Factors which influence the sedimentation characteristics of Torulopsis glabrata after growth in a recirculation system

Summary Some environmental affects on cell aggregation described in the literature are briefly summarized. By means of a biomass recirculation culture (Contact system), using the yeast Torulopsis glabrata, the aggregation behavior of cells in static and in dynamic test systems is described. Sedimentation times required to obtain 50 g · l^–1 yeast dry matter in static systems were always higher than in dynamic ones.In addition to, influencing the biomass yield, the specific growth rate of the yeast also affected cell aggregation. The specific growth rate and therefore the aggregation could be regulated by the biomass recirculation rate as well as by the sedimenter volume.Abbreviations f_o Overflow flow rate (l·h^–1) - f_R Recycle flow rate (l·h^–1) - f_t0t Total flow rate through the fermenter (l·h^–1) - g Gram - h Hour - D_R Fermenter dilution rate due to recycle (h^–1) - D_S Fermeter dilution rate due to substrate (h^–1) - D_tot Total fermenter dilution rate (h^–1) - l Liter - Specific growth rate (h^–1) - P_F Fermenter productivity (g·l^–1·h^–1) - P_FS Overall productivity (g·l^–1·h^–1) - R_pM Rates per minute - RS Residual sugar content in the effluent with respect to the substrate concentration (%) - Y Yield of biomass with respect to sugar concentration (%) - Sed 50 Sedimentation time to reach a YDM of 50 g·l^–1 (min) - V Volume (l) - V_F Fermenter volume (l) - V_Sed Sedimenter volume (l) - VVM Volumes per volume and minute - X_F YDM in the fermenter (g·l^–1) - X_F YDM in the recycle (g·l^–1) - X_S Yeast dry matter due to substrate concentration (g·l^–1) - YDM Yeast dry matter (g·l^–1)     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Regulation of pH in the isolated perfused gills of the shore crabCarcinus maenas

Isolated posterior gills (no. 7) of shore crabsCarcinus maenas acclimated to brackish water of a salinity of 10 S were bathed and perfused with 50% sea water (200 mmol·l^-1 Na⁺), and the internal perfusate collected during subsequent periods of 5 min. During a single passage through the gill the pH of the perfusion medium decreased from ca. 8.1 to ca. 7.7, a result implying that the gill possesses structures which recognize unphysiologically high pH values in the haemolymph and regulates them down to physiological values of ca. 7.7. The calculated apparent proton fluxes from the epithelial cells into the haemolymph space amounted to 17.9 mol·g fw^-1·h^-1, a value of only 3.8% of net Na⁺ fluxes observed under comparable conditions. When 0.1 mmol·l^-1 KCN, an inhibitor of mitochondrial cytochrome oxidase, or 5 mmol·l^-1 ouabain, a specific inhibitor of Na⁺/K⁺-ATPase were applied in the internal perfusate, down-regulation of pH was no longer observed and the gill was completely depolarized, i.e. transepithelial potential differences dropped from-7.8 to 0 mV (haemolymph space negative to bath). Regulation of pH was completely inhibited by antagonists of carbonic anhydrase (0.1 mmol·l^-1 acetazolamide or 0.01 mmol·l^-1 ethoxyzolamide) applied in the perfusate. Inhibitors of Na⁺/H⁺ exchange, 0.1 mmol·l^-1 amiloride applied in the external bathing medium or in the internal perfusate, and symmetrical 0.01 mmol·l^-1 5-(N-ethyl-N-isopropyl)amiloride, as well as inhibitors of Cl^-/HCO₃ ^- exchange and Na⁺/HCO₃ ^- cotransport, 0.5 mmol·l^-1 4,4-diisothiocyanatostilbene-2,2-disulphonate or 0.3 mmol·l^-1 4-acetamido-4-isothiocyanatostilbene 2,2-disulphonate applied on both sides of the gill, and inhibitors of H⁺-ATPase, 0.05 mmol·l^-1 N-ethylmaleimide and 0.1 mmol·l^-1 N,N-dicyclohexylcarbodiimide —applied on both sides of the gill — did not alter the acidification of the perfusate observed in controls. Using artificial salines buffered to pH 8.1 with 0.75 mmol·l^-1 tris (hydroxymethyl) aminomethane instead of 2 mmol·l^-1 HCO₃ ^-, apparent proton fluxes were reduced to 11% of controls, a result suggesting that pH regulation by crab gills needs the presence of HCO₃ ^-. The findings obtained suggest that pH regulation by crab gills depends on the oxidative metabolism of the intact branchial epithelium and that carbonic anhydrase plays a central role in this process. Na⁺/H⁺ exchange, anion exchange or cotransport and active proton secretion seem not to be involved. While unimpaired active ion uptake is a prerequisite for pH regulation, ion transport itself is independent of it.Abbreviations acetazolamide (N-[sulphamoyl-1, 3, 4-thiadiazol-2-yl]-acetamide) - amiloride 3,5-diamino-6-chloropyrazinoyl-guanidine - CA carbonic anhydrase - DBI dextrane-bound inhibitor thiadiazolesulphonamide - DCCD N N dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanato-stilbene-2,2-disulphonate - EIPA 5-(N-ethyl-N-isopropyl) amiloride - ethoxyzolamide 6-ethoxy-2-benzothiazole-sulphonamide - fw fresh weight - J _H ⁺ apparent proton flux - NEM N-ethylmaleimide - PD transepithelial potential difference - PEG-STZ polyethylene-glycol-thiadiazolesulphonamide - STTS 4-acetamido-4-isothiocyanatostibene 2,2-disulphonate - SW sea water - TRIS tris(hydroxymethyl)aminomethane     

Mechanisms of fluid and ion secretion by the parotid gland of the kangaroo,Macropus rufus,assessed by administration of transport-inhibiting drugs

Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05–0.5 mmol·l^-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0±0.04 to 0.22±0.024 ml·min^-1 (mean±SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135±1.6 to 67±1.7 mmol·l^-1 and 272±7.6 to 15±2.6 mol·min^-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11±1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol·l^-1), bumetanide (up to 0.2 mmol·l^-1) and ethacrynate (1 mmol·l^-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25–1.0 mmol·l^-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid at 0.1 mmol·l^-1 was without effect, whereas at 0.5 mmol·l^-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (>90%) by secretion of bicarbonate which is formed in the endpiece cells from CO₂ delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na⁺-K⁺-2Cl^- symports, Na⁺-Cl^- symports or Cl^-/HCO ₃ ^- antiports.Abbreviations CA carbonic anhydrase - CAI carbonic anhydrase inhibitors - MAP mean arterial blood pressure - PAH p-aminohippurate - SITS 4-acetamido-4-isothiocyanatostilbene-2,2 disulphonic acid     

Secretion of electrolytes,protein and urea by the mandibular gland of the common wombat (Vombatus ursinus)

Saliva was collected from the mandibular glands of anaesthetized common wombats (Vombatus ursinus) to ascertain maximal flow rates, salivary compostion and possible adaptations, particularly PO₄ ^3- secretion, to assist digestion. After temporary catheterization of the main duct through its oral opening, salivary secretion was evoked at flow rates ranging from 0.02±0.002 (±SEM) ml·min^-1 (0.7±0.07 l·min^-1·kg body weight^-1) to 0.4±0.05 ml·min^-1(14±1.9 l·min^-1·kg body weight^-1) by ipsilateral intracarotid infusion of acetylcholine. The [Na⁺] (15±5.1 to 58±8.6 mmol·l^-1) and [HCO₃ ^-] (35±1.9 to 60±1.9 mmol·l^-1) were positively correlated with salivary flow rate. The [K⁺] (58±5.2 to 30±2.4 mmol·l^-1), [Ca²⁺] (10.4±1.67 to 4.1±0.44 mmol·l^-1), [Mg²⁺] (0.94±0.137 to 0.17±0.032 mmol·l^-1), [Cl^-] (71±9.2 to 45±6.0 mmol·l^-1), [urea] (9.3±0.79 to 5.1±0.54 mmol·l^-1), H⁺ activity (29±1.6 to 17±1.6 nEq·l^-1) and amylase activity (251±57.4 to 92±23.3 kat·l^-1) were negatively correlated with flow. Both concentration and osmolality fell with increasing flow at the lower end of the flow range but osmolality always increased again by maximal flow whereas the relation between protein and flow was not consistent at the higher levels of flow and stimulation. Salivary [PO₄ ³⁺] was not correlated with flow and at 3–14% of the plasma concentration was extremely low. Thus, in contrast to its nearest relative, the koala (Phascolarctos cinereus), the wombat secretes little PO₄ ³⁺ presumably because it does not need high levels of PO₄ ³⁺ in its saliva to facilitate microbial digestion of plant fibre.Abbreviations bw body weight - ww wet weight     

Use of formate gradients for improving biomass yield of Pichia pinus growing continuously on methanol

Summary Investigations were made into the improvement of growth yield (Y) of Pichia pinus MH 4 growing continuously on methanol by feeding formate so as to create an increasing concentration gradient (transient state). Under particular formate supply conditions, Y could be increased from 0.37 g·g^-1 on methanol alone to 0.55 and 0.47 g·g^-1 in the presence of formate at dilution rates (D) of 0.045 and 0.075 h^-1, respectively. These differences could be explained as being due to a limiting formate consumption rate of 50–60 nmol·min^-1·g^-1 dry wt., coupled to a net-energy generation independent of D. Any further formate oxidation proceeded without energy gain. Deviations from optimum conditions of biomass increase are discussed in terms of different formate oxidizing systems and uncoupling properties of formate itself. These results are compared to and confirmed by steady-state considerations.Abbreviations a steepness of the formate gradient (g·l^-1·h^-1) - a acceleration of change of formate concentration in the fermenter (g·l^-1·h^-2) - D dilution rate (h^-1) - Ft formate - S₁ and S₂ initial and final formate concentration of the gradient (g·l^-1) - Y growth yield in g·g^-1 methanol     

Influence of pH,lactose and lactic acid on the growth of Streptococcus cremoris: a kinetic study

Summary The effect of various culture conditions on growth kinetics of an homofermentative strain of the lactic acid bacterium Streptococcus cremoris were investigated in batch cultures, in order to facilitate the production of this organism as a starter culture for the dairy industry. An optimal pH range of 6.3–6.9 was found and a lactose concentration of 37 g·l^-1 was shown to be sufficient to cover the energetic demand for biomass formation, using the recommended medium. The study of the effect of lactic acid concentration on growth kinetics revealed that the end-product was not the sole factor affecting growth. The strain was characterized for its tolerance towards lactic acid and a critical concentration of 70 g·l^-1 demonstrated. With the product yield of 0.9 g·g^-1 at non-lactose limiting conditions the lactic acid concentration of 33 g·l^-1 could not explain the low growth rates obtained, implicating a nutritional limitation.Symbols t _f fermentation duration (h) - X Biomass concentration (g·l^-1) - X _m maximum biomass concentration (g·l^-1) - S lactose concentration (g·l^-1) - S _r residual lactose concentration (g·l^-1) - P produced lactic acid concentration (g·l^-1) - P _a added lactic acid concentration (g·l^-1) - P _c critical lactic acid concentration (g·l^-1) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - R _x/S biomass yield (g·g^-1) calculated when =0 - R _P/S product yield (g·g^-1)     

Kinetics of α-amylase production in a batch and fed-batch culture of Bacillus subtilis with caseinate as nitrogen source and starch as carbon source

Summary Analysis of a large number of experimental data from the cultivation of Bacillus subtilis formed the basis for a kinetic model of the process explaining the effect of composition of the culture medium and of the growth rate on the rate of enzyme production. The resulting rate of formation of -amylase (EC 3.2.1.1) reflects the sum of the rate of enzyme production and the rate of its degradation as affected by the environment. The kinetic dependence confirms the previously described mechanism of regulation of enzyme biosynthesis. The mathematical model of the process served here to determine the optimal conditions for enzyme biosynthesis which were then verified in a fed-batch cultivation. The production of the enzyme in fed-batch culture was found to be twice that found in a batch cultivation.Symbols X biomass concentration, g·l^-1 - t time, h - S ₁ caseinate concentration, g·l^-1 - S ₂ starch concentration, g·l^-1 - P product concentration, U·ml^-1 - r _P specific rate of product formation, U·g^-1·h^-1 - R _P total rate of product formation, U·l^-1·h^-1 - Y yield coefficient - specific growth rate, h^-1     

Determination of specific monoclonal antibody secretion rate during very slow hybridoma growth

A total cell recycling suspension perfusion reactor has been constructed for investigation of specific monoclonal antibody secretion rate of the 9.2.27 murine hybridoma under conditions of a very low growth rate. By rapidly recycling hybridoma cells using a thermostated tangential flow filter, 3.6 mg cell dry weight/cm³ could be maintained at growth rate of <0.05 _max for over 250 h. Under these conditions, secretion of lactate, ammonia and l-alanine were directly related to the rate of l-glutamine supply. Monoclonal antibody accumulated in the reactor to levels in excess of 1400 g/ cm³. Surprisingly, as specific growth rate decreased, the specific immunoglobulin secretion rate remained constant, implying that monoclonal antibody synthesis could be uncoupled from growth.List of Symbols CMF cm³/(min · cm²) cross membrane flow rate - D h^–1 dilution rate - DOT % air saturation dissolved oxygen tension - F _R cm³/min perfusion rate - GlcPR mg/min glucose provision rate - GlnPR mg/min l-glutamine provision rate - N _A mmoles O₂/(dm³ · h) oxygen transfer rate - q _ala mmoles/h l-alanine secretion rate - q _MAB mg MAB · 10^–8 viable cells ^–1 · day^–1 specific MAB secretion rate by viable cells - ¯q _MAB (dimensionless) ¯q _MAB/¯q _{MAB MAX} - ¯q _{NH 3} mmoles/h ammonia secretion rate - S _R mg/cm³ limiting substrate concentration - h^–1 specific growth rate - _app h^–1 apparent growth rate - ¯ (dimensionless) / _MAX - VC cells/cm³ viable cell number     

Cell recovery by continuous flotation

Summary Cell recovery by means of continuous flotation of the Hansenula polymorpha cultivation medium without additives was investigated as a function of the cultivation conditions as well as of the flotation equipment construction and flotation operational parameters. The cell enrichment and separation is improved at high liquid residence times, high aeration rates, small bubble sizes, increasing height of the aerated column, and diameter of the foam column. Increasing cell age and cultivation with nitrogen limitation reduce the cell separation.Symbols C_P cell mass concentration in medium g·l^–1 - C_R cell mass concentration in residue g·l^–1 - C_S cell mass concentration in foam liquid g·l^–1 - V equilibrium foam volume cm³ - V gas flow rate through the aerated liquid column cm³·s^–1 - V_F feed rate to the flotation column ml/min - ¹ V _S/V foaminess s - mean liquid residence time in the column s     

The effect of carbonic anhydrase inhibitors on secretion by the parotid and mandibular glands of red kangaroos Macropus rufus

Summary The effects of carbonic anhydrase inhibitors on secretion by macropodine parotid and mandibular glands were investigated using anaesthetized red kangaroos. In the parotid gland, acetazolamide (500 mol·l^-1) reduced a stable acetylcholine-evoked, half-maximal flow rate of 2.02±0.034 to 0.27±0.023 ml·min^-1 (87% reduction). Concurrently, salivary bicarbonate concentration and secretion fell (129.4±1.46 to 80.9±1.63 mmol·l^-1 and 264.8±7.96 to 22.3±2.30 mol·min^-1, respectively), phosphate and chloride concentrations rose (14.0±0.79 to 27.6±0.85 mmol·l^-1 and 5.6±0.25 to 27.5±1.32 mmol·l^-1, respectively), sodium concentration and osmolality were unaltered, and potassium concentration fell (8.8±0.33 to 6.4±0.29 mmol·l^-1). High-rate cholinergic stimulation during acetazolamide blockade was unable to increase salivary flow beyond 11±0.9% of that for equivalent unblocked control stimulation. However, superimposition of isoprenaline infusion on the acetylcholine stimulation caused a three-fold increase in the blocked flow rate. These treatments were accompanied by small increases in salivary phosphate and chloride concentrations but not bicarbonate concentration. Methazolamide infusion caused similar changes in parotid secretion. In the mandibular gland, acetazolamide infusion had no effect on salivary flow rate during either low- or high-level acetylcholine stimulation. Acetazolamide caused no alterrations in salivary electrolyte secretion at low flow rates, but curtailed the rise in bicarbonate concentration associated with high-level acetylcholine stimulation. Acetazolamide administration did not affect the increase in salivary flow rate associated with isoprenaline infusion, but did block the concomitant increase in bicarbonate concentration and secretion substantially. It was concluded that neither cholinergic nor adrenergic stimulation of mandibular fluid secretion depends on secretion of bicarbonate derived from catalysed hydration of CO₂, but a substantial proportion of the increase in bicarbonate secretion during isoprenaline administration, which is probably ductal in origin, is so dependent. In contrast to other salivary glands, including the ovine parotid, fluid secretion by the kangaroo parotid gland during cholinergic stimulation is largely dependent (about 90%) on secretion of bicarbonate derived from hydration of CO₂ catalysed by glandular carbonic anhydrase. Fluid secretion during adrenergic stimulation is not bicarbonate dependent.Abbreviations b.w. body weight - PAH p-aminohippurate - PCO₂ partial pressure carbon dioxide - PCO₂ partial pressure of oxygen     

Renal response to saline infusion in chicks of Leach's storm petrel (Oceanodroma leucorhoa)

The renal response to infusion of three different saline solutions was studied in chicks of Leach's storm petrel (Oceanodroma leucorhoa). Each of the solutions (125 mmol·1^-1 NaCl at 5.3 ml·h^-1, 250 mmol·l^-1 NaCl at 2.6 ml·h^-1, and 550 mmol·l^-1 NaCl at 1.2 ml·h^-1) provided the same delivery of Nacl but in different volumes of water. Birds infused with 125 mmol·l^-1 NaCl had a glomerular filtration rate of 25.7 ml·h^-1, a urine flow rate of 4.4 ml·h^-1, and excreted 71% of the infused Na⁺ load in the urine. With infusion of 250 mmol·l^-1 NaCl, the glomerular filtration rate was unchanged (23.3 ml·h^-1), but urine flow rate was reduced to 0.93 ml·h^-1 and only 35% of the Na⁺ load was excreted in the urine. Infusion of 550 mmol·l^-1 NaCl induced a sharp decrease in glomerular filtration rate (to 3.8 ml·h^-1) and urine flow rate (to 0.08 ml·h^-1), and only 1.4% of the infused Na⁺ was excreted in the urine. The contribution of different nephron populations to filtration was assessed by the pattern of staining of glomeruli by alcian blue infused during the last 30 min of the saline infusion. The numbers of stained glomeruli did not differ between birds infused with 125 and 250 mmol·l^-1 NaCl (59000 and 55000 glomeruli per kidney, respectively), and the patterns of staining were similar for birds in these two groups. Birds infused with 550 mmol·l^-1 NaCl had lighter staining overall and fewer stained glomeruli (37000 per kidney). This absence of staining was predominant in the smaller size classes of glomeruli, suggesting a selective shutdown of smaller (reptilian-type) nephrons during times of osmotic challenge in these birds. This may be part of an overall suite of water-conserving strategies employed by these chicks during their long confinement with irregular feeding in the nesting burrow.Abbreviations ADH antidiuretic hormone - GFR glomerular filtration rate - MT mammalian-type - P plasma inulin concentration - RT reptilian-type - U urine inulin concentration - V urine flow rate     

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.     

Conversion of cellulose into fungal cell mass

Summary Chaetomium cellulolyticum (ATCC 32319) was cultivated on glucose, Avicel and/or Sigmacell in a 20-1 stirred tank batch reactor. The substrate (cellulose) concentration, the cell mass concentration (through protein and/or nitrogen content), reducing sugar concentration, the enzyme activity, the alkali consumption rate, the dissolved O₂ and CO₂ concentrations in the outlet gas were measured. The specific growth rate, the substrate yield coefficient, cell productivity, the oxygen consumption rate, the CO₂ production rate and the volumetric mass transfer coefficient were determined. At the beginning of the growth phase the oxygen utilization rate exhibits a sharp maximum. This maximum could be used to start process control. Because of the long lag phase periodic batch operation is recommended.Symbols CP cell protein concentration (g l^–1) - FPA FP enzyme activity (IU l^–1) - GP dissolved protein concentration (g l^–1) - IU international unit of enzyme activity - k_La volumetric mass tranfer coefficient (h^–1) - LG alkali (1 n NaOH) consumption (ml) - LGX specific alkali consumption rate per cell mass (ml g^–1 h^–1) - P cell mass productivity (g l^–1 h^–1) - specific oxygen consumption rate per cell mass (g g^–1 h^–1) - Q aeration rate (volumetric gas flow rate per volume of medium, vvm) (min^–1) - N impeller speed (revolution per minute, rpm) (min^–1) - S substrate concentration (g l^–1) - S⁰ S at t_F=0 (g l^–1) - S₀ S in feed (g l^–1) - SR acid consumption (ml) - TDW total dry weight (g l^–1) - T temperature (° C) - t_F cultivation time (h) - U substrate conversion - X cell mass concentration (g l^–1) - Y_X/S vield coefficient - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1)     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Organic reduction of tapioca wastewaters using modified RBC

A modified Rotating Biological Contactor (RBC) was used for the treatability studies of synthetic tapioca wastewaters. The RBC used was a four stage laboratory model and the discs were modified by attaching porous nechlon sheets to enhance biofilm area. Synthetic tapioca wastewaters were prepared with influent concentrations from 927 to 3600 mg/l of COD. Three hydraulic loads were used in the range of 0.03 to 0.09 m³·m^–2·d^–1 and the organic loads used were in the range of 28 to 306 g COD· m^–2·d^–1. The percentage COD removal were in the range from 97.4 to 68. RBC was operated at a rotating speed of 18 rpm which was found to be the optimal rotating speed. Biokinetic coefficients based on Kornegay and Hudson models were obtained using linear analysis. Also, a mathematical model was proposed using regression analysis.List of Symbols A m² total surface area of discs - d m active depth of microbial film onany rotating disc - K _s mg ·l^–1 saturation constant - P mg·m^–2·^–1 area capacity - Q l·d^–1 hydraulic flow rate - q m³·m^–2·d^–1 hydraulic loading rate - S ₀ mg·l^–1 influent substrate concentration - S _e mg·l^–1 effluent substrate concentration - w rpm rotational speed - V m³ volume of the reactor - X _f mg·l^–1 active biomass per unit volume ofattached growth - X _s mg·l^–1 active biomass per unit volume ofsuspended growth - X mg·l^–1 active biomass per unit volume - Y _s yield coefficient for attachedgrowth - Y _A yield coefficient for suspendedgrowth - Y yield coefficient, mass of biomass/mass of substrate removed Greek Symbols hr mean hydraulic detention time - (_max)_A d^–1 maximum specific growth rate forattached growth - (_max)_s d^–1 maximum specific growth rate forsuspended growth - _max d^–1 maximum specific growth rate - d^–1 specific growth rate - _v mg·l^–1·hr^–1 maximum volumetric substrateutilization rate coefficient     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

The fermentation of hexose and pentose sugars by Candida shehatae and Pichia stipitis

Summary The fermentation by Candida shehatae and Pichia stipitis of xylitol and the various sugars which are liberated upon hydrolysis of lignocellulosic biomass was investigated. Both yeasts produced ethanol from d-glucose, d-mannose, d-galactose and d-xylose. Only P. stipitis fermented d-cellobiose, producing 6.5 g·l^-1 ethanol from 20 g·l^-1 cellobiose within 48 h. No ethanol was produced from l-arabinose, l-rhamnose or xylitol. Diauxie was evident during the fermentation of a sugar mixture. Following the depletion of glucose, P. stipitis fermented galactose, mannose, xylose and cellobiose simultaneously with no noticeable preceding lag period. A similar fermentation pattern was observed with C. shehatae, except that it failed to utilize cellobiose even though it grew on cellobiose when supplied as the sole sugar. P. stipitis produced considerably more ethanol from the sugar mixture than C. shehatae, primarily due to its ability to ferment cellobiose. In general P. stipitis exhibited a higher volumetric rate and yield of ethanol production. This yeast fermented glucose 30–50% more rapidly than xylose, whereas the rates of ethanol production from these two sugars by C. shehatae were similar. P. stipitis had no absolute vitamin requirement for xylose fermentation, but biotin and thiamine enhanced the rate and yield of ethanol production significantly.Nomenclature _max Maximum specific growth rate, h^-1 - Q _p Maximum volumetric rate of ethanol production, calculated from the slope of the ethanol vs. time curve, g·(l·h)^-1 - q _p Maximum specific rate of ethanol production, g·(g cells·h) - Y _p/s Ethanol yield coefficient, g ethanol·(g substrate utilized)^-1 - Y _x/s Cell yield coefficient, g biomass·(g substrate utilized)^-1 - E Efficiency of substrate utilization, g substrate consumed·(g initial substrate)^-1·100     

Characterization of the Na+-dependent taurine influx in flounder erythrocytes

About 92% of the taurine influx in flounder erythrocytes at physiological conditions in vitro (330 mosmol·l^-1, 145 mmol·l^-1 Na⁺, 0.30 mmol·l^-1 taurine) is Na⁺-dependent. This influx is highly specific for taurine. The -amino compounds hypotaurine and -alanine were the only compounds which mimicked the inhibitory effect of taurine on influx of [¹⁴C]taurine, the former more than the latter. Counterexchange of taurine was also mediated by the taurine transporters. Reduction of osmolality per se did not affect the activity of these transporters. Non-linear regression analysis of the influx values revealed the presence of two different influx systems: a system with high affinity and low capacity and another with low affinity and high capacity. However, we cannot exclude the possibility that this influx of taurine was mediated by only one transporter which operated in different modes depending on the extracellular Na⁺ concentration. On the assumption that the Na⁺-dependent influx was mediated by two separate systems, the maximal velocity of the low capacity system was 2.55 nmol·g dry weight^-1·min^-1 at 145 mmol·ll^-1 extracellular Na⁺. This capacity was about 50% lower than that of the high capacity system. The Michaelis constants were 0.013 and 1.34 mmol·l^-1, respectively. Reduction of the extracellular Na⁺ concentration reduced maximal velocity and the affinity to taurine of both transport systems. At 10 mmol·l^-1 Na⁺ or lower concentrations the high capacity system did not seem to operate. The activation method suggested that each taurine molecule transported by the high capacity system was accompanied by two Na⁺. The stoichiometry of the low capacity system was 1 taurine: 1 Na⁺. The Hill-coefficient for both transport systems was 1.00.Abbreviations cpm counts per minute - dw dry weight - GABA -amino-n-butyric acid - K _m Michaelis constant - pK _b basic dissociation constant - SD standard deviation - -ABA Dl--amino-n-butyric acid - V _max maximal velocity - ww wet weight     

Growth of Thermoanaerobium brockii in batch and continuous culture at supraoptimal temperatures

Summary Thermoanaerobium brockii was grown in batch and continuous culture at supraoptimal temperatures (>65° C). Specific growth rates were lower in batch (_max>1.0 h^-1) than in continuous cultures (_max1.2–1.4 h^-1). Acetone addition to the medium did not increase critical dilution rate significantly. The media used contained significantly less organic material and sulfide than previously reported media; however, yeast extract requirements were shown to be exceptionally high (60% of the glucose concentration used). Organic substrates inhibited growth and product formation in chemostat cultures whereas the slow formation of acetic acid was observed in batch cultures, but also with virtually no growth. The inhibiting concentration was found to be approximately 15 g organic carbon·l^-1. The maintenance requirements of T. brockii were in the same range as expected of aerobic extreme thermophiles (m_s0.5 g·g^-1·h^-1) and could be met only by glucose and not by yeast extract. Maintenance was obviously not independent of specific growth rate. Production of the stereospecific alcohol-aldehyde/ketone oxidore-ductase was strictly growth associated and its formation was not affected by acetone added to medium.