Multi-substrate growth kinetics of Pseudomonas putida for phenol removal

The biodegradation of phenol by a pure culture of Pseudomonas putida was investigated in a continuously fed stirred-tank reactor, under aerobic conditions. The dilution rate was varied between 0.0174 h⁻¹ and 0.278 h⁻¹, covering a wide range of dissolved oxygen and the inhibition region of phenol. Through non-linear analysis of the data, a dual-substrate growth kinetics, Haldane kinetics for phenol and Monod kinetics for oxygen, was derived with high correlation coefficients. Respective biokinetic parameters were evaluated as μ_m = 0.569 h⁻¹, K _p = 18.539 mg/l, K _i = 99.374 mg/l, K _o = 0.048 mg/l, Y _x/p = 0.521 g microorganism/g phenol and Y _x/o = 0.338 g microorganism/g oxygen, being in good agreement with other studies in the literature. Maintenance factors for both phenol and oxygen were calculated for the first time for P. putida while the saturation coefficient for oxygen, K _o, was genuinely evaluated from the constructed model, not imported or adapted from other studies as reported in the literature. All pertinent biokinetic parameters for P. putida have been calculated from continuous system data, which are most appropriate for use in continuous bioprocess applications. Received: 29 July 1996 / Received revision: 18 November 1996 / Accepted: 23 November 1996     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52°C for 4.5 min), ethanol (20% v/v for 30 min), H₂O₂ (0.3 M for 60 min), rapid freezing (−196°C for 20 min, cooling rate 200°C min⁻¹), slow freezing (−20°C for 24 h, cooling rate 3°C min⁻¹), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H₂O₂ stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H₂O₂ and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H₂O₂ tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast. Received 10 October 1995/ Accepted in revised form 10 September 1996     

Pentachlorophenol biodegradation kinetics of an oligotrophic fluidized-bed enrichment culture

A fluidized-bed reactor (FBR) was used to enrich an aerobic chlorophenol-degrading microbial culture. Long-term continuous-flow operation with low effluent concentrations selected oligotrophic microorganisms producing good-quality effluent for pentachlorophenol(PCP)-contaminated water. PCP biodegradation kinetics was studied using this FBR enrichment culture. The results from FBR batch experiments were modeled using a modified Haldane equation, which resulted in the following kinetic constants: q _max = 0.41 mg PCP mg protein⁻¹ day⁻¹, K _S = 16 μg l⁻¹, K _i = 5.3 mg l⁻¹, and n = 3.5. These results show that the culture has a high affinity for PCP but is also inhibited by relatively low PCP concentrations (above 1.1 mg PCP l⁻¹). This enrichment culture was maintained over 1 year of continuous-flow operation with PCP as the sole source of carbon and energy. During continuous-flow operation, effluent concentrations below 2 μg l⁻¹ were achieved at 268 min hydraulic retention time (t _HR) and 2.5 mg PCP l⁻¹ feed concentration. An increase in loading rate by decreasing t _HR did not significantly deteriorate the effluent quality until a t _HR decrease from 30 min to 21 min resulted in process failure. Recovery from process failure was slow. Decreasing the feed PCP concentration and increasing t _HR resulted in an improved process recovery. Received: 10 October 1996 / Received revision: 21 January 1997 / Accepted: 24 January 1997     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

An electrochemical method for the measurements of substrate-oxidizing activity of acetic acid bacteria using a carbon-paste electrode modified with immobilized bacteria

In order to measure the substrate-oxidizing activity of intact cells of Acetobacter pasteurianus no. 2, a given amount of the bacterial cells was immobilized on a carbon-paste electrode, and the current at the electrode was measured in a buffer solution. When Fe(CN)³⁻ ₆ was added to the buffer solution, an anodic current was observed at 0.5 V (against Ag/AgCl). Further, when ethanol was added to the solution, the current started to increase to reach a steady-state within 3 min. The electrode had a good response to acetaldehyde and lactic acid as well as ethanol. Culture conditions affected the current response to various substances; the response of the electrode modified with the cells grown in static culture was much higher than that of the electrode with the cells grown in shaking culture, and the electrode with ethanol-grown cells had a high response to ethanol and acetaldehyde compared with that of the electrode with glucose-grown cells. The increase in the amount of the current after the addition of ethanol (ΔI _EtOH) was linearly proportional to the total number of immobilized cells per electrode in the range 1.0 × 10⁴–1.0 × 10⁸ cells. The ΔI _EtOH values were measured with the electrode prepared with a fixed volume of the cell suspensions taken from the culture at 6-h intervals; the dependence of the ΔI _EtOH value on time agreed well with the cell growth measured by colony counting and turbidity in the lag and logarithmic phase. After the logarithmic phase, the value of ΔI _EtOH sharply decreased, resembling to the growth measured by colony counting, rather than by turbidity. Received: 30 October 1998 / Received revision: 2 February 1999 / Accepted: 5 February 1999     

The allelopathy and allelopathic mechanism of phenolic acids on toxic <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Cyanobacterial contamination of water has been a serious problem in recent years. Thus, the effective control of undesired cyanobacteria has become an urgent issue. We studied therefore the effects of ρ-coumaric acid and vanillic acid on toxic Microcystis aeruginosa and the allelopathic mechanisms. The results showed that the growth of toxic M. aeruginosa was significantly inhibited by ρ-coumaric acid and vanillic acid, with an EC₅₀ of 0.26 ± 0.07 and 0.34 ± 0.05 mmol L⁻¹, respectively. Our data also demonstrated that both ρ-coumaric acid and vanillic acid triggered the generation of superoxide anion radicals (O₂ ^•−). The O₂ ^•− might induce a lipid peroxidation which may change cell membrane penetrability, thereby leading to the eventual death of M. aeruginosa. Our current studies further provide evidence that some phenolic acids such as ρ-coumaric acid and vanillic acid may be a potential effective solution for aquatic management.     

High alcohol production by repeated batch fermentation using an immobilized osmotolerant Saccharomyces cerevisiae

A repeated batch fermentation system was used to produce ethanol using an osmotolerant Saccharomyces cerevisiae (VS₃) immobilized in calcium alginate beads. For comparison free cells were also used to produce ethanol by repeated batch fermentation. Fermentation was carried for six cycles with 125, 250 or 500 beads using 150, 200 or 250 g glucose L⁻¹ at 30°C. The maximum amount of ethanol produced by immobilized VS₃ using 150 g L⁻¹ glucose was only 44 g L⁻¹ after 48 h, while the amount of ethanol produced by free cells in the first cycle was 72 g L⁻¹. However in subsequent fed batch cultures more ethanol was produced by immobilized cells compared to free cells. The amount of ethanol produced by free cells decreased from 72 g L⁻¹ to 25 g L⁻¹ after the fourth cycle, while that of immobilized cells increased from 44 to 72 g L⁻¹. The maximum amount of ethanol produced by immobilized VS₃ cells using 150, 200 and 250 g glucose L⁻¹ was 72.5, 93 and 87 g ethanol L⁻¹ at 30°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 222–226. Received 16 September 1999/ Accepted in revised form 22 December 1999     

Denitrifying and methanogenic bacteria in the biofilm of a fixed-film reactor operated with methanol/nitrate demonstrated by immunofluorescence and microscopy

 A denitrifying bacterial biofilm population established on a polypropylene substratum of a fixed-film reactor was characterized by microscopy, scanning electron microscopy and immunofluorescence after 120 days of operation. The reactor, operated at pH 7.0, 22°C, and −180 mV with synthetic wastewater containing methanol/nitrate, achieved a denitrification rate of 0.24 mol NO^- ₃ l^-1 day^-1 with a removal efficiency for nitrate of 95%–99% at an organic loading rate of 0.325 mol methanol l^-1 day^-1. The gas produced contained 2%–3% (v/v) methane and 3%–4% (v/v) carbon dioxide in addition to nitrogen. The biofilm contained mainly cells of Methanobrevibacter arboriphilus antigenically related to strain DC, short, flagellated, gram-negatively staining rods of Pseudomonas sp. antigenically related to Pseudomonas stutzeri strain AN11, non-identified pink-pigmented rods and small lemon-shaped cells with mono- and bipolar appendages resembling prosthecate Hyphomicrobium sp. The biofilm analysis provided evidence for a syntrophy between the denitrifying, methylotrophic, bacterial consortium and hydrogenotrophic methanogens, which were identified by antigenic fingerprinting with 17 antibody probes. Received: 11 July 1994/Received revision: 23 September 1994/Accepted: 28 September 1994     

Haemoglobin components and oxygen transport in relation to habitat distribution in triplefin fishes (Tripterygiidae)

Haemoglobin components were analysed for nine species of New Zealand triplefins and their isoelectric points (pI) ranged from 5.1 to 7.0. The number of well-expressed isohaemoglobins was larger in shallow-water and tidal pool species, ranging from four in Grahamina signata to eight in Grahamina capito, and were relatively cathodal. Two strongly anodal isohaemoglobins were expressed in the mid-depth species Ruanoho decemdigitatus and Ruanoho whero, and one in the deeper water species Karalepis stewarti and Forsterygion malcolmi. The red blood cell oxygen-binding properties were determined at 15 °C and 25 °C in the pH range 6.7–7.9 for the shallow-water species G. capito, the shallow to mid-depth species Forsterygion varium, and the deep-water species F. malcolmi. Oxygen affinity was highest for G. capito and the magnitude of the Bohr effect lower (Δlog P ₅₀/ΔpH = −0.37 at 25 °C, where P ₅₀ is the half-saturation coefficient) compared to the two Forsterygion species (Δlog P ₅₀/ΔpH = −0.52 to −0.59). Further, the cooperativity factor, n ₅₀, was lower in G. capito thus maintaining oxygen transport over a wide range of environmental oxygen pressures. Oxygen binding was similarly influenced by temperature in both G. capito and F. malcolmi (maximum heat of oxygenation ΔH_max = −27 kJ mol⁻¹ and −37 kJ mol⁻¹, respectively). Thus, triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodally migrating isohaemoglobins, and their red blood cells have a higher oxygen affinity and reduced cooperativity which is less sensitive to changes in pH than do species occurring in more stable, deeper water habitats. Our analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where interpretation of experimental results is confounded by phylogeny. Accepted: 18 March 1999     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Respiratory and metabolic responses of the Australian Yabby Cherax destructor to progressive and sustained environmental hypoxia

The Australian Yabby, Cherax destructor, inhabits occasionally hypoxic water. The respiratory gas, acid-base, metabolite and energetic status of this crayfish was assessed during progressive hypoxia and during 3 h at a water PO₂ of 1.33 kPa. The O₂ affinity of haemocyanin from C. destructor was increased by lactate (Δlog P ₅₀/Δlog[lactate] = −0.111) and by Ca (Δlog P ₅₀/Δlog[Ca] = −0.62) but not by urate. While the non-bicarbonate buffering capacity was low (Δ[HCO₃ ⁻]/ ΔpH=−4.89) the haemocyanin had a low sensitivity to pH changes (ϕ = −0.33). The crayfish showed a compensatory hyperventilation, which induced a respiratory alkalosis, until the water O₂ partial pressure declined below 2.67 kPa, after which the O₂ uptake rate was approximately 10% of normoxic rates. The high haemocyanin-O₂ affinity maintained haemolymph O₂ content during progressive hypoxia despite the normally low arterial O₂ partial pressure of C. destructor. During severe hypoxia, pH decreased but increased lactate aided in maintaining haemocyanin-O₂ saturation. The importance of regulated haemocyanin-O₂ affinity in hypoxic C. destructor was reduced by lowered metabolism, including reduced cardiac output, and the consequent reduction in O₂ requirement. Anaerobiosis became important only at very low PO₂ but thereafter proceeded rapidly, supported by a marked hyperglycaemia. There was no depletion of adenylates, even after 3 h of severe hypoxia. The tail muscle of C. destructor held small amounts of glycogen which would sustain anaerobiosis for a only a few hours. Hypometabolism seems an important hypoxic response but severe hypoxia may encourage the crayfish to breathe air. Accepted: 26 February 1998     

Variations in leaf β13C along a vertical profile of irradiance in a temperate Japanese forest

The vertical profile of stable carbon isotope ratios (δ¹³C) of leaves was analyzed for 13 tree species in a cool-temperate deciduous forest in Japan. The vertical distribution of long-term averaged δ¹³C in atmospheric CO₂ (δ_a) was estimated from δ¹³C of dry matter from NADP-malic enzyme type C₄ plant (Zea mays L. var. saccharata Sturt.) grown at a tower in the forest for 32␣days, assuming constant Δ value (3.3‰) in Z. mays against height. The δ_a value obtained from δ¹³C in Z.␣mays was lowest at the forest floor (−9.30 ± 0.03‰), increased with height, and was almost constant above 10␣m (−7.14 ± 0.14‰). Then leaf Δ values for the tree species were calculated from tree leaf δ¹³ C andδ_a. Mean leaf Δ values for the three tall deciduous species (Fraxinus mandshurica, Ulmus davidiana, and Alnus hirsuta) were significantly different among three height levels in the forest: 23.1 ± 0.7‰ at the forest floor (understory), 21.4 ± 0.5‰ in lower canopy, and 20.5 ± 0.3‰ in upper canopy. The true difference in tree leaf Δ among the forest height levels might be even greater, because Δ in Z. mays probably increased with shading by up to ∼‰. The difference in tree leaf Δ among the forest height levels would be mainly due to decreasing intercellular CO₂ (C _i) with the increase in irradiance. Potential assimilation rate for the three tree species probably increased with height, since leaf nitrogen content on an area basis for these species also increased with height. However, the increase in stomatal conductance for these tree species would fail to meet the increase in potential assimilation rate, which might lead to increasing the degree of stomatal limitation in photosynthesis with height. Received: 30 September 1995 / Accepted: 25 October 1996     

Biodegradation of propanol and isopropanol by a mixed microbial consortium

The aerobic biodegradation of high concentrations of 1-propanol and 2-propanol (IPA) by a mixed microbial consortium was investigated. Solvent concentrations were one order of magnitude greater than any previously reported in the literature. The consortium utilized these solvents as their sole carbon source to a maximum cell density of 2.4 × 10⁹ cells ml⁻¹. Enrichment experiments with propanol or IPA as carbon sources were carried out in batch culture and maximum specific growth rates (μ_max) calculated. At 20 °C, μ _max values were calculated to be 0.0305 h⁻¹ and 0.1093 h⁻¹ on 1% (v/v) IPA and 1-propanol, respectively. Growth on propanol and IPA was carried out between temperatures of 10 °C and 45 °C. Temperature shock responses by the microbial consortium at temperatures above 45 °C were demonstrated by considerable cell flocculation. An increase in propanol substrate concentration from 1% (v/v) to 2% (v/v) decreased the μ _max from 0.1093 h⁻¹ to 0.0715 h⁻¹. Maximum achievable biodegradation rates of propanol and IPA were 6.11 × 10⁻³% (v/v) h⁻¹ and 2.72 × 10⁻³% (v/v) h⁻¹, respectively. Generation of acetone during IPA biodegradation commenced at 264 h and reached a maximum concentration of 0.4% (v/v). The results demonstrate the potential of mixed microbial consortia in the bioremediation of solvent-containing waste streams. Received: 14 December 1999 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Ethanol adaptation in a thermotolerant yeast strain Kluyveromyces marxianus IMB3

The maximum ethanol concentration produced from glucose in defined media at 45°C by the thermotolerant yeast Kluyveromyces marxianus IMB3 was 44 g L⁻¹. Acclimatisation of the strain through continuous culture at ethanol concentrations up to 80 g L⁻¹, shifted the maximum ethanol concentration at which growth was observed from 40 g L⁻¹ to 70 g L⁻¹. Four isolates were selected from the continuous culture, only one of which produced a significant increase in final ethanol concentration (50 ± 0.4 g L⁻¹), however in subsequent fermentations, following storage on nutrient agar plates, the maximum ethanol concentration was comparable with the original isolate. The maximum specific ethanol production rates (approximately 1.5 g (gh)⁻¹) were also comparable with the original strain except for one isolate (0.7 g (gh)⁻¹). The specific ethanol productivity decreased with ethanol concentration; this decrease correlated linearly (rval 0.92) with cell viability. Due to the transience of induced ethanol tolerance in the strain it was concluded that this was not a valid method for improving final ethanol concentrations or production rates. Received 18 July 1997/ Accepted in revised form 19 February 1998     

Intracellular pH regulation and buffer capacity in CO2/HCO− 3-buffered media in cultured epithelial cells from rainbow trout gills

The influence of a CO₂/HCO⁻ ₃-buffered medium on intracellular pH regulation of gill pavement cells from freshwater rainbow trout was examined in monolayers grown in primary culture on glass coverslips; intracellular pH (pH_i) was monitored by continuous spectrofluorometric recording from cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxy-fluoroscein. When cells in HEPES-buffered medium at normal pH=7.70 were transferred to normal CO₂/HCO⁻ ₃-buffered medium {P _CO2=3.71 mmHg, [HCO⁻ ₃]= 6.1 mmol l⁻¹, extracellular pH (pH_e)=7.70}, they exhibited a brief acidosis but subsequently regulated the same pHi (∼7.41) as in HEPES. Buffer capacity (β) increased by the expected amount (5.5–8.0 slykes) based on intracellular [HCO⁻ ₃], and was unaffected by most drugs and treatments. However, after transfer to high P _CO2=11.15 mmHg, [HCO⁻ ₃]= 18.2 mmol l⁻¹ at the same pH_e=7.70, the final regulated pHi was elevated (∼7.53). The rate of correction of alkalosis caused by washout of this high P _CO2, high-HCO⁻ ₃ medium was unaffected by removal of extracellular Cl⁻. Removal of extracellular Na⁺ lowered resting pH_i and greatly inhibited the rate of pH_i recovery from acidosis. Bafilomycin A₁ (3 μmol l⁻¹) had no effect on these responses. However amiloride (0.2 mmol l⁻¹) inhibited recovery from acidosis caused by washout of an ammonia prepulse, but did not affect resting pH_i, the latter differing from the response in HEPES where amiloride also lowered resting pH_i. Similarly 4-acetamido-4′- isothiocyanatostilbene-2,2′-disulfonic acid, sodium salt (0.1 mmol l⁻¹) did not affect resting pH_i but slowed the rate of recovery from acidosis, though to a lesser extent than amiloride. Removal of extracellular Cl⁻ also slowed the rate of recovery but greatly increased β by an unknown mechanism; when this was taken into account, H⁺ extrusion rate was unaffected. These results are consistent with the presence of Na⁺-(HCO⁻ ₃)_N co-transport and/or Na⁺-dependent HCO⁻ ₃/Cl⁻ exchange, in addition to Na⁺/H⁺ exchange, as mechanisms contributing to “housekeeping” pH_i regulation in gill cells in CO₂/HCO⁻ ₃ media, whereas only Na⁺/H⁺ exchange is seen in HEPES. Both Na⁺-independent Cl⁻/HCO⁻ ₃ exchange and V-type H⁺-ATPase mechanisms appear to be absent from these cells cultured in isotonic media. Accepted: 30 November 1999     

Characterization of a putative pheromone biosynthesis-activating neuropeptide (PBAN) receptor from the pheromone gland of Heliothis peltigera

The binding of [³H]tyrosyl-PBAN28-33NH₂ to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO₃ ⁻ ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents did not have any effect on binding. Binding was found to be saturable, with a K_d of 5.73 ± 1.05 × 10⁻⁶ M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH₂ and PBAN28-33ΝΗ₂ with a K_i of 4.3 ± 1.1 × 10⁻⁶ M and 4.9 ± 2.6 × 10⁻⁶ M, respectively. Accepted: 4 February 1999     

Detection of the change point in oxygen uptake during an incremental exercise test using recursive residuals: relationship to the plasma lactate accumulation and blood acid base balance

The purpose of this study was to develop a method to determine the power output at which oxygen uptake (V˙O₂) during an incremental exercise test begins to rise non-linearly. A group of 26 healthy non-smoking men [mean age 22.1 (SD 1.4) years, body mass 73.6 (SD 7.4) kg, height 179.4 (SD 7.5) cm, maximal oxygen uptake (V˙O_2max) 3.726 (SD 0.363) l · min⁻¹], experienced in laboratory tests, were the subjects in this study. They performed an incremental exercise test on a cycle ergometer at a pedalling rate of 70 rev · min⁻¹. The test started at a power output of 30 W, followed by increases amounting to 30 W every 3 min. At 5 min prior to the first exercise intensity, at the end of each stage of exercise protocol, blood samples (1 ml each) were taken from an antecubital vein. The samples were analysed for plasma lactate concentration [La]_pl, partial pressure of O₂ and CO₂ and hydrogen ion concentration [H⁺]_b. The lactate threshold (LT) in this study was defined as the highest power output above which [La⁻]_pl showed a sustained increase of more than 0.5 mmol · l⁻¹ · step⁻¹. The V˙O₂ was measured breath-by-breath. In the analysis of the change point (CP) of V˙O₂ during the incremental exercise test, a two-phase model was assumed for the 3rd-min-data of each step of the test: X _i =at _i +b+ɛ _i for i=1,2,…,T, and E(X _i )>at _i +b for i =T+1,…,n, where X ₁, … , X _n are independent and ɛ _i ∼N(0,σ²). In the first phase, a linear relationship between V˙O₂ and power output was assumed, whereas in the second phase an additional increase in V˙O₂ above the values expected from the linear model was allowed. The power output at which the first phase ended was called the change point in oxygen uptake (CP-V˙O₂). The identification of the model consisted of two steps: testing for the existence of CP and estimating its location. Both procedures were based on suitably normalised recursive residuals. We showed that in 25 out of 26 subjects it was possible to determine the CP-O₂ as described in our model. The power output at CP-V˙O₂ amounted to 136.8 (SD 31.3) W. It was only 11 W – non significantly – higher than the power output corresponding to LT. The V˙O₂ at CP-V˙O₂ amounted to 1.828 (SD 0.356) l · min⁻¹ was [48.9 (SD 7.9)% V˙O_{2 max} ]. The [La⁻]_pl at CP-V˙O₂, amounting to 2.57 (SD 0.69) mmol · l⁻¹ was significantly elevated (P<0.01) above the resting level [1.85 (SD 0.46) mmol · l⁻¹], however the [H⁺]_b at CP-V˙O₂ amounting to 45.1 (SD 3.0) nmol · l⁻¹, was not significantly different from the values at rest which amounted to 44.14 (SD 2.79) nmol · l⁻¹. An increase of power output of 30 W above CP-V˙O₂ was accompanied by a significant increase in [H⁺]_b above the resting level (P=0.03). Accepted: 25 March 1998     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.