Ethanol acetylation by mycelium-bound carboxylesterase of Aspergillus oryzae: estimation of thermodynamic parameters and integral productivity

The results collected at different temperatures for ethanol acetylation by cell-bound carboxylesterase from lyophilized cells of Aspergillus oryzae have been used to investigate the kinetics and thermodynamics of this esterification in n-heptane. The occurrence of reversible unfolding followed by irreversible denaturation of the enzyme has been proposed to explain the increase in the starting rate of ethyl acetate formation with temperature observed up to 55 °C and the consequent fall beyond this threshold. The Arrhenius model has been used to estimate the apparent activation enthalpies of both the acetylation reaction (H = 29–33 kJ mol^–1) and reversible enzyme unfolding (H _u = 56–63 kJ mol^–1). The results of residual activity tests performed with cells previously exposed at different temperatures for variable times enabled us also to estimate the first-order rate constant of irreversible denaturation (2.40 × 10^–3 h^–1 < k _d < 8.11 × 10^–3 h^–1) as well as the related thermodynamic parameters (H _d = 22 kJ mol^–1; S _d = –0.29 kJ mol^–1 K^–1). This last phenomenon proved particularly slow for the system under consideration, probably because the biocatalyst link to the mycelium was able to improve its thermostability. In view of future continuous application, the effects of operating time, starting substrate concentration and temperature on the theoretical integral productivity of a fixed-bed column filled with this biocatalyst have been investigated.     

High density fed-batch cultures for hybridoma cells performed with the aid of a kinetic model

Hybridoma fed-batch cultures with either standard medium as feed or concentrated medium as feed and removal of toxic metabolites through dialysis were performed by using model calculations for a priori determination of process parameters. In a first step a kinetic model for specific growth and death rate, respectively as well as for substrate uptake and metabolite production rates was formulated. In a bed-batch culture with standard medium as feed the appropriate time for start of the feeding pump and the increase of feed rate were determined a priori. The glutamine concentration was controlled at 0.04 mmoll^–1. A priori calculation and course of the culture coincided rather well. A cell concentration of 3.2×10⁶ cells ml^–1, a MAb-concentration of 54 mg _MAb l^–1 and a MAb-time-space-yield of 0.53 mg _MAb l^–1h^–1 were obtained.For further increase of the efficiency a high density fed-batch process was developed, where concentrated medium is fed to the cells and the accumulating toxic low molecular weight metabolites are removed through a dialysis membrane into a dialyizng fluid. In a membrane dialysis reactor consisting of a culture chamber and a dialyzing chamber, which are separated by a cylindrical dialysis membrane, again model calculations were used to determine feed rate and exchange rate of dialyzing fluid. A viable cell density of 1.2×10⁷ cells ml^–1 and a MAb concentration of 425 mg l^–1 were reached in a culture with stepwise feeding of 10 x concentrated medium and exchange of dialyzing fluid for removal of low molecular metabolites. The course of the culture could be predicted a priori rather well. The MAb-time-space-yield was 2.47 mg _MAb l^–1h^–1, appr. 5 times higher compared to fed-batch cultures with standard medium as feed.List of Symbols A membrane area m² - c _i substrate or product concentration in culture chamber mmoll^–1 - c _a substrate or product concentration in dialyzing chamber mmoll^–1 - c _0i substrate or product concentration in the feed of culture chamber mmoll^–1 - c _0a substrate or product concentration in the feed of dialyzing chamber mmoll^–1 - c _Gln glutamine concentration mmoll^–1 - c _Amm ammonia concentration mmoll^–1 - c _MAb MAb concentration mmoll^–1 - D _a dilution rate in dialyzing chamber d^–1 - F _i feed rate during fed-batch to the culture chamber mlh^–1 - V _a volume of dialyzing chamber l - V _i volume of culture chamber l - P membrane permeability coefficient cm min^–1 - q specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q _Gln spec. glutamine uptake rate mmol cell^–1 h^–1 - q _MAb spec. MAb production rate mmol cell^–1 h^–1 - t time h - X _v viable cell concentration cells ml^–1 - _MAb MAb-time-space-yield mgl^–1 h^–1 - specific growth rate h^–1 - _d specific death rate h^–1 Financial support from the Volkswagen-Stiftung, Germany, grand nr. I/69 359 is gratefully acknowledged.The concentrated medium was kindly provided by SERVA, Heidelberg, Germany. The hybridoma cell line was donated by Prof. fil. dr. Volker Kasche, Technische Universität Hamburg-Harburg, Germany.We express our special thanks to Andreas Schütt, Ralf Gassner, Katja Herbers and Thomas Schäfer for their help in this project.     

Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide

The responses of photosynthesis, Rubisco activity, Rubisco protein, leaf carbohydrates and total soluble protein to three carbon dioxide treatments were studied in winter wheat [Triticum aestivum (L.)] and barley [Hordeum vulgare (L.)]. Barley and wheat plants were grown in small field plots during 1995 and 1996 in clear, acrylic chambers (1.2–2.4 m²) and were provided with continuous carbon dioxide fertilization at concentrations of 350, 525 and 700 mol mol^–1. Photosynthetic rates of barley penultimate leaves and wheat flag leaves measured at growth carbon dioxide concentrations decreased with leaf age in all three CO₂ treatments during 1995 and 1996. Photosynthetic acclimation to elevated CO₂ was observed on seven of eight measurement dates for barley and ten of eleven measurement dates for wheat over both years. Initial Rubisco activity, total soluble protein and Rubisco protein in barley penultimate leaves and wheat flag leaves also decreased with leaf age. Total Rubisco activity was not used because of enzyme degradation. There was a significant CO₂ treatment effect on initial Rubisco activity, total soluble protein and Rubisco protein for wheat in 1995 and 1996 and for barley in 1995. Responses of barley penultimate leaf Rubisco activity and leaf protein concentrations to elevated carbon dioxide were nonsignificant in 1996. A significant CO₂ treatment effect also was detected when means of Rubisco activity, soluble protein and Rubisco protein for wheat flag leaves were combined over harvests and years. These three flag leaf parameters were not significantly different in the 350 and 525 mol mol^–1 CO₂ treatments but were decreased during growth in 700 mol mol^–1 CO₂ relative to the other two CO₂ treatments. Ratios of photosynthesis at 700 and 350 mol mol^–1 were compared to ratios of Rubisco activity at 700 and 350 mol mol^–1 using wheat flag leaf data from 1995 and 1996. Regression analysis of these data were linear [y = 0.586 + 1.103t x (r² = 0.432)] and were significant at P 0.05. This result indicated that photosynthetic acclimation was positively correlated with changes of initial Rubisco activity in wheat flag leaves in response to CO₂ enrichment. Effects of elevated CO₂ on wheat leaf proteins during 1995 and 1996 and on barley during 1995 were consistent with an acceleration of senescence.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Opioid receptor binding in rat spinal cord

The interaction of various radioligands with spinal opioid receptors has been characterized under variable experimental conditions. Binding to , , and sites was measured in all (cervical, thoracic, lumbar) segments. The apparent affinity constant (K) of [³H]Ethylketocyclazocine (EKC) was similar in Tris, 2.09 (±1.06)×10⁸ M^–1, and phosphate buffer, 2.16 (±0.02)×10⁸ M^–1, when its interaction with and sites was blocked. Without blocking ligands, EKC binding was resolved in two components:K ₁=1.01 (±0.21)×10⁹ M^–1 andK ₂=0.95 (±0.61)×10⁷ M^–1. Likewise, the binding of [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAGO) or [D-Ala², D-Leu⁵]-enkephalin (DADLE) alone was represented by a 2-site model. By adjusting the radioligand and receptor concentration or by the addition of blocking ligands, binding was represented by a 1-site model for DAGO,K=4.35 (±1.41)×10⁸ M^–1, and DADLE,K=2.44 (±0.08)×10⁸ M^–1.The abbreviations used are DADLE [D-Ala², D-Leu⁵]enkephalin - DAGO [D-Ala², MePhe⁴, Gly(ol)⁵]enkephalin - EKC ethylketocyclazocine - DYN dynorphin (1–17)     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Modeling the cucumber fermentation: Growth ofLactobacillus plantarum

Summary Specific growth rate models of product-inhibited cell growth exist but are rarely applied to fermentations beyond ethanol and large-scale antibiotic production. The present paper summarizes experimental data and the development of a model for growth of the commercially important bacterium,Lactobacillus plantarum, in cucumber juice. The model provides an excellent correlation of data for the influence on bacterial growth rate of NaCl, protons (H⁺), and the neutral, inhibitory forms of acetic acid and the fermentation product, lactic acid. The effects of each of the variables are first modeled separately using established functional forms and then combined in the final model formulation.Nomenclature [C] inhibitory component concentration, mM - [C]_max concentration of the inhibitory component where the specific growth rate is zero, mM, determined by model fitting - [H⁺] hydrogen ion concentration, mM - [HLa] undissociated lactic acid concentration, mM - [La^–] dissociated lactic acid concentration, mM - [La_t] total lactic acid ([HLa]+[La^–]) concentration, mM - [HAc] undissociated acetic acid concentration, mM - [Ac^–] dissociated acetic acid concentration, mM - [Ac_t] total acetic acid ([HAc]+[Ac^–]) concentration, mM - [NaCl] sodium chloride concentration, %, w/v - specific growth rate, h^–1 - _max maximum specific growth rate, h^–1 - ₀ specific growth rate, h^–1, at 0 concentration of additive - K _ij inhibition coefficient - , ,K _m coefficients determined by model fitting Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable.     

The interactive effects of elevated CO2 and O3 concentration on photosynthesis in spring wheat

This study investigated the interacting effects of carbon dioxide and ozone on photosynthetic physiology in the flag leaves of spring wheat (Triticum aestivum L. cv. Wembley), at three stages of development. Plants were exposed throughout their development to reciprocal combinations of two carbon dioxide and two ozone treatments: [CO₂] at 350 or 700 mol mol^–1, [O₃] at < 5 or 60 nmol mol^–1. Gas exchange analysis, coupled spectrophotometric assay for RuBisCO activity, and SDS-PAGE, were used to examine the relative importance of pollutant effects on i) stomatal conductance, ii) quantum yield, and iii) RuBisCO activity, activation, and concentration. Independently, both elevated [CO₂] and elevated [O₃] caused a loss of RuBisCO protein and V_cmax. In combination, elevated [CO₂] partially protected against the deleterious effects of ozone. It did this partly by reducing stomatal conductance, and thereby reducing the effective ozone dose. Elevated [O₃] caused stomatal closure largely via its effect on photoassimilation.     

Transinhibition and Voltage-gating in a Fungal Nitrate Transporter

We have applied enzyme kinetic analysis to electrophysiological steady-state data of Zhou et al. (Zhou, J.J., Trueman, L.J., Boorer, K.J., Theodoulou, F.L., Forde, B.G., Miller, A.J. 2000. A high-affinity fungal nitrate carrier with two transport mechanisms. J. Biol. Chem. 275:39894–9) and to new current-voltage-time records from Xenopus oocytes with functionally expressed NrtA (crnA) 2H⁺-NO ₃ ^– symporter from Emericella (Aspergillus) nidulans. Zhou et al. stressed two Michaelis-Menten (MM) mechanisms to mediate the observed nitrate-induced currents, I _{NO 3} ^– . We show that a single straightforward reaction cycle describes the data well, pointing out that during exposure to external substrate, S = (2H⁺+NO ₃ ^– )_o, the product concentration inside, [P] = [H⁺] _i ² · [NO ₃ ^– _i, may rise substantially near the plasma membrane, violating the condition [P] [S] for MM kinetics. Here, [P] and its changes during experimentation are treated explicitly. K _1/2 20 µM for I _{NO 3} ^– at pH_o from Zhou et al. is confirmed. According to our analysis, NrtA operates between about 0.2 and 0.6 of the electrical distance in the membrane (outside 0, inside 1). In absence of thermodynamic gradients, the predominant orientation of the binding site(s) is probably inwards. The activity of the enzyme is sensitive to the transmembrane voltage, V, with an apparent gating charge of +1.0 ± 0.5 for inactivation, and transition probabilities of 0.3–1.3 s^–1 at V = 0. This gating mode impedes loss of cellular NO ₃ ^– during depolarization.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Parameters influencing transient and stable transformation of barley (Hordeum vulgare L.) protoplasts

Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml^–1 or kanamycin sulphate at 250 g ml^–1. Absolute transformation frequencies of 28.9×10^–5 and 21.3×10^–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10^–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA abscisic acid - AC-CAP acetylated chloramphenicol - BAP 6-benzylaminopurine - cat chloramphenicol acetyltransferase gene - CAT chloramphenicol acetyltransferase activity - CaMV cauliflower mosaic virus - CAP chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - G418 Geneticin - gus -glucuronidase gene - HEPES (N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid]) - IAA indole acetic acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid - npt II neomycin phosphotransferase gene - NPTH neomycin phosphotransferase activity - PEG polyethylene glycol - SCV settled cell volume     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Independent and contrasting effects of elevated CO2 and N-fertilization on root architecture in Pinus ponderosa

The effects of elevated CO₂ and N-fertilization on the architecture of Pinus ponderosa Dougl. ex P. Laws & C. Laws fine roots and their associated mycorrhizal symbionts were measured over a 4-year period using minirhizotron tubes. The study was conducted in open-top field-exposure chambers located near Placerville, Calif. A replicated (3 replicates), 3×3 factorial experimental design with three CO₂ concentrations [ambient air (354 mol mol^–1), 525 mol mol^–1, and 700 mol mol^–1] and three rates of N-fertilization (0, 100 and 200 kg ha^–1 year^–1) was used. Elevated CO₂ and N treatment had contrasting effects on the architecture of fine roots and their associated mycorrhizae. Elevated CO₂ increased both fine root extensity (degree of soil exploration) and intensity (extent that roots use explored areas) but had no effect on mycorrhizae. In contrast, N-fertilization had no effect on fine root extensity or intensity but increased mycorrhizal extensity and intensity. To better understand and model the responses of systems to increasing CO₂ concentrations and N deposition/fertilization it is necessary to consider these contrasting root architectural responses.     

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 g ml^–1 CdCl₂ in the presence or absence of 0.1–1000 ng ml^–1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17-estradiol (E₂) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 g ml^–1 CdCl₂ suppressed P accumulation to 65 and 10%, respectively; accumulation of E₂ (at 5 g ml^–1 CdCl₂) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 g ml^–1 CdCl₂ was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 g ml^–1 CdCl₂, whereas basal E₂ production of granulosa cells was markedly diminished. It is concluded that CdCl₂ suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Influence of soil temperature on methane emission from rice paddy fields

Methane emission rates from an Italian rice paddy field showed diel and seasonal variations. The seasonal variations were not closely related to soil temperatures. However, the dieL changes of CH₄ fluxes were significantly correlated with the diel changes of the temperature in a particular soil depth. The soil depths with the best correlations between CH4 flux and temperature were shallow (1–5cm) in May and June, deep (10–15cm) in June and July, and again shallow (1–5 cm) in August. Apparent activation energies (E_a) calculated from these correlations using the Arrhenius model were relatively low (50–150 kJ mol^–1) in May and June, but increased to higher values (80–450 kJ mol^–1) in August. In the laboratory, CH₄ emission from two rice cultures incubated at temperatures between 20 and 38°C showed E . values of 41 and 53 kJ mol^–1) Methane production in anoxic paddy soil suspensions incubated between 7 and 43°C showed E values between 53 and 132 kJ mol^–1 with an average value of 85 kJ mol^–1) and in pure cultures of hydrogenotrophic methanogenic bacteria E _a values between 77 and 173 (average 126) kJ mol^–1. It is suggested that diel changes of soil properties other than temperature affect CH₄ emission rates, e.g. diel changes in root exudation or in efficiency of CH⁴ oxidation in the rhizosphere.     

Optimization of cellulase-free xylanase production by a novel yeast strain

Summary A novel yeast strain, NCIM 3574, isolated from a decaying wood produced up to 570 IU ml^–1 of xylanolytic enzymes when grown on medium containing 4% xylan. The yeast strain also produced xylanase activity (40–50 IU ml^–1) in the presence of soluble carbon sources like xylose or arabinose. No xylanase activity was detected when the organism was grown on glucose. The crude xylanase preparation showed no activity towards cellulolytic substrates but low levels of -xylosidase (0.1 IU ml^–1) and -l-arabinofuranosidase (0.05 IU ml^–1) were detected. The temperature and pH optima for the crude xylanase preparation were 55°C and 4.5 respectively. The crude xylanase produced mainly xylose from xylan within 5 min. Prolonged hydrolysis of xylan produced xylobiose and arabinose, in addition to xylose, as the end products. The presence of arabinose as one of the end products in xylan hydrolysate could be due to the low levels of arabinofuranosidase enzyme present in the crude fermentation broth.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

The relationship between nutrient feed rate and specific growth rate in fed batch cultures

Summary Equations are described which relate nutrient feed rate to specific microbial growth rate in fed batch culture. Fed batch cultures are classified into three types: 1) those allowing constant specific microbial growth rate, 2) those in which the rate of change of flow rate is constant and 3) those in which the nutrient flow rate is constant. The basic properties of these three types are described.Symbols F medium flow rate, L³ T^–1 - F _o medium flow rate at zero time, L³ T^–1 - g rate of change of flow rate with time, L³ T^–2 - K _v volume constant, being the total cell weight at zero time divided by the product of the yield coefficient and growth-limiting substrate concentration in the feed, L³ - s _r growth limiting substrate concentration in the feed, ML^–3 - V volume of liquid in the growth vessel, L³ - V _f volume of medium fed to the growth vessel, L³ - V _o volume of liquid in the growth vessel at zero time, L³ - X total weight of cells, M - x concentration of cells, ML^–3 - X _g total weight of cells grown, M - X _o total weight of cells at zero time, M - Y yield coefficient, weight of cells grown per unit weight of growth-limiting substrate - specific microbial growth rate, T^–1     

CO2 gas exchange of benthic microalgae during exposure to air: a technique for the rapid assessment of primary production

A method of measuring CO₂gas exchange (caused, for example, by microalgal photosynthesis on emersed tidal mudflats) using open flow IR gas analyzers is described. The analyzers are integrated in a conventional portable photosynthesis system (LI-6400, LI-COR, Nebraska, USA), which allows manipulation and automatic recording of environmental parameters at the field site. Special bottomless measuring chambers are placed directly on the surface sediment. Measurements are performed under natural light conditions and ambient CO₂concentrations, as well as under different CO₂concentrations in air, and various PAR radiation levels produced by a LED light source built into one of the measurement chambers. First results from tidal channel banks in a north Brazilian mangrove system at Bragança (Pará, Brazil) under controlled conditions show a marked response of CO₂assimilation to CO₂concentration and to irradiance. Photosynthesis at 100molmol^–1CO₂in air in one sample of a well-developed algal mat was saturated at 309mol photons m^–2s^–1, but increased with increasing ambient CO₂concentrations (350 and 1000mol mol^–1CO₂) in the measuring chamber. Net CO₂assimilation was 0.8mol CO₂m^–2s^–1at 100mol mol^–1CO₂, 5.9mol CO₂m^–2s^–1at 350mol mol^–1CO₂and 9.8mol CO₂m^–2s^–1at 1000mol mol^–1CO₂. Compensation irradiance decreased and apparent photon yield increased with ambient CO₂concentration. Measurements under natural conditions resulted in a quick response of CO₂exchange rates when light conditions changed. We recommend the measuring system for rapid estimations of benthic primary production and as a valuable field research tool in connection with certain ecophysiological aspects under changing environmental conditions.