Cat Heart Muscle in Vitro : II. The steady state resting potential in quiescent papillary muscles

The steady state transmembrane resting potential difference (V_m) has been measured in quiescent papillary muscles. V_m was determined as a function of the external K concentration in Cl and SO₄ solutions and compared with the K equilibrium potential. Other measurements were made after replacement of external Na by choline, K by Rb and Cs, and Cl by SO₄, CH₃SO₄, and NO₃. Effects on V_m of albumin, temperature, and variation in internal K concentration are described.     

The Influence of Sodium-Free Solutions on the Membrane Potential of Frog Muscle Fibers

The membrane potential of frog sartorius muscle fibers in a Cl- and Na-free Ringer's solution when sucrose replaces NaCl is about the same as that in normal Ringer's solution. The K⁺ efflux is also about the same in the two solutions but muscles lose K and PO₄ in sucrose Ringer's solutions. The membrane potential in sucrose Ringer's solution is equal to that given by the Nernst equation for a K⁺ electrode, when corrections are made for the activity coefficients for K⁺ inside and outside the fiber. For a muscle in normal Ringer's solution, the measured membrane potential is within a few millivolts of E_K. This finding is incompatible with a 1:1 coupled Na-K pump. It is consistent with either no coupling of Na efflux to K influx, or a coupling ratio of 3 or greater.     

A MICRO INJECTION STUDY ON THE PERMEABILITY OF THE STARFISH EGG

The experiments with the NH₄Cl are similar to, and corroborate micro injection experiments performed in connection with some work on mustard gas in which the writer collaborated. Eggs immersed in sea water containing decomposed mustard gas, at a certain low concentration are not affected. If, however, the solution be injected, the egg quickly cytolyzes owing to the free HCl present. A similar impermeability of the protoplasmic surface film to certain substances was also encountered in injection work on Amœba. Amœbœ immersed in an aqueous solution of eosin will not take the stain till after death. On the other hand, the eosin, when injected into the Amœba, quickly permeates the protoplasm, to be arrested only at the surface. The semipermeability of a living cell appears primarily to be a function of its surface film. It is immaterial whether this film be that of the original cortex of the cell, a film newly formed over a cut surface, or a film that surrounds an artificially induced vacuole within the cell. As long as such a surface film exists neither the acid group of the NH₄Cl nor the alkaline group of the NaHCO₃ can, within certain concentration limits, penetrate the protoplasm. These solutions, if injected beneath the surface film, however, will produce their characteristic effects upon the protoplasm.     

ACCUMULATION OF BRILLIANT CRESYL BLUE IN THE SAP OF LIVING CELLS OF NITELLA IN THE PRESENCE OF NH3

When the living cells of Nitella are placed in a solution of brilliant cresyl blue containing NH₄Cl, the rate of accumulation of the dye in the sap is found to be lower than when the cells are placed in a solution of dye containing no NH₄Cl and this may occur without any increase in the pH value of the cell sap. This decrease is found to be primarily due to the presence of NH₃ in the sap and seems not to exist where NH₃ is present only in the external solution at the concentration used.     

The Control of the Membrane Potential of Muscle Fibers by the Sodium Pump

Frog sartorius muscles were made Na-rich by immersion in K-free sulfate Ringer's solution in the cold. The muscles were then loaded with Na²⁴ and the extracellular space cleared of radioactivity. When such Na-rich muscles were transferred to lithium sulfate Ringer's solution at 20°C, Na efflux was observed to increase with time, to reach a maximum about 15 minutes after the transfer of the muscles to Li₂SO₄, and then to decline. The decline in efflux from these muscles was proportional to ([Na]_i)⁸ over a considerable range of [Na]_i. The membrane potential of Na-rich muscles was about -48 mv in K-free sulfate Ringer's at 4°C but changed to -76 mv in the same solution at 20°C and to -98 mv in Li₂SO₄ Ringer's at 20°C. By contrast, muscles with a normal [Na]_i showed a fall in membrane potential when transferred from K-free sulfate Ringer's to Li₂SO₄ Ringer's solution. The general conclusions from this study are (a) that Na extrusion is capable of generating an electrical potential, and (b) that increases in [Na]_i lead to reversible increases in P_Na of muscle fibers.     

Effect of Ammonium Chloride and Methionine Sulfoximine on the Acetylene Reduction of Detached Root Nodules of Peas (Pisum sativum)

Acetylene-reducing activity of detached pea nodules was determined by submerging the nodules in buffer solution [tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4] containing 100 mM sodium succinate and incubating under a gas phase of 90% O₂ and 10% C₂H₂. The nitrogenase activity was 4 to 8 μmol of C₂H₄ formed per g of nodule fresh weight per h and remained constant for at least 4 h. Addition of NH₄Cl to the buffer solution (at a concentration of 10 mM or more) resulted in a significant decrease of nitrogenase activity, which was more pronounced at higher concentrations of ammonium chloride. The inhibition of nitrogenase activity by NH₄Cl was reversible; when the NH₄Cl-containing buffer solution was replaced by buffer without NH₄Cl, the original activity was partly restored. Treatment of the nodules with NH₄Cl had almost no effect on the amount of nitrogenase, as measured by the acetylene-reducing activity of ethyl-enediaminetetraacetate-toluene-treated bacteroid suspensions. The effect of NH₄Cl was largely eliminated by simultaneous addition of 10 mM methionine sulfoximine to the assay solution. This suggests that the assimilation of ammonium ions by glutamine synthetase controls the functioning of nitrogenase activity in the nodules. However, no effect of glutamine, glutamate, or aspartate on the acetylene reduction by detached nodules could be detected.     

Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

The Control of Na Uptake into Nitella translucens

Movement of Na into cells of Nitella translucens is a ‘downhill’process; the ions move across the plasmalemma down an electrochemicalpotential gradient. Nevertheless, measurements of Na influxesunder a wide range of experimental conditions have shown thatthere must be links between Na uptake and processes controlledby metabolism. When Ca ions are present in the bathing solution,Na influxes are greatly increased by light under conditionswhere photosynthesis can proceed (i.e. when both photosystemsare active). In the presence of Ca, the influx of Na increasesonly slightly when the external Na concentration is raised above1 mM, and the light-promoted Na influx is considerably inhibitedwhen Cl is removed from the bathing solution. When the Cl concentrationis kept constant, the Na influx in light is determined by theconcentrations of other cations present in solution (K, Ca,or NH₄). In the absence of Ca from the cell wall and solution,the influx is stil enhanced by light, but does not saturatewhen the external Na concentration is raised above 1 mM. Itis suggested that the Na influx in light is partly linked tothe inward Cl pump, but there is also a separate (Cl-independent)effect of light on the permeability of the plasmalemma to Na.Links between Na and Cl uptake could be maintained by effectsof Cl on electrochemical driving forces controlling Na entry;alternatively, chemical coupling between the two processes maybe involved.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

New Culture Medium Containing Ionic Concentrations of Nutrients Similar to Concentrations Found in the Soil Solution

A new growth medium which closely approximates the composition of the soil solution is presented. This soil solution equivalent (SSE) medium contains the following components (millimolar): NO₃, 2.5; NH₄, 2.5; HPO₄, 0.005; Na, 2.5; Ca, 4.0; Mg, 2.0; K, 0.503; Cl, 4.0; SO₄, 5.0; ethylenediamine-di(o-hydroxyphenylacetic acid), 0.02; and MES [2-(N-morpholino)ethanesulfonic acid] (to maintain the pH at 6.0), 10, plus 0.1% arabinose. The advantages of the SSE medium are discussed.     

Bicarbonate and fast-twitch muscle: Evidence for a major role in pH regulation

Summary Internal pH (pH_i) was analyzed in rat extensor digitorum longus (Edl) muscle at 30°C with single-barrel liquid ionselective electrodes. Average pH_i in 284 cells was 7.197±0.006. Increases in CO₂ from nominally 0 to 5% produced an acidification from which recovery took place. In different groups of cells, recovery from the 5% CO₂ acidification was significantly inhibited by 100 m 4,4 diisothiocyanatostilbene 2,2 disulfonic acid (DIDS), Cl removal, Na removal and 2mm amiloride. Prepulsing with 20mm NH₄ in the presence of CO₂/HCO₃ typically reduced pH_i to only about neutral, whereas 50mm reduced pH_i to 6.7–6.8. In the nominal absence of CO₂/HCO₃, 20mm NH₄ reduced pH_i to about 6.7 from which recovery took place at about 58% of the rate seen in different cells in the presence of CO₂/HCO₃. In the presence of CO₂/HCO₃, cells prepulsed with 50mm NH₄ had fully recovered to an average pH_i of 7.22±0.04 about 90 min after removal of NH₄. However, 90 min after removal of 20mm NH₄ in the absence of CO₂/HCO₃, average pH_i was significantly less (7.05±0.03). Intrinsic buffering capacity ( _i ) was obtained during pulses of CO₂, acetic acid or after an NH₄ pulse, _i was significantly reduced in the absence of HCO₃, Cl or Na and HCO₃. The data provide significant support for an important role of HCO₃ in the control of pH_i in fast-twitch muscle.     

Regulation of the Na+-K+(NH)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH₄Cl, and the variations of the intracellular concentration of calcium ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured using fura-2 and 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH₄Cl significantly increased the [Ca²⁺]_i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH₄Cl, the cells acidified immediately. At 30 mmol/l, NH₄Cl first alkalinized the cells and the pH_i subsequently dropped. This drop reflects the uptake of NH ions that dissociate to NH₃ and H⁺ in the cytosol. These protons are exchanged for extracellular sodium by the Na⁺/H⁺ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH₄Cl. Ouabain partly blocked the uptake of NH. In the combined presence of ouabain and bumetanide (an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter), 1 mmol/l NH₄Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na⁺-K⁺-2Cl⁻ cotransporter in the uptake of NH was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β-adrenergic agonist. ATP increased the release of ³H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na⁺-K⁺-2Cl⁻ cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley-Liss, Inc.     

CHARACTERISTICS OF SALTS SECRETED BY TAMARIX APHYLLA

The composition and concentration of salts secreted by the salt glands of Tamarix aphylla L. grown under controlled nutrient conditions were determined. Eight ions, Na, K, Mg, Ca, Cl, NO₃, HCO₃, and SO₄, constituted 99 % + of the dry weight of salts secreted by plants grown on half-strength Hoagland's solution. The divalent cations Mg and Ca accounted for most of the cations; HCO₃ comprised about 60 % of the anions. The micronutrients B, Mn, Cu, Zn, and Mo were present in enriched concentrations in the secretion. The composition of the secretions was highly dependent on the composition of the root environment. The predominating cation in the saline culture solutions was also the predominant cation secreted. The accompanying anion in the culture solution influences the cation composition of the secreted salt. The concentration of the salt gland secretion averaged 0.5n , a 50-fold increase in concentration over the nutrient solution in which the plants were grown.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

Effects of NH<Subscript>4</Subscript>CL application and removal on astrocytes and endothelial cells

Background

Hepatic encephalopathy (HE) is a complex disorder associated with increased ammonia levels in the brain. Although astrocytes are believed to be the principal cells affected in hyperammonemia (HA), endothelial cells (ECs) may also play an important role by contributing to the vasogenic effect of HA.

Methods

Following acute application and removal of NH₄Cl on astrocytes and endothelial cells, we analyzed pH changes, using fluorescence imaging with BCECF/AM, and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i), employing fluorescence imaging with Fura-2/AM. Using confocal microscopy, changes in cell volume were observed accompanied by changes of [Ca²⁺]_i in astrocytes and ECs.

Results

Exposure of astrocytes and ECs to 1 – 20 mM NH₄Cl resulted in rapid concentration-dependent alkalinization of cytoplasm followed by slow recovery. Removal of the NH₄Cl led to rapid concentration-dependent acidification, again followed by slow recovery. Following the application of NH₄Cl, a transient, concentration-dependent rise in [Ca²⁺]_i in astrocytes was observed. This was due to the release of Ca²⁺ from intracellular stores, since the response was abolished by emptying intracellular stores with thapsigargin and ATP, and was still present in the Ca²⁺-free bathing solution. The removal of NH₄Cl also led to a transient concentration-dependent rise in [Ca²⁺]_i that resulted from Ca²⁺ release from cytoplasmic proteins, since removing Ca²⁺ from the bathing solution and emptying intracellular Ca²⁺ stores did not eliminate the rise. Similar results were obtained from experiments on ECs. Following acute application and removal of NH₄Cl no significant changes in astrocyte volume were detected; however, an increase of EC volume was observed after the administration of NH₄Cl, and EC shrinkage was demonstrated after the acute removal of NH₄Cl.

Conclusions

This study reveals new data which may give a more complete insight into the mechanism of development and treatment of HE.

     

The chemistry of surface water in prairie ponds

Water analyses from 80 small prairie ponds, 0.17 to 89.8 hectares, in Manitoba and Saskatchewan between 1967 to 1972 exhibited two basic ionic dominance patterns HCO₃ > SO₄ > Cl and SO₄ > HCO₃ > Cl. The order of SO₄ and Cl were reversed in 40 percent of the bicarbonate ponds below 500 µmhos/cm. At salinities above 12,000 µmhos/cm, Cl exceeds SO₄ in several wetlands. Temporary wetlands were characterised by Ca > K > Mg > Na / HCO₃ > Cl > SO₄, semi-permanent ponds Mg > Ca > Na > K / SO₄ > HCO₃ > Cl while the permanent pond structure was Ca > Mg > Na > K / HCO₃ > SO₄ > Cl.Conductivities of the wetlands studied ranged from 47 to 23,000 µmhos / cm.Seasonal changes in salinity varied within and between pond types as well as from year to year. The average salinity increase within season in temporary ponds was 67 percent, 63 percent in semi-permanent ponds and 20 percent for permanent ponds. These changes were affected by evaporation, transpiration, see-page and precipitation patterns. Ionic dominance patterns did not change within a season although Mg, K and HCO₃ increased at higher rates than Ca, Na, CI and SO₄.Temporary wetlands are slightly acidic, averaging pH 6.8 while semi-permanent and permanent ponds were alkaline, pH range 7.1 to 9.2. No stratification of pH with depth and minimal diurnal variation was recorded.At two study areas, Bradwell and St. Denis fhe following ranges (micrograms/litre) were recorded: 3 to 630 PO₄-P, 10 to 650 NO₃-N, 5 to 630 TDFe, 1 to 13 TDCu and 1 to 27 TDZn. Colour was 5 to 190 Hazen units at St. Denis and 10 to 110 for Bradwell.Two herbicides, 2,4-D and 2,4,5-T were detected at levels of 4 to 111 and 1.4 to 13.5 micrograms/litre, respectively, from 15 ponds at St. Denis.     

The Concentration Dependence of Sodium Efflux from Muscle

Frog sartorius muscles subjected to overnight loading with Na⁺ in K-free Ringer in the cold were subsequently labeled with Na²⁴ and then immersed in choline Ringer and the efflux of Na²⁴ followed for 4 hours. The initial efflux of Na⁺ appeared to be 17 pmole/cm² sec.; this value was maintained for 20 minutes and was followed by an abrupt decline to about 9 pmole/cm² sec. This latter rate was maintained for the next 20 minutes of efflux. The efflux then declined gradually with time and reached values of the order of 0.1 pmole/cm² sec. The back addition of counts lost from muscles enabled one to calculate the relationship between efflux and [Na]_i for muscle. This roughly approximates an S-shaped curve with a value at half-saturation of about 17 mmole Na per liter of fiber water. The efflux-concentration curve is closely described by assuming that 3 Na⁺ are transported per carrier cycle.     

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.