Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Effects of culture conditions on the production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in media containing a high concentration of alpechín (wastewater from olive oil mills) as primary carbon source

Large amounts of homopolymers containing beta-hydroxybutyrate (PHB) and copolymers containing beta-hydroxyvalerate (P[HB-co-HV]) are produced by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechín (wastewater from olive oil mills) as the sole carbon source. Copolymer was formed when valerate (pentanoate) was added as a precursor to the alpechín medium, but it was not formed with the addition of propionate as a precursor. A. chroococcum formed homo- and copolymers of polyhydroxyalkanoates (PHAs) up to 80% of the cell dry weight, when grown on NH(4)(+)-medium supplemented with 60% (v/v) alpechín, after 48 h of incubation at 100 rev min(-1) and 30 degrees C. Production of PHAs by strain H23 using alpechín looks promising, as the use of a cheap substrate for the production of these materials is essential if bioplastics are to become competitive products.     

Production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in wastewater from olive oil mills (alpechin)

We describe the production of large amounts of homo- and copolymers of polyhydroxyalkanoates (PHAs) by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechin. A. chroococcum grown on NH₄⁺-medium supplemented with alpechin formed PHAs up to 50% of the cell dry weight after 24h. The results show that alpechin supports the growth of strain H23 and also that this waste could be utilized as a carbon source. Production of PHAs by using alpechin looks promising, since the use of inexpensive feed-stocks for PHAs is essential if bioplastics are to become competitive products.     

Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24

Methylobacterium sp. ZP24, isolated from a local pond, is able to grow in a medium containing 12 g l⁻¹ lactose as a sole source of carbon, giving 5·25 g l⁻¹ biomass yield and poly-3-hydroxybutyrate (PHB) up to 59% of its dry weight in 40 h. The isolate was also able to utilize cheese whey and produce 1·1 g l⁻¹ PHB. Addition of ammonium sulphate increased the production of PHB from whey 2·5-fold. The potential of Methylobacterium sp. ZP24 in PHB production from cheese whey is discussed.     

Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria

The effect of the herbicide glyphosate ( N -(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha^-1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O₂ h^-1 g^-1 dry weight for A. chroococcum and 12.09 mmol h^-1 g^-1 for A. vinelandii. When 20 kg ha^-1 was used with A. vinelandii , respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha^-1. Nitrogen fixation dropped drastically 80% with 20 kg ha^-1 in A. vinelandii and 98% with 28 kg ha^-1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.     

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.     

A spectrophotometric method for the determination of the kinetic parameters of NH+4 uptake by yeast cells

Abstract The kinetic parameters of NH⁺₄-uptake in yeast cells were determined by a method that is based on the following changes in the external NH⁺₄ concentration in cell suspensions by using NADH-dependent glutamate formation from NH⁺₄ and 2-oxoglutarate. The kinetics of the observed NADH oxidation were analyzed by computer and enabled an estimation of V _max and K _m of the NH⁺₄-uptake system of the cells.     

Effect of a constant supply of different nitrogen sources on protein and carbohydrate content and enzyme activities of Anabaena variabilis cells

The effect of the nitrogen source on carbohydrate and protein contents and on several enzymatic activities involved in the carbon and nitrogen metabolism was studied in Anabaena variabilis ATCC 29413 cells grown under a constant supply of either N, NO⁻₃ or NH⁺₄ at different concentrations. An enhancement of protein content accompanied by a parallel decrease of carbohydrates was observed with increasing NO⁻₃ or NH⁺₄ concentrations in the medium. In cultures containing 0.1 m M NO⁻₃ or 0.1 m M NH⁺₄ nitrogenase (EC 1.18.6.1) activity was 74 and 66%, respectively, of that found in N₂-grown cells. This activity was still present with 1 m M NO⁻₃ or 1 m M NH⁺₄ in the medium and even with 10 m M NO⁻₃, but it was completely inhibited by 5 m M NH⁺₄. Ferredoxin-nitrate reductase (EC 1.7.7.2) activity was detected only in NO⁻₃ grown cells and simultaneously with nitrogenase activity. Increasing concentrations of combined nitrogen in the medium, especially NH⁺₄, promoted a concomitant decline of glutamine synthetase (EC 6.3.1.2), NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42), and NAD⁺-malate dehydrogenase (EC 1.1.1.37) activities, suggesting that these enzymes play an important role in the regulation of carbon-nitrogen metabolism in cyanobacteria.     

Poly(β-hydroxyalkanoate) biosynthesis and accumulation by different Rhizobium species

Abstract Synthesis and accumulation of poly(β-hydroxy-alkanoate) in Rhizobium leguminosarum , R. leguminosarum biovar. trifolii, Rhizobium 'galega', Rhizobium 'hedysarum' and R. meliloti were studied.
Poly(β-hydroxybutyrate) is accumulated up to 55% of the cell dry weight. At 30–50% air saturation R. meliloti accumulates 50% of its biomass (5.5 g·1⁻¹ dry weight) as PHB after 90 h of batch fermentation. At 90% air saturation maximum accumulation (37.5%) of PHB occurs after 70 h cultivation.
R. meliloti strain 41 is able to synthesize the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) at concentrations up to 58% of the biomass dry weight, containing up to 22 mol%β-hydroxyvalerate. Different concentrations of both copolymer and hydroxyvalerate were obtained when the microorganism was grown, in batch culture, in the presence of propionate or valerate and with glucose, sucrose or succinate as main carbon source.     

Interface-mediated death of unconditioned Tetrahymena cells: effect of the medium composition

ABSTRACT We have previously shown that the cell death of Tetrahymena thermophila in low inocula cultures in a chemically-defined medium is not apoptotic. The death is caused by a cell lysis occurring at the medium-air interface and can be prevented by the addition of insulin or Pluronic F-68. Here, we report that cell death can also be caused by the medium. The specific effects of several medium constituents were tested in the presence and absence of an interface. Four of the 19 amino acids (arginine, aspartic acid, glutamic acid, and histidine in millimolar concentration) as well as Ca²⁺ (68 μM) and Mg²⁺ (2 mM) and trace metal ions (micromolar concentrations) are all sufficient to induce the interface-mediated death. The effect of the amino acids and the salt ions Ca²⁺ and Mg²⁺ can be abolished by the addition of insulin (10^-6 M) or Pluronic F-68 (0.01% w/v), whereas insulin/Pluronic F-68 only postpones the death induced by trace metal ions. On the basis of our findings, a new recipe for a chemically-defined medium has been formulated. Single cells can grow in this medium in the presence of medium-air interface without any supplements.     

Cyanate is transported by the nitrate permease in Azotobacter chroococcum

Abstract Azotobacter chroococcum cells exhibiting the capacity to take up nitrate actively could transport [¹⁴C]cyanate. This activity was dependent on the nitrogen source present in the culture medium, ammonium acting as a repressor and nitrate as an inducer. The uptake of cyanate required metabolic energy and was absent from A. Chroococcum TR1, a mutant strain lacking the nitrate transport system, but was present at wild-type levels in A. chroococcum E4, a mutant strain deficient in nitrate reductase. These results show that cyanate is transported by the nitrate permease in A. chroococcum and therefore [¹⁴C]cyanate may be useful as a nitrate analogue for studies on nitrate transport.     

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.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Uptake regions of inorganic nitrogen in roots of carob seedlings

Three-week-old seedlings of carob ( Ceratonia siliqua L. cv. Mulata) were grown for 9 weeks under different root temperatures (20, 30 and 40°C) at pH values of 5, 7 and 9 with nitrate or ammonium as nitrogen source. Nitrogen uptake rates were determined by depletion from the medium and decreased with distance from the apex. The decline of nitrogen uptake rates along the roots depended on the form of inorganic nitrogen in the medium as well as on pH and temperature, such that the NO⁻₃ and NH⁺₄ ions were taken up essentially by the root tips (0–2 cm) through processes requiring energy. The uncharged NH₃ species entered passively, through the mature parts of the root (2–10 cm). Root zone temperature and pH affect the NH⁺₄/NH³ equilibrium in the nutrient solution and, consequently, the uptake areas of the root for these ions. Furthermore. while root tip uptake of nitrogen is energy dependent, uptake through mature root areas is essentially passive and seems to depend on a well developed apparent free space.     

Influence of nitrate and ammonium on the growth and 2,4-diaminobutyric acid composition of flatpea (Lathyrus sylvestris L.)

Abstract. Presence of 2.4-diaminobutyric acid (A₂bu), a neurotoxin, in tissues of flatpea ( Lathyrus sylvestris L.) necessitates a thorough understanding of the regulation of this nonprotein amino acid before the species can be recommended to livestock producers for forage applications. To determine how different concentrations and ratios of NO₃ and NH⁺₄ in growth media influence the levels of A₂bu and other free amino acids in the 'Lathco'flatpea cultivar, plants were grown hydroponically in controlled environments. The concentration of A₂bu was highest in tissues when the NO₃ to NH⁺₄ ratio in the nutrient solution was low. Responses of amides and other nonprotein amino acids, especially in the roots, followed a similar trend. Free protein amino acids in leaves and stems were generally unaffected by changes in NO₃ to NH⁺₄ ratios. In roots, protein amino acids increased as the NO₃ to NH⁺₄ ratio in the growth medium increased. Ammonium inhibited shoot and root growth; NO₃ alleviated the toxic effects of NH⁺₄. Soluble protein concentrations were higher in the shoots of NO₃-fed plants and in the roots of plants supplied with NH⁺₄. These results suggest that accumulation of A₂bu and other nonprotein amino acids, as well as asparagine and glutamine, plays a role in detoxification of NH⁺₄ and storage of N.     

Isolation and characterization of Azotobacter chroococcum from the roots of Zea mays

Abstract Bacteria showing rapid growth on a nitrogenfree medium and acetylene-reducing activity were isolated from maize roots collected from agricultural soils in Spain. The isolates were Gram-negative motile rods and were identified as Azotobacter chroococcum . Acetylene-reducing activity and microbial counts were determined on root segments from 7- and 30-day-old plants. Rates obtained were in the range of 0.0053–0.848 nmol C₂H₂· g⁻¹· h⁻¹. Root populations were 1.4–6.0 × 10⁴ micro-organisms · g⁻¹. These results showed that there was an association between A. chroococcum strains and roots of maize planted in some Spanish soils.     

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

Azotobacter chroococcum MAL-201, when grown under nitrogen-free conditions with excess glucose, accumulated poly-β-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 β-hydroxybutyrate and butyrate, but PHB production was inhibited in general. Supplementation of medium with these compounds enhanced the onset of encystment, and only β-hydroxybutyrate increased PHB accumulation significantly. Received: 23 April 1997 / Accepted: 31 May 1997     

Low external pH prevents cell elongation but not multiplication of embryogenic carrot cells

Embryogenic cultures of cultivated carrot ( Daucus crota cv. Scarlet Nantes) were initiated from seedling hypocotyls on hormone-containing nutrient medium and from wounded zygotic embryos on hormone-free medium. Both of these cultures were maintained with continuous multiplication as unorganized, embryogenic cell masses on hormone-free medium at pH 4.0, containing NH⁺₄ as the sole nitrogen source. When grown on hormone-free medium at pH 4.0, neither culture contained any elongated cells. Virtually all cells were densely cytoplasmic and nearly spherical. Some cells were enlarged, not densely cytoplasmic, but always spherical. When either culture was transferred to an auxin-containing medium at pH 5.8, numerous elongated cells were produced. Elongated cells were observed when either naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was used, and whether the nitrogen source was NH⁺₄ alone or a combination of NH⁺₄ and NO⁻₃. Elongated cells were more abundant when a combined nitrogen source was used. When cultures containing elongated cells were transferred to and multiplied on hormone-free or hormone-containing medium buffered at pH 4.0, all elongated cells disappeared after 2 weeks. No elongated cells were observed in any of the lines tested at pH 4.0. These results clearly show that it was the pH of the culture medium and not the presence or absence of an auxin or the nitrogen source(s) that permitted or prevented cell elongation in the embryogenic cultures tested.     

Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA mutants in microaerobiosis

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% +/- 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% +/- 3%. This strain grew in a simple medium at a specific growth rate of 0.69 +/- 0.07 h(-1), whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 +/- 0.06 h(-1). The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.     

Potential of Bacillus sp. to produce polyhydroxybutyrate from biowaste

Aim:  To test the Bacillus strains for their abilities to produce polyhydroxybutyrate (PHB) from different sugars and biowaste (Pea-shells).
Methods and Results:  Six Bacillus strains were checked for their ability to produce PHB from GM2 medium supplemented with different sugars at the rate of 1% (w/v) and from biowaste and GM2 (BW : M) combinations (3 : 7, 1 : 1, 7 : 3). Glucose supplemented GM2 medium resulted in maximum PHB production of 435 mg l⁻¹ constituting 31–62% w/w of the total cell dry mass. Substituting GM2 medium to the extent of 50% with biowaste (pea-shell slurry) resulted in 945–1205 mg l⁻¹ PHB (55–65% w/w). Optimization for additional nitrogen supplementation, inoculum size resulted in a final PHB production of 3010–3370 mg l⁻¹ equivalent to 300 g kg⁻¹ biowaste (dry wt).
Conclusion:  The Bacillus strains were able to produce PHB from biowaste (Pea-shells) as cheap source of substrate.
Significance and Impact of the Study:  This is the first report on usage of pea-shells as feed for PHB production, opening new possibilities for its use for production of PHB and Bacillus as potential candidate for the purpose.