Continuous degradation of phenol(s) by Pseudomonas putida P8 entrapped in polyacrylamidehydrazide

Summary The continuous degradation of phenol by immobilized cells of Pseudomonas putida P8 under different conditions was investigated. The bacterial cells were entrapped in polyacrylamidehydrazide (PAAH) and cultivated in a columnar fluidized-bed bioreactor. Working with a dilution rate of 0.067 h^-1 the phenol content in the feed was varied to find the capacity of an one-stage system with complete phenol degradation.Under sterile conditions and with phenol as the sole carbon source a degradation rate of 7.2 g·l^-1·d^-1 was reached whereas in non-sterile waste water only 3.12 g·l^-1·d^-1 were degraded. In any case the immobilized cells showed a stable phenol degradation activity and even simultaneously fed cresols or 4-chlorophenol were utilized completely.     

Biodegradation of 4-chlorophenol by adsorptive immobilized Alcaligenes sp. A 7-2 in soil

Summary Alcaligenes sp. A 7-2 immobilized on granular clay has been applied in a percolator to degrade 4-chlorophenol in sandy soil. Good adsorption rates on granular clay were achieved using cell suspensions with high titres and media at pH 8.0. The influence of various parameters such as aeration rate, pH, temperature, concentration of 4-chlorophenol and size of inoculum on the degradation rate were investigated. During fedbatch fermentations under optimal culture conditions, concentrations of 4-chlorophenol up to 160 mg·1^–1 could be degraded. Semicontinuous culture experiments demonstrated that the degradation potential in soil could be well established and enhanced by the addition of immobilized bacteria. Continuous fermentation was performed with varying 4-chlorophenol concentrations in the feed and different input levels. The maximum degradation rate was 1.64 g·1^–1·day^–1. Offprint requests to: H.-J. Rehm     

Adaptation of hybridoma cells to higher ammonia concentration

Using two mouse-mouse hybridoma cell lines, the response to ammonia step and serial changes was investigated in batch and continuous cultures with serum-free medium. The inhibitory effect of ammonia on cell growth depended on the cultivation mode, and differed markedly between cell lines. The cell line, 4C10B6 producing IgG monoclonal antibody against Pseudomonas, showed a high adaptation ability to ammonia. The 4C10B6 cells could grow under ammonia concentration as high as 21 mmol/l NH₄Cl with a viability of 80% in the continuous culture with serial increase in ammonia concentration. Whereas, in the batch culture with ammonia step change the cell growth completely ceased at 12 mmol/l NH₄Cl. The other cell line, TO-405 producing IgG monoclonal antibody against hepatitis B surface antigen, could not adapt to ammonia, and the cell growth did not occur at 9 mmol/l NH₄Cl even under the ammonia serial change.List of symbols D_Feed d^-1 Dilution rate of fresh feed medium (=F_o/V) - D_Out d^-1 Dilution rate of cell suspension (=F₁/V) - F₁ ml·d^-1 Volumetric discharge rate of cell suspension - F₀ ml·d^-1 Volumetric flow rate of fresh feed medium - k_D h^-1 Specific death rate - P mmol·l^-1 Product concentration - S mmol·l^-1 Substrate concentration in culture broth - S₀ mmol·l^-1 Substrate concentration in feed medium - t d Cultivation time - V ml Working volume of reactor - X₀ cells·ml^-1 Total cell density - X_V cells·ml^-1 Viable cell density - Y_P/S mmol·mmol^-1 Yield of product from substrate - Y_X/S cells·mmol^-1 Yield of cells from substrate - mmol·cell^-1·h^-1 Specific production rate - h^-1 Specific growth rate - mmol·cell^-1·h^-1 Specific consumption rate of substrate     

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     

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     

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     

Isolation of l-phenylalanine dehydrogenase from Rhodococcus sp. M4 and its application for the production of l-phenylalanine

Summary l-Phenylalanine dehydrogenase [l-phenylalanine: NAD⁺-oxidoreductase (deaminating)] of Rhodococcus sp. strain M4 was studied emphasizing its application for the production of l-phenylalanine. A high enzyme level (30,000 U·l^-1, 25–30 U·mg^-1 in the crude extract) could be reached during aerob degradation of l-phenylalanine (10 g·l^-1) under optimized growth coditions. A partial purification of the intracellular enzyme by liquid-liquid extraction, and DEAE-cellulose led to a specific activity of more than 1300 U·mg^-1. The continuous production of l-phenylalanine in an enzyme-membrane-reactor for 350h resulted in a space-time yield of 456 g·l^-1·d^-1 with a mean substrate conversion of 95%. Consumption of phenylalanine dehydrogenase was 1,500 U·kg Phe^-1.Abbreviations BSA bovine serum albumine - pheDH l-phenylalanine dehydrogenase - phepyr phenylpyruvate - OD optical density - FDH formate dehydrogenase     

Biodegradation of 2,4-dichlorophenol by a <Emphasis Type="Italic">Bacillus</Emphasis> consortium

As part of our effort at establishing microbial consortia of relevance for the bioremediation of xenobiotics polluted environments in Mexico, we assessed the aerobic biodegradation of 2,4-dichlorophenol (2,4-DCP) by a consortium of four Bacillus species that were isolated from a polluted soil by enrichment using a mixture of chlorophenols. The bacterial consortium effectively biodegraded 2-chlorophenol, 3-chlorophenol and 2,4-dichlorophenol at degradation rates of between 1.7 and 6.7 μmoles l⁻¹ h⁻¹. In the presence of NH₄Cl or KNO₂ as nitrogen sources_, 2,4-DCP was variously degraded. Under both conditions, cell biomass attained highest values of 350 and 450 mg l⁻¹ respectively, while the amounts of 2,4-DCP metabolized in 21 days reached peak values of 2.1 and 2.5 mM representing between 70 and 85% degradation respectively. Chloride releases during the same period were highest at 4.7 mM and 5.3 mM in the presence of the two nitrogen sources. The presence of free-chloride in the culture medium had a significant impact on the catabolism of 2,4-dichlorophenol.     

Effect of phenoxyalkanoic acid herbicides on the fermentative production of l-lysine

Summary The effect of the herbicides MCPA, MCPB, mecoprop, dichlorprop, 2,4-D, 2,4-DB, and 2,4,5-T on l-lysine fermentation was investigated using a lysine-producing mutant of Corynebacterium glutamicum. Stimulation of l-lysine production by 6% to 36% was observed in shaken flask experiments when the test herbicides were added at a concentration of 5 · 10^-4 M to growing cultures after 24 h of cultivation. The most effective stimulators were MCPA, mecoprop and dichlorprop.Detailed studies of the effect of MCPA (5 · 10^-6 M to 5 · 10^-3 M) showed that the degree of stimulation depended on medium composition and aeration. In the synthetic medium, maximum production of 50 g · l^-1 lys · HCl occurred at 5 · 10^-4 M MCPA and an oxygen transfer rate (OTR) of 1.97 g O₂ · l^-1 · h^-1, while 61.7 g · l^-1 of lys · HCL was formed at 5 · 10^-3 M MCPA and an OTR of 3.75 g O₂ · l^-1 · h^-1. In the amino-nitrogen rich medium, maximum production of 42 g · l^-1 lys · HCl was observed at 5 · 10^-6 M MCPA and an oxygen transfer rate of 1.5 g O₂ · l^-1 · h^-1. Results from batch l-lysine fermentation in a fermenter showed similar stimulatory effects, with an optimal concentration of MCPA for l-lysine production of 5 · 10^-5 M. Without herbicide addition, the test strain produced 16.25 g · l^-1 of product and with addition of 5 · 10^-5 M MCPA, the same strain produced 52.1 g · l^-1 lys · HCl after 72 h of fermentation.Abbreviations MCPA 2-methyl-4-chlorophenoxyacetic acid - MCPB 2-methyl-4-chlorophenoxybutyric acid - mecoprop 2-methyl-4-chlorophenoxypropionic acid - dichlorprop 2,4-dichlorophenoxypropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4-DB 2,4-dichlorophenoxybutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Application of a Doehlert experimental design to the optimization of microbial degradation of crude oil in sea water by continuous culture

Summary Crude oil was degraded by a mixed bacterial community grown in continuous culture on sea water. The fermentation process included an emulsification step prior to the introduction of the substrate in the reactor, with external cell recycling by a tangential-flow filtration system. Optimization of the fermentation technique was achieved by using the surface response methodology (Doehlert experimental design). Besides reducing the number of experiments, this approach allowed optimal experimental conditions to be chosen, for the particular goal: percent degradation of crude oil (80%), biomass (7.6 g·l^-1) and degradation rate (0.73 g·l^-1·h^-1). This biodegradation process could be used as a tool to fight against pollutions by petroleum products.     

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

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     

Ethanol tolerance of Pichia stipitis and Candida shehatae strains in fed-batch cultures at controlled low dissolved oxygen levels

Summary Fed-batch cultivations of Pichia stipitis and strains of Candida shehatae with d-xylose or d-glucose were conducted at controlled low dissolved oxygen tension (DOT) levels. There were some marked differences between the strains. In general growth was inhibited at lower ethanol concentrations than fermentation, and ethanol levels of up to 47 g·l^-1 were produced at 30°C. Ethanol production was mainly growth associated. The yeast strains formed small amounts of monocarboxylic acids and higher alcohols, which apparently did not enhance the ethanol toxicity. The maximum ethanol concentration obtained on d-xylose could not be increased by using a high cell density culture, nor by using d-glucose as substrate. The latter observation suggested that the low ethanol tolerance of these xylose-fermenting yeast strains was not a consequence of the metabolic pathway used during pentose fermentation. In contrast with the C. shehatae strains, it was apparent with P. stipitis CSIR-Y633 that when the ethanol concentration reached about 28 g·l^-1, ethanol assimilation exceeded ethanol production, despite cultivation at a low DOT of 0.2% of air saturation. Discontinuing the aeration enabled ethanol accumulation to proceed, but with concomitant xylitol production and cessation of growth.     

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     

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     

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     

Intracellular and luminal ion concentrations in sea turtle salt glands determined by x-ray microanalysis

Summary The lachrymal salt glands of hatchlings of the green sea turtle (Chelonia mydas) secrete a hyperosmotic (up to 2000 mosmol·kg^–1) NaCl solution. X-ray microanalysis of frozen-hydrated glands showed that during secretion intracellular Na⁺ concentration in the principal cells increased from 13 to 34 mmol·l^–1 of cell water, whilst Cl^– and K⁺ concentrations remained unchanged at 81 mmol·l^–1 and 160–174 mmol·l^–1, respectively. The high Cl^– concentration and the change in Na⁺ concentration are consistent with the prevailing paradigm for secretion by the structurally and functionally similar elasmobranch rectal gland. Concentrations of Na⁺, Cl^– and K⁺ in the lumina of secretory tubules of secreting (Na⁺ 122, Cl^– 167, K⁺ 38 mmol·l^–1) and non-secreting (Na⁺ 114, Cl^–1 174, K⁺ 44 mmol·l^–1) glands were similar and the fluid was calculated to be approximately isosmotic with blood. In the central canals Na⁺ and Cl^– concentrations were similar but K⁺ concentration was lower (11–15 mmol·l^–1). It is concluded that either a high transepithelial NaCl gradient in secretory tubules and central canals is very rapidly dissipated during the short time between gland excision and freezing, or that ductal modification of an initial isosmotic secretion occurs.     

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K−Cl cotransport

Pathways of K⁺ movement across the erythrocyte membrane of frog Rana temporaria were studied using ⁸⁶Rb as a tracer. The K⁺ influx was significantly blocked by 0.1 mmol·l^-1 ouabain (by 30%) and 1 mmol·l^-1 furosemide (by 56%) in the red cells incubated in saline at physiological K⁺ concentration (2.7 mmol·l^-1). Ouabain and furosemide had an additive effect on K⁺ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K⁺ influx saturated as f(K⁺)_e with apparent K _m values for external K _e ⁺ concentration of 0.96±0.11 and 4.6±0.5 mmol·l^-1 and V _max of 0.89±0.04 and 2.8±0.4 mmol·l cells^-1·h^-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of K _e ⁺ medium concentration. Total K⁺ influx was significantly reduced when frog erythrocytes were incubated in NO _- ³ medium. Furosemide did not affect K⁺ transport in frog red cells in NO ₃ ^- media. At the same K _e ⁺ concentration the ouabain-furosemide-insensitive K⁺ influx in Cl^- medium was significantly greater than that in NO _- ³ medium. We found no inhibitory effect of 1 mmol·l^-1 furosemide on Na⁺ influx in frog red cells in Cl^- medium. K⁺ loss from the frog erythrocytes in a K⁺-free medium was significantly reduced (mean 58%) after replacement of Cl^- with NO _- ³ . Furosemide (0.5 mmol·l^-1) did not produce any significant reduction in the K⁺ loss in both media. The Cl^--dependent component of K⁺ loss from frog red cells was 5.7±1.2 mmol·l^-1·h^-1. These results indicate that about two-thirds of the total K⁺ influx in frog erythrocytes is mediated by a K–Cl cotransport which is only partially blocked by furosemide.Abbreviations DMSO dimethyl sulphoxide - K _e ⁺ external concentration of K⁺ - K _m apparent Michaelis constant for external - K⁺ K _e ⁺ at V _max/2 - RBC red blood cell(s) - V _max maximal velocity of the unidirectional K⁺ influx - TRIS tris(hydroxymethyl)aminomethane     

Ion movements associated with chorion elevation during egg laying in trout (Oncorhynchus mykiss) in fresh water

Within 1 min of transfer from coelomic fluid to fresh water, eggs of rainbow trout (Oncorhynchus mykiss) underwent a transient loss of Na⁺ and K⁺ coupled with an elevation of the chorionic envelope. Both mechanisms were blocked by adding a monovalent cation Li⁺ or K⁺ (140 mmol·l^-1) to the fresh water, but the divalent ion Mg²⁺ (100 mmol MgCl₂·l^-1) or elevating the osmotic pressure to 300 mOsmol·l^-1 with glycine had no inhibitory effect. The blocking of Na⁺ loss occurred at external monovalent cation (LiCl) concentrations above 70 mmol·l^-1. A 20-s exposure of eggs to fresh water was sufficient to trigger Na⁺ loss and chorion elevation, even when the eggs were subsequently transferred to fresh water containing 140 mmol LiCl·l^-1. Eggs placed in a medium containing 140 mmol LiCl·l^-1 and 2 mmol Ca(NO₃)₂·l^-1 showed chorion elevation and associated Na⁺ loss after addition of calcium ionophore (20 mol·l^-1 A.23187). This activation by calcium ionophore was supressed in a Ca²⁺-free medium containing 5 mmol EGTA·l^-1.     

Production of volatile fatty acids from bagasse by rumen bacteria

Summary Conditions are described for converting bagasse lignocellulose to volatile fatty acids (VFA) by anaerobic fermentation. Although yields of VFA were as high as 74% by weight of digestible organic matter (or 54% of dry bagasse), limitations were imposed by both fermenter design and fibre digestibility. All fermentations were substrate-limited up to the maximum initial concentration examined of 50 g bagasse · l^-1 and no product inhibition was evident (up to 260 mM VFA produced). Maximum VFA productivities of 0.25 to 0.65 g · l^-1 · h^-1 were obtained in batch fermentations and this is greater than those previously reported using lignocellulosic substrates. Batch fermentations neared completion after 66 h.