated at various developmental stages of growth and sporulation. Electron microscopy of thin sections showed that most of the granules were dispersed throughout the cytoplasm. Chemical analysis and electron microscopy showed that the granules were poly-beta-hydroxybutyrate (PHB). The polymer began to accumulate after 8 h of growth, reaching 9 and 13% of the cell dry weight in the sporogenic and asporogenic strains, respectively, during early stationary phase. (14)C-acetate was readily incorporated into PHB. The rate of assimilation paralleled the production and utilization of PHB, indicating that the acetate served as its precursor. (14)C-butyric acid was not utilized to any significant extent. Most of the PHB which had accumulated in the sporogenic strain was catabolized during the development of the spore, but in the asporogenic mutant it remained essentially unchanged. The findings suggest that the PHB provides endogenous carbon and energy for the synthesis of spore-specific components required for spore maturation.     

Occurrence of Poly-β-Hydroxybutyrate in the Azotobacteriaceae

Poly-beta-hydroxybutyrate (PHB) from various representative strains of the genera Azotobacter, Beijerinckia, and Derxia was isolated and characterized. During growth in shake culture, with glucose as a carbon and energy source, and molecular nitrogen as a nitrogen source, increase in dry weight appeared linear, and PHB formed a constant percentage of the dry weight. In a medium containing 1% (w/v) glucose, PHB declined with the onset of the stationary phase of growth; with 2% (w/v) glucose, an increase in PHB content during stationary phase was noted in the case of some strains, before a subsequent decline. The decrease in PHB as a percentage of dry cellular weight (not of total amount present in the culture) during growth of some strains with 2% as opposed to 1% (w/v) glucose may be ascribed to a greater production of capsular polysaccharide. PHB content could not be used as a taxonomic criterion. Strain differences were as great as or greater than species differences. The only strain of Beijerinckia fluminensis obtained contained PHB, but it could not be grown on the nitrogen-free medium used. Two species of the genus Azotomonas, reported to be aerobic, nonsymbiotic nitrogen-fixers, did not grow on the nitrogen-free medium used and did not produce PHB during growth with a combined nitrogen source.     

Determination of Poly-β-Hydroxybutyrate and Poly-β-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography

A convenient gas-liquid chromatography procedure to quantify poly-β-hydroxybutyrate and poly-β-hydroxyvalerate in activated sludge was developed by combining lyophilization of the samples, purification of the chloroform phase by water reextraction, and the use of capillary columns. With a flame ionization detector the sensitivity was estimated at 10⁻⁵ g/liter.     

Recovery of Poly-β-Hydroxybutyrate from Estuarine Microflora

Poly-β-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-¹⁴C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Floc Formation by Azospirillum lipoferum Grown on Poly-β-Hydroxybutyrate

Azospirillum lipoferum RG6xx was grown under conditions similar to those resulting in encystment of Azotobacter spp. A. lipoferum produced cells of uniform shape when grown on nitrogen-free β-hydroxybutyrate agar. Cells accumulated poly-β-hydroxybutyrate and often grew as chains or filaments that eventually lost motility and formed capsules. Within 1 week, vegetative A. lipoferum inocula were converted into microflocs arising from filaments or chains. Cells within microflocs were pleomorphic, contained much poly-β-hydroxybutyrate, and were encapsulated. Some cells had a cystlike morphology. Up to 57% of the dry weight of encapsulated flocs was poly-β-hydroxybutyrate, whereas vegetative cells grown in broth with combined nitrogen had only 3% of their dry weight as poly-β-hydroxybutyrate. Neither encapsulated cells in flocs nor nonencapsulated vegetative cells were significantly desiccation resistant. Under starvation conditions (9 days) only 25% of encapsulated cells remained viable, whereas vegetative cells multiplied severalfold. In short-term germination experiments with encapsulated flocs, nitrate, ammonium, and soil extract promoted formation of motile vegetative cells. Most cells in treatments lacking combined nitrogen eventually depleted their visible poly-β-hydroxybutyrate reserves without germinating. The remaining cells retained the reserve polymer and underwent size reduction.     

Hyperproduction of Poly-β-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-β-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N₂, respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-β-hydroxybutyrate, but O₂-limited growth was required for these optimal production values. Polymer production was not dependent on O₂ limitation in strain UWD, but the efficiency of conversion of glucose to poly-β-hydroxybutyrate was enhanced in O₂-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-β-hydroxybutyrate per mg of glucose consumed under vigorous- and low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to from poly-β-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.     

Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly-β-Hydroxybutyrate

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-β-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH₄⁺, but PHB synthesis stopped when NH₄⁺ was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH₄⁺ depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH₃ (pH 11.4) at 45°C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 × 10⁶ to 2.0 × 10⁶) and dispersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH₃ extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.     

Purification of Poly-β-Hydroxybutyrate by Density Gradient Centrifugation in Sodium Bromide

Fractionation of fully sporulated cultures of Bacillus thuringiensis by density gradient centrifugation in NaBr produced two bands which were identified as poly-β-hydroxybutyrate. This technique generated high yields of membrane-bound and unbound granules of exceptional purity and degree of polymerization.     

rRNA and Poly-β-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast and Famine Cycles

Feast and famine cycles are common in activated sludge wastewater treatment systems, and they select for bacteria that accumulate storage compounds, such as poly-β-hydroxybutyrate (PHB). Previous studies have shown that variations in influent substrate concentrations force bacteria to accumulate high levels of rRNA compared to the levels in bacteria grown in chemostats. Therefore, it can be hypothesized that bacteria accumulate more rRNA when they are subjected to feast and famine cycles. However, PHB-accumulating bacteria can form biomass (grow) throughout a feast and famine cycle and thus have a lower peak biomass formation rate during the cycle. Consequently, PHB-accumulating bacteria may accumulate less rRNA when they are subjected to feast and famine cycles than bacteria that are not capable of PHB accumulation. These hypotheses were tested with Wautersia eutropha H16 (wild type) and W. eutropha PHB-4 (a mutant not capable of accumulating PHB) grown in chemostat and semibatch reactors. For both strains, the cellular RNA level was higher when the organism was grown in semibatch reactors than when it was grown in chemostats, and the specific biomass formation rates during the feast phase were linearly related to the cellular RNA levels for cultures. Although the two strains exhibited maximum uptake rates when they were grown in semibatch reactors, the wild-type strain responded much more rapidly to the addition of fresh medium than the mutant responded. Furthermore, the chemostat-grown mutant culture was unable to exhibit maximum substrate uptake rates when it was subjected to pulse-wise addition of fresh medium. These data show that the ability to accumulate PHB does not prevent bacteria from accumulating high levels of rRNA when they are subjected to feast and famine cycles. Our results also demonstrate that the ability to accumulate PHB makes the bacteria more responsive to sudden increases in substrate concentrations, which explains their ecological advantage.     

Poly-β-Hydroxybutyrate Accumulation as a Measure of Unbalanced Growth of the Estuarine Detrital Microbiota

The procaryotic endogenous storage material poly-β-hydroxybutyrate (PHB) can be induced to accumulate in the estuarine detrital microbiota under conditions which suggest unbalanced growth, such as limitation of a critical factor(s) in the presence of carbon and energy sources. Changes in PHB-to-lipid phosphate ratios detected in field samples can be mimicked in the laboratory with common estuarine stresses. Acute anoxia or low pH induces conditions of no growth with depression of both the synthesis and catabolism of PHB without change in the lipid phosphate. Balanced growth induced by nutrients increases the lipid phosphate, depresses PHB synthesis, and stimulates PHB catabolism, resulting in a low ratio of PHB to lipid phosphate. Unbalanced growth induced to a small extent by high salinity or much more readily by dark upland runoff water results in rapid accumulation of PHB and slowing of PHB catabolism with little change in lipid phospate. Unbalanced growth conditions result in high PHB-to-lipid phosphate ratios in the detrital microbiota.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

Formation of Crystalline δ-Endotoxin or Poly-β-Hydroxybutyric Acid Granules by Asporogenous Mutants of Bacillus thuringiensis

Parental strains and asporogenous mutants of Bacillus thuringiensis subspp. kurstaki and aizawai produced high yields of δ-endotoxin on M medium, which contained 330 μg of potassium per ml, but not on ST and ST-a media, each of which contained only 11 μg of potassium per ml. On ST and ST-a media, refractile granules were formed instead. These granules had no insecticidal activity against silkworms and were isolated and identified as poly-β-hydroxybutyric acid. Supplementation of the potassium-deficient ST-a medium with 0.1% KH₂PO₄ (3.7 mM) led to the formation of crystalline δ-endotoxin. The replacement of KH₂PO₄ with equimolar amounts of KCl, KNO₃, and potassium acetate or an equivalent amount of K₂SO₄ had a similar effect, whereas the addition of an equimolar amount of NaH₂PO₄ or NH₄H₂PO₄ did not cause the endotoxin to form. An asporogenous mutant, B. thuringiensis subsp. kurstaki strain 290-1, produced δ-endotoxin on ST-a medium supplemented with 3 mM or more potassium but formed only poly-β-hydroxybutyric acid granules on the media containing ≤1 mM potassium. These results clearly indicate that a certain concentration of potassium is essential for the fermentative production of δ-endotoxin by these isolates of B. thuringiensis. Manganese could not be substituted for potassium. Phosphate ions stimulated poly-β-hydroxybutyric acid formation by strain 290-1. The sporulation of B. thuringiensis and several other Bacillus strains was suppressed on the potassium-deficient ST medium. This suggests that potassium plays an essential role not only in Bacillus cell growth and δ-endotoxin formation but also in sporulation.     

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.     

Fine Structure of Poly-β-Hydroxybutyric Acid Granules in a Blue-Green Alga, Chlorogloea fritschii

Granules characterized as poly-beta-hydroxybutyric acid are described in Chlorogloea fritschii, a blue-green alga. They are generally spherical inclusions of slight electron density which are limited by a membrane approximately 3 nm in thickness.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

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

The potential of Pseudomonas pseudoflava to produce poly-β-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-β-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⁻¹ on glucose, 0.13 h⁻¹ on xylose, and 0.10 h⁻¹ on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g⁻¹ h⁻¹ on arabinose to 0.11 g g⁻¹ h⁻¹ 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 β-hydroxybutyric and β-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter⁻¹. The β-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter⁻¹.