Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Fluorescence of 3-keto-steroids in aqueous solution

The physiologically important 3-keto-steroids are non-fluorescent or only weakly fluorescent in protic as well as in aprotic solvents. In contrast, the 4,6,8(14)-triene-3-one steroids are highly fluorescent in aqueous solution but they do not appreciably fluoresce in other solvents. Evidence is presented that the introduction of double bonds into the skeleton of the 3-keto-steroids leads to a decrease of the energy of the lowest – ^* state, bringing this level into the neighbourhood of the non-fluorescent n – ^* state. As a consequence, for two states of approximately the same energy, relatively small perturbations such as those due to solvent interactions, protein binding and micelle formation, will then determine whether a system will fluoresce ( – ^* state lowest) or not (n – ^* state lowest). When the fluorescent 3-keto-steroids, having three conjugated double bonds, bind to proteins, the fluorescence intensity becomes almost zero, making these compounds useful as probes for steroid-protein interactions. This quenching of the fluorescence is explained by a decrease in energy of the n – ^* state relative to the – ^* state of the steroids due to hydrophobic interactions with the proteins.Abbreviations 6,8-BDT 6,8-bisdehydrotestosterone; DMSO, dimethylsulfoxide - HPLC high pressure liquid chromatography This work was presented in part at the Annual Meeting of the Gesellschaft für Biologische Chemie, September 26–29, 1983, in Göttingen. For an abstract see: Hoppe-Seyler's Z. Physiol. Chem. (1983) 364: 1151–1152Dedicated to Prof. Dr. F.-W. Zilliken on the occasion of his 65th birthday     

The role of β-dimethylsulphoniopropionate,glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis

The tertiary sulphonium compound, -dimethylsulphoniopropionate (DMSP) and the quaternary ammonium compounds glycine betaine and homarine are important osmotica in Platymonas subcordiformis cells. Following hypersaline stresses the compounds were accumulated after a lag period of 3 h and equilibrium concentrations were reached 6 h later. In contrast to these organic solutes, mannitol was synthesised immediately and equilibrium concentrations were reached within 90 min. Hyposaline stresses induced losses of the organic solutes from the cells. The ions K⁺, Na⁺, Cl^- and the above organic solutes can account for the osmotic balance of the cells.Abbreviations DMSP -dimethylsulphoniopropionate - _i intracellular osmolality - _o extracellular osmolality     

A knowledge based system for diagnosing microbial activities during a fermentation process

A knowledge based system, LAexpert, was developed to diagnose microbial activities during a fermentation process on the basis of specific rates determined on-line. The LAexpert is a supervisor for a process control system and assists operators in fault diagnosis. The LAexpert was implemented using a fuzzy expert system shell based on the object oriented programming tool Smalltalk V/Mac running in a Macintosh II computer. The shell can handle uncertainties both in the measurements and knowledge by fuzzy reasoning.List of Symbols X g/l biomass dry weight (g/l) - S g/l substrate concentration (g/l) - P g/l product concentration (g/l) - _c, _c, _c 1/h specific rates calculated from on-line measured data of X, S and P (1/h) - _d, _d, _d 1/h specific rates read from database of BIOACS (1/h)     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Influence of intracellular and extracellular tonicities on water permeability in Characean cells

Summary The effect of the concentration of the central vacuolar sap on water permeability previously demonstrated onNitella internode (Tazawa and Kamiya 1966), has been further studied. By using a technique of vacuole perfusion the ionic concentration of the cell sap has been modified independently of its tonicity. Transcellular water permeability has been measured by means of a double-chamber osmometer.When the tonicities of artificial saps were adjusted to that of the natural cell sap, wide variations in the concentration of K⁺, Na⁺, or Ca⁺⁺ in the vacuole did not bring about any change in the magnitude of water permeability. On the other hand, water permeability was strongly influenced by varying the tonicity of the vacuolar medium by addition of mannitol. It increased when the tonicity was lowered from the normal level, while it decreased when tonicity was heightened. Water permeability was also decreased by increase in the tonicity of the external medium.Analysis of the results showed that the specific resistance to water flow across the plasmalemma and the tonoplast in series (the reciprocal of the water permeability k_p) was related to the osmotic pressures of the intracellular ( _i) and the extracellular ( ₀) medium by the empirical formula, l/k_p=0.088 + 0.015 . + 0.0074 ₀. Thus, intra- and extracellular tonicities influence the water permeability of theNitella internode independently of each other. The decrease in water permeability by increase in tonicity of the intra- or extracellular medium may be explained in terms of the effect of these tonicities on hydration of the cell membranes.The water permeability ofLamprothamnium, a brackish water Characeae was only one fourth that ofNitella, a fresh water Characeae. The lower permeability inLamprothamnium may be accounted for in terms of the high tonicities of its cell sap and external medium.     

Identification of a glutathione-S-transferase effector domain for inhibition of jun kinase, by molecular dynamics

We have recently found that the glutathione-S-transferase -isozyme (GST-), a cellular detoxification enzyme, potently and selectively inhibits activation of jun protein by its upstream kinase, jun kinase (JNK). This newly identified regulatory activity of GST- is strongly inhibited by a group of agents that inhibit its enzymatic activity. Since loss of enzymatic activity in general does not correlate with loss of regulatory activity, it is likely that inhibitor binding induces changes in the structure of one or more domains of GST that block its interaction with JNK. To identify regions of GST that change conformation on the binding of inhibitors, we have performed molecular dynamics calculations on GST- to compute its average structure in the presence and absence of the inhibitor, glutathione sulfonate. Superposition of the two average structures reveals that several regions change local structure depending upon whether the inhibitor is bound or not bound. Two of these regions, residues 36–50 and 194–201, are highly exposed. We have synthesized peptides corresponding to these two segments and find that the 194–201 sequence strongly inhibits the ability of GST- to block the in vitro phosphorylation of jun by JNK. These results suggest that this region of GST- is critical to its functioning as a newly discovered regulator of signal transduction.     

Hypo-osmotic stimulation of active Na+ transport in frog muscle: Apparent upregulation of Na+ pumps

Summary The purpose of this work was to determine if hypotonicity, in addition to the stimulation of active Na⁺ transport (Venosa, R.A., 1978,Biochim. Biophys. Acta 510:378–383), promoted changes in (i) active K⁺ influx, (ii) passive Na^– and K⁺ fluxes, and (iii) the number of³H-ouabain binding sites.The results indicate that a reduction of external osmotic pressure () to one-half of its normal value (=0.5) produced the following effects: (i) an increase in active K⁺ influx on the order of 160%, (ii) a 20% reduction in Na⁺ influx and K⁺ permeability (P _K), and (iii) a 40% increase in the apparent density of ouabain binding sites. These data suggest that the hypotonic stimulation of the Na⁺ pump is not caused by an increased leak of either Na⁺ (inward) or K⁺ (outward). It is unlikely that the stimulation of active Na⁺ extrusion and the rise in the apparent number of pump sites produced by hypotonicity were due to a reduction of the intracellular ionic strength. It appears that, at least in part, the stimulation of active Na⁺ transport takes place whenever muscles are transferred from one medium to another of lower tonicity even if neither one was hypotonic (for instance =2 to =1 transfer). Comparison of the present results with those previously reported indicate that in addition to the number of pump sites, the cycling rate of the pump is increased by hypotonicity. Active Na⁺ and K⁺ fluxes were not significantly altered by hypertonicity (=2).     

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new -tubulin isoform from sunflower we named -tubulin. -tubulin is the most divergent higher-plant -tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that -tubulin expression is restricted to the male gametophyte. -tubulin mRNA represents 90% of -tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, -tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that -tubulin is specific to this family. Our results suggest that -tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that -tubulin is found in a complex with -tubulin in mature sunflower pollen.     

Prediction of protein secondary structure from amino acid sequence

The conformational parametersP _k for each amino acid species (j=1–20) of sequential peptides in proteins are presented as the product ofP _i,k , wherei is the number of the sequential residues in thekth conformational state (k=-helix,-sheet,-turn, or unordered structure). Since the average parameter for ann-residue segment is related to the average probability of finding the segment in the kth state, it becomes a geometric mean of (P _k )_av=(P _i,k ) ^1/n with amino acid residuei increasing from 1 ton. We then used ln(P_k)_av to convert a multiplicative process to a summation, i.e., ln(P _k ) _av =(1/n)P _i,k (i=1 ton) for ease of operation. However, this is unlike the popular Chou-Fasman algorithm, which has the flaw of using the arithmetic mean for relative probabilities. The Chou-Fasman algorithm happens to be close to our calculations in many cases mainly because the difference between theirP _k and our InP _k is nearly constant for about one-half of the 20 amino acids. When stronger conformation formers and breakers exist, the difference become larger and the prediction at the N- and C-terminal-helix or-sheet could differ. If the average conformational parameters of the overlapping segments of any two states are too close for a unique solution, our calculations could lead to a different prediction.     

Anionradicals from 5-halouracil substituted oriented DNA after X-irradiation at low temperatures

Conclusions The model of two initial radiation - induced radical components in oriented fibers of DNA,T ^– andG ⁺ [1], should be amended, for 5-halouracil substituted DNA by the finding that the primary anion is formed on the 5-halouracil base specifically but differentiates, depending on the halogen substituent, into*- and*-forms. The occurrence of the latter appears, from theoretical calculations, to be connected with a C-halogen bond elongation. The DNA matrix apparently allows for this whereas in the corresponding single crystals of 5-bromodeoxyuridine [1] or 5-iododeoxyuridine [4] the crystal packing prevents the elongation thus allowing for*-anion formation only.     

Effect of the dnaN mutation on the growth of small DNA phages

Summary The effect of the dnaN mutation on the growth of single-stranded DNA phages was studied by burst experiments. In HC138 dnaN cells exposed to 42.5° C at 5 min before infection, growth of spherical (microvirid or isometric) phages such as 3, Kh-1 and X174 was partially reduced at the nonpermissive temperature. When infection was performed at 30 min after temperature shift-up, viral replication was completely inhibited at 42.5° C in the dnaN strain but not in a dna ⁺ revertant. At 41° C, multiplication of filamentous (inovirid) phages M13 and fd was restricted specifically in HC138 F⁺ dnaN bacteria. When dnaN cells lysogenic for i²¹ were grown at 42.5° C for 60 min and then shifted down to 33° C, a burst of i²¹ occurred with concomitant cellular lysis, manifesting induction of the prophage development.     

Pre-clinical studies of the negative pi-meson beam at TRIUMF

Summary The ^– flux from the biomedical channel at TRIUMF increases with increasing channel momentum, while the contaminating electron flux decreases. Since the electrons appear to result from conversion of the high energy-rays produced by ⁰ decay in the production target, the electron contamination can be reduced further by target configurations which minimize gamma conversion.The attenuation of ^– beams by in-flight interactions was found to decrease from an initial value of 1.67 ± 0.02 % per g/cm² at zero depth to 1.48 ± 0.02 % per g/cm² near the stopping peak.The inactivation of cultured CHO cells by an extended-peak ^– dose distribution has been measured using the gel technique. The survival data have been fitted by a model which characterizes the physical quality of the dose profile by means of measured star densities. This model provides a convenient method of analysis for large sets of survival data and may be useful for prediction of the biological effect of new ^– dose distributions.The RBE value for 50% survival measured at the centre of a 7 cm extended peak was found to be approximately 1.4, in reasonable agreement with recent values obtained at LAMPF and SIN.     

Visualization ofπ − andπ + stopping density distributions in one and two dimensions

Summary A technique suitable for mapping ^± stopping density distributions in patients or phantoms is described. As a position sensitive detector a multiwire proportional chamber with a slit or a hole collimator in front was applied. Results using a water and a Rando phantom are presented for various momenta and momentum band widths of the ^± beam. To our knowledge the two-dimensional visualization of a ^– stopping density distribution was realized for the first time.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Structure and properties of nonclassical polymers X. Heteroatomic polymers with degenerate half-filled band

It is shown that the group of -conjugated, nonclassical (non-Kekulé) homonuclear, alternative organic polyradicals and polymers with degenerate NBMOs can be essentially extended to a large class of heterocyclic analogues having a set of degenerate MOs. The presence of a set of degenerate MOs (DMOs) results from the molecular topology of the system. The conditions of occurrence of DMOs are determined by the generalized Coulson-Rushbrooke-Longuet-Higgins theorem. The character of spin-exchange interaction of -electrons in the half-filled band (HFB) of a large group of model polymers, analogues of poly(meta-anilines), has been investigated. It is shown that the main component of the ferromagnetic exchange interaction is the potential (Coulomb) exchange and a smaller contribution of the indirect exchange among the HFB electrons via the delocalized -electrons in the occupied bands. The theoretical method used, which predicts the existence of a set of DMOs, may serve as a guiding principle in the design of narrow-band, high-spin organic polymers in which cooperative magnetic phenomena can arise.Part IX:Theoret. Chim. Acta 86, 353–367 (1993).     

Correlation between changes in photosynthetic activity and changes in total protoplast volume in leaf tissue from hygro-, meso- and xerophytes under osmotic stress

Rates of photosynthesis of leaf slices from various hygro-, meso- and xerophytes were measured in the absence of stomatal control in various stages of osmotic dehydration. The external osmotic potential _° for a 50% inhibition of photosynthesis varied between 20 bar in some hygrophytes up to 50 bar in xerophytes. The response of photosynthetic enzymes to increased salt concentrations in the reaction medium was similar in leaf extracts from hygro-, meso- and xerophytes. The total protoplast volume in vacuum-infiltrated leaf discs from various plants was measured as the difference between ³H₂O-labeled space and [¹⁴C]sorbitol-labeled space. In all plants, the protoplast volume could be reduced to about 55% of the maximum volume of tissue in equilibrium with water, without decreasing photosynthesis. Reduction of the maximal protoplast volume below 55% decreased photosynthesis in all tissues to the same decreased photosynthesis in all tissues to the same degree. At 20% maximal volume, photosynthesis of all plants was completely inhibited. The differential decrease of protoplast volumes of various leaf tissues in response to changes in _° was mainly due to the different osmotic potential of the cell sap (_cs). The relative contribution of sugars to the overall osmolarity of the cell sap was up to nineteen times higher in xerophytes than in hygrophytes. Short-term recovery of photosynthesis after hypertonic stress was good in xerophytes, incomplete in mesophytes and absent in hygrophytes. There was also a large discrepancy between the partial recovery of protoplast volumes and the complete absence of a recovery of photosynthesis in hygrophytes.     

Ecophysiological analysis of woody species in contrasting temperate communities during wet and dry years

This study employed an intensive sampling regime in which leaf gas exchange and tissue-water relations were measured simultaneously on the same leaf at midday on 19 tree species from three distinct forest communities during wet (1990) and dry (1991) growing seasons. The study sites were located on a xeric barrens, a misic valley floor, and a wet-mesic floodplain in central Pennsylvania, United States. The xeric, mesic, and wetmesic sties had drought-related decreases in gravimetric soil moisture of 53, 34 and 27%, respectively. During the wet year, xeric and mesic communities had high seasonal mean photosynthetic rates (A) and stomatal conductance of water vapor (g _wv) and low midday leaf water potential (), whereas the wet-mesic community had low A and g _wv and high midday . The mesic and wet-mesic communities had dry year decreases in predawn , g _wv and A with the greatest drought effect occurring in the mesic community. Regression analysis indicated that species from each site that exhibited high wet-year A and g _wv tended to have low midday . This trend was reversed only in the mesic community in the drought year. Despite differences in midday , all three communities had similar midday leaf turgor pressure (_p) in the wet year attributable to lower osmotic potential at zero turgor ( ⁰ ) with increasing site droughtiness. Lower wet year ⁰ in the xeric community was due to low symplast volume rather than high solute content. Species with the lowest ⁰ in the wet year often did not have the lowest ¹⁰⁰ possibly related to differences in tissue elasticity. Moreover, increased elasticity during drought may have masked osmotic adjustment in ¹⁰⁰ but not in ⁰ , via dilution of solutes at full hydration in some species. Despite the sampling regime used, there were no relationships between gas exchange and osmotic and elastic parameters that were consistently significant among communities or years. This result questions the universal, direct effect of osmotic and elastic adjustments in the maintenance of photosynthesis during drought. By including a large number of species, this study provided new insight to the ecophysiology of contrasting forest communities, and the community-wide impact of drought on contrasting sites.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Structure and nonlinear optical properties of copper phthalocyanine thin films

This paper describes a joint study of the structure and nonlinear optical properties of vacuum evaporated thin films of copper phthalocyanine (CuPc for brevity). Film thickness ranges from 50 to 500 nm. The anisotropic paramagnetic resonance of Cu⁺⁺ ions reveals that the Pc rings lie almost parallel to the substrate plane with however a large angular distribution (30° FWHM). Third harmonic optical generation measurements performed at 1.064 m and 1.907 m fundamental wavelengths give respectively an average value of the cubic susceptibility ⁽³⁾(-3,)=(4±0.4)·10^–12 e.s.u. and (2.1+-0.2) · 10^-12 These values, although significantly higher than for a common ionic crystal, are about one order of magnitude lower than in conjugated 1-D systems, which shows that the 2-D -electron delocalization is less profitable than the 1-D one. Besides third harmonic, we have also observed second harmonic generation. Its polarization dependence is characteristic of a quadratic susceptibility enhanced in one direction, almost perpendicular to the substrate, withd _eff comprised between 30 and 60 · 10^-9 e.s.u. The possible origins ofd _eff are discussed.