A new sensor,the “filtration probe,” for quantitative characterization of the penicillin fermentation. II. The monitor of mycelial growth

Biomass concentration during penicillin production can be estimated rapidly and with reasonable accuracy using the “filtration probe.” The basis for the measurement is the volume of the filter cake which, in contrast to the conventional “packed-cell volume” measured by centrifugation, is only moderately affected by the morphology of the mycelia. The influence of the mycelial morphology on the specific filter cake volume [ υ (cm³ cake/g dry cells)] can additionally, to a large extent, be predicted from the (specific) filtration resistance of the broth. During the rapid (exponential) growth period, the specific cake volume is, however, more variable and typically exhibits a transient maximum value. The presence of such a maximum can be explained by a simple model for exponential growth of single mycelial particles. this model is based upon conventional kinetics for hyphae elongation and branching and applies well to experimental data (filtration behavior as well as microscopic studies) provided hyphae fragmentation is negligible (i.e., at high growth rates, μ ? μ_max).     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.     

Estimation of cell volume and biomass of penicillium chrysogenum using image analysis

A methodology for the estimation of biomass for the penicillin fermentation using image analysis is presented. Two regions of hyphae are defined to describe the growth of mycelia during fermentation: (1) the cytoplasmic region, and (2) the degenerated region including large vacuoles. The volume occupied by each of these regions in a fixed volume of sample is estimated from area measurements using image analysis. Areas are converted to volumes by treating the hyphae as solid cylinders with the hyphal diameter as the cylinder diameter. The volumes of the cytoplasmic and degenerated regions are converted into dry weight estimations using hyphal density values available from the literature. The image analysis technique is able to estimate biomass even in the presence of nondissolved solids of a concentration of up to 30 gL(-1). It is shown to estimate successfully concentrations of mycelia from 0.03 to 38 gL(-1). Although the technique has been developed for the penicillin fermentation, it should be applicable to other (nonpellected) fungal fermentations.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

Dopamine for “Wanting” and Opioids for “Liking”: A Comparison of Obese Adults With and Without Binge Eating

Obesity research suffers from an overinclusion paradigm whereby all participants with a BMI beyond a certain cutoff value (e.g., 30) are typically combined in a single group and compared to those of normal weight. There has been little attempt to identify meaningful subgroups defined by their salient biobehavioral differences. In order to address this limitation, we examined genetic and psychological indicators of hedonic eating in obese adults with (n = 66) and without (n = 70) binge eating disorder (BED). Our analyses focused on dopamine (DA) and opioid genetic markers because of their conjoint association with the functioning of brain reward mechanisms. We targeted three functional polymorphisms related to the D2 receptor (DRD2) gene, as well as the functional A118G polymorphism of the mu‐opioid receptor (OPRM1) gene. We found that significantly more obese controls had the “loss‐of‐function” A1 allele of Taq1A compared to their BED counterparts, whereas the “gain‐of‐function” G allele of A118G occurred with greater frequency in the BED group. A significant gene–gene combination χ² analysis also indicated that of those participants with the gain‐gain genotype (G⁺ and A1), 80% were in the BED group whereas only 35% with the loss‐loss genotype (G^? and A1⁺) were in this group. Finally, BED subjects had significantly higher scores on a self‐report measure of hedonic eating. Our findings suggest that BED is a biologically based subtype of obesity and that the proneness to binge eating may be influenced by a hyper‐reactivity to the hedonic properties of food—a predisposition that is easily exploited in our current environment with its highly visible and easily accessible surfeit of sweet and fatty foods.     

Aggregates and other particles as the origin of the “extraordinary” diffusional phase in polyelectrolyte solutions

The “extraordinary” diffusional phase (EP) at low ionic strength, and the conditions for 1 its removability by filtration were investigated for dilute solutions of the following linear polyelectrolytes: poly(L -lysine), heparin, chondroitin-6-sulfate, hyaluronate, polystyrene sulfonate, and variably ionized polyacrylamide. The EP was not present for all the different types studied, and for heparin, for example, the phase was present only for samples from certain sources. In all cases the phase was removable by filtration through sufficiently small pore-size membranes. Once filtered, the EP remained absent for over one week. It is concluded that the extraordinary diffusional phase consists of fairly stable polyelectrolyte aggregates, and sometimes also includes other very small particulate impurities. These aggregates and other small particles are thought to be present, or at least nascent, in the dry polyelectrolyte material, so that their properties may depend critically on the manner in which such dry material is produced. Tests for “reversibility” of the EP by cycling between high and low C_s by dialysis further confirm these conclusions. The evidence is thus against the EP representing any type of temporal aggregates or local ordering, at least for the linear polyelectrolytes studied in this work Rather, due to the extremely feeble scattering of ordinary polyelectrolytes at low ionic strength, the weak scattering from residual aggregates and other particles, not removed by ordinary filtration and centrifugation procedures, give autocorrelable scattering signals with long decay times. The “loss” of the extraordinary phase as ionic strength increases appears to be due simply to the weak EP scattering signal getting buried in the sharply increasing scattering from the ordinary polyelectrolyte phase. Model calculations based on experimental data support this latter conclusion. © 1992 John Wiley & Sons, Inc.     

A stable, “near-haploid” mammalian cell line (Dipodomys ordii)

An established SV40-transformed cell line of Dipodomys ordii was cloned for selective loss of chromosomal material. A clone is described which has a modal chromosome number of 50 (in the normal diploid 2n=72), and has about 66% of the DNA content of normal diploid cells. Karyotype analysis shows that, although some chromosome rearrangement has taken place, 23 chromosomes are completely unpaired and 7 chromosomes are partially unpaired. Buoyant density analysis in neutral CsCl and Cs₂SO₄+Ag gradients of the DNA of this clone shows that there has been no selective retention or loss of any of the satellite DNA components present in D. ordii during the elimination of 34% of the genetic material.     

Physiological energetics of the zebra musselDreissena polymorpha in lakes II. Food uptake and gross growth efficiency

Food uptake ofDreissena polymorpha from two shallow water sites and one site at a 9 m depth in lakes was monitored over the course of 1 1/2 years. Feeding was characterized by the filtration capacity and ingestion capacity (i.e. the unrestricted maximum rates) at ambient temperature. The available food was estimated as seston dry weight, as seston ash free dry weight and as seston volume (sum of the volumes of the particles in the water column). The filtration capacity and ingestion capacity varied strongly during the course of the year by more than 1:10, with maximum values during late spring and early summer. The filtration capacity was correlated with water temperature, the ingestion capacity with temperature and seston concentration. However, at identical water temperatures both capacities were higher in spring than in autumn. At the 9 m site, filtration and ingestion capacity was also significantly influenced by lack of oxygen caused by stratification during summer. In the allometric equations by which both capacities (C) were related to body weightW (C=a W ^b ), an exponent b of 0.89±0.14 (mean±95% confidence interval) was calculated for the filtration capacity and 1.00±0.18 for the ingestion capacity. Both exponents did not vary significantly during the course of the year. The seasonal trend of the incipient limiting concentration (i.e. the seston concentration at which the intestine is filled to its capacity when the animal filters at maximum rate) was less pronounced. It showed a significantly positive correlation with seston concentration and a negative correlation with temperature. Except during the spring bloom, the incipient limiting concentration was in the range of the seston volume registered at the sites. Gross growth efficiencies were highly variable. An annual average between 5 and 20% was calculated. Growth efficiencies were lower at the shallow water sites with the highest seston concentrations.     

“Ecological Armageddon” – more evidence for the drastic decline in insect numbers

The efficient use of nitrogen by crops can minimise environmental risks and maximise returns to farmers. Under organic farming systems, this can be achieved by adjusting the fertilisation management and/or using genetic variability. Seven durum wheat (Triticum durum) cultivars and three emmer (Triticum dicoccum) cultivars were assessed under an organic farming system over a non‐consecutive 4‐year period (2005–11) in Foggia (southern Italy). The objectives were to investigate the agronomic and qualitative characteristics, and to evaluate the agronomic efficiency and adaptability according to three N fertilisation levels (0, 40, 80 kg N ha^?1). A split‐plot design was used, with three replications in each year. Nine traits were investigated: heading time, plant height, seed yield, number of spikes m^?2, harvest index, specific weight, 1000‐seed weight, and protein and gluten contents. Increasing N to 80 kg ha^?1 increased plant height (+7.3%), seed yield (+22.1%), spike density (+16.6%), and protein (+9.8%) and gluten (+2.1%) contents. The emmer genotypes and the oldest durum wheat ‘Cappelli’ showed the highest protein and gluten contents (mean, 13.9%, 11.2%, respectively). The maximum N agronomic efficiency [AE(N)] and N recovery efficiency [RE(N)] were seen for the modern durum wheat cultivars under 40 kg ha^?1 N treatment: ‘Duilio’, ‘Iride’ and ‘Varano’. The significant correlation between AE(N) and RE(N) and the year of release of the durum wheat cultivars (r = 0.53, P < 0.05, F = 4.7 for AE(N) and r = 0.57, P < 0.01, F = 5.7 for RE(N), respectively; n = 14) showed that the genetic breeding of this species for high grain yield is associated with an increase in the efficiency of N fertiliser use under organic farming. The highly significant effect for the Genotype × Environment interaction (F = 18.1, P < 0.001) of seed yield indicate the possibility to select for stable genotypes across environments. Based on the additive main effects and multiplicative interaction analysis, and the yield stability index, the varieties ‘Iride’ and ‘Varano’ show high‐stability responses and good seed yields under all N fertiliser treatments. Therefore, these varieties can be recommended for organic farming systems in Mediterranean areas.     

Estimating the effective clearance rate and refiltration by zebra mussels, Dreissena polymorpha, in a stratified reservoir

1. The grazing impact of zebra mussels, Dreissena polymorpha Pallas, is often evaluated by applying the individual filtration rate measured in the laboratory to the field abundance and then by comparing the total volume of water filtered with the whole lake volume. Since this approach overlooks refiltration, it overestimates the grazing impact of zebra mussels. To deal with this problem, the present authors developed an in situ method for collecting faeces and pseudofaeces to measure the actual volume of water that is cleared of suspended particles by Dreissena in a unit time under a given set of environmental conditions. This is termed the effective clearance rate (ECR). 2. The experiment was conducted in Hargus Lake, OH, U.S.A., a small thermally stratified reservoir, to test the effects of spatial aggregation, mussel density and the concentration of particulate inorganic matter (PIM) on the effective clearance rate of Dreissena. 3. Over 40 measurements, the ECR values ranged from 15.3 to 68.6 mL ind^??1 h^??1. Much of the variation can be explained by colony form, mussel density and seston concentration. The effects of these variables were all statistically significant. The average ECR for isolated individuals was higher than that for those in clumps (40.4 versus 32.8 mL ind^??1 h^??1), which is attributed to increased refiltration in the cores of the clumps. The ECR decreased with increased zebra mussel density because of intensified competition for food particles within the group. The ECR increased with increased PIM concentration in the lake water, which may be interpreted as a result of enhanced water mixing which ultimately caused increases in both sediment resuspension and particle delivery to the mussels. 4. Taking the filtration rates for a 20-mm mussel to be between 116 and 234 mL ind^??1 h^??1, based on data from the literature, the clumped mussels under the present experimental conditions would have a refiltration ratio between 3.4 and 6.9. 5. The present authors developed an areal clearance model which predicts that seston removal by the Dreissena population is limited by the particle delivery from the ambient water to the mussel bed and will reach a maximum value beyond which no further increase will occur with increased population density. 6. It is concluded that the direct grazing impact of zebra mussel on phytoplankton in thermally stratified lakes is much less effective than predicted from simple filtration rate estimation.     

Distribution, biomass and production of the freshwater mussel, Hyridella menziesi (Gray), in Lake Taupo, New Zealand

SUMMARY.

• 1 A study of the distribution, biomass and production of the freshwater mussel Hyridella menziesi (Gray) was carried out at six sites in Tapuaeharuru Bay, Lake Taupo, New Zealand.
• 21 H. menziesi is common in the littoral zone, with clean sand and angle of slope being the most important environmental variables measured which are positively associated with mussel density. These variables are related to the accumulation of fine material which can clog the filtering mechanism of mussels. The angle of slope may also influence the movement and supply of food.
• 3 Density (5.6 m^-2), biomass (2.8 g dry weight shell free tissue m^-2) and potential production (0.50 g dry weight shell free tissue m^-2 yr^-1) are high compared to values found for mussels from other oligotrophic lakes.
• 4 The age structure of Hyridella suggests recruitment has declined in recent years. A periodicity in generations is proposed.

     

An image analysis technique to estimate the cell density and biomass concentration of Trichoderma reesei

Aim:  The objective is to develop an automated image analysis protocol to quantify the cell volume fraction of filamentous fungi ( Trichoderma reesei ) and estimate the biomass concentration.
Methods and Results:  Both dry weight and image analyses were performed on samples collected periodically from 7-l stirred tank fermentations. Using the projected area of lactophenol blue-stained hyphae, the fraction occupied by the cells in a given volume was estimated. Combined with the biomass dry weight obtained by filtration, the method was used to estimate the density of filamentous fungi. Knowing the density of fungi, the algorithm was employed to quantitatively assess the biomass evolution during the course of fermentation even in the presence of solid particles.
Conclusions:  A density of 0.334 g dry weight cm⁻³ was found for T. reesei RUT C-30. The image analysis protocol allowed successful estimation of biomass concentration in the presence or absence of solid particles.
Significance and Impact of the Study:  Methods to quantify biomass during the industrial production of cellulase with T. reesei are often limited due to the presence of solid substrates. The image analysis protocol presented here offers a quick and easy way to estimate biomass concentration of filamentous micro-organisms in insoluble medium.     

Microscopical determination of the filtration permeability of the mucosal surface of the goldfish intestinal epithelium

Summary The rate of shrinkage of the mucosal folds of goldfish intestine in response to mucosal hypertonicity was measured by microscopic means. Because of the geometry of the intestinal folds the rate of shrinkage could be directly related to the loss of volume from the fold through the brush border membranes and tight junctions. Experimentally a wide range of velocities was observed, reflecting the difficulty of rapidly estabilishing a uniform osmotic gradient at the preparation's mucosal surface. The initial velocity of volume loss provided a measure of the filtration permeability (P _f ) of the mucosal surface. From the highest velocities observed the filtration permeability was estimated to be approximately 14×10^–3 cm/sec related to the folded mucosal surface and 65×10^–3 cm/sec related to the straight serosal surface. Consideration of the experimental errors and unstirred layer effects make it probable that the latter value is still an underestimate of the trueP _f . The series barriers of the epithelium cause the total tissueP _f to be less than theP _f of the mucosal surface alone. In addition theP _f measured in the presence of an osmotic gradient may differ substantially from the tissue filtration permeability which exists in the absence of a change in osmolarity.     

Prescribed “Breaks” as a Means to Disrupt Weight Control Efforts

Objective: Research on long‐term maintenance of weight loss is hampered by the fact that relapse typically does not occur until after 6 months. We sought to determine whether relapses could be induced earlier by intentionally interrupting the momentum of weight loss during a treatment program and thus provide a model for weight maintenance research. Research Methods and Procedures: Subjects (N = 142) were recruited at two centers and randomly assigned to a control group, a long break group (LB), or a short break group (SB). The control group received a standard behavioral weight loss program with 14 weekly sessions; the LB and SB groups received the same. However, the LB group took a 6‐week break after lesson 7, and the SB group took a 2‐week break after lessons 3, 6, and 9. Participants were instructed to stop all weight loss efforts during breaks. Results: Participants seemed to take breaks as prescribed, with interruptions in self‐monitoring, dietary adherence, and self‐weighing (but not in exercise). However, participants quickly resumed these behaviors after the break. Breaks produced a slowing of weight loss or slight regain, but weight losses during the breaks were not significantly different from the control group. Importantly, overall weight losses (0 to 5 months or 0 to 11 months) did not differ between conditions. Discussion: This study was not successful in developing a method to experimentally produce weight loss relapses. However, the finding that prescribed breaks do not have adverse effects may have clinical application.     

Periphyton productivity and radionuclide accumulation in the Columbia River,Washington, U.S.A.

Summary An investigation of the periphyton of the Columbia River below the Hanford Atomic Works, Washington, was conducted to study the relationships between productivity, radionuclide uptake, and environmental influences.Best correlations between the four biomass measurements were between dry weight, ash weight, and chlorophyll a. Net Production Rate varied from 0.005 to 0.070 mg dry weight/cm²/day and was closely related to chlorophyll a and also to solar energy.The accumulation of ³²P and ⁶⁵Zn was highly related to dry and ash weight and chlorophyll a. Low correlations were found between radionuclide accumulation and the radioisotope burden of the river. The data suggest that adsorption was the dominant mode of uptake.This paper is based on work performed under United States Atomic Energy Commission Contract AT(45-1)-1830.     

Naltrexone reduces weight gain, alters “β-endorphin”, and reduces insulin output from pancreatic islets of genetically obese mice

Naltrexone, an opiate antagonist, was administered to young obese (ob/ob) and lean mice for five weeks. Animals had continuous access to food and received 10 mg/kg SC twice daily with equivalent volumes of saline given to controls. The effects on body weight, and pituitary and plasma levels of β-endorphin-like material were measured. Naltrexone-injected obese animals gained weight more slowly over the first three weeks while the weight gain of lean animals was not affected by naltrexone. Plasma levels of β-endorphin were shown to be significantly higher in untreated ob/ob mice and this difference increased with age (4–20 weeks). With naltrexone treatment, plasma levels in +/? mice rose and exceeded those in ob/ob. Saline treatment appeared to be a stress, and pituitary β-endorphins rose 4–6 fold in ob/ob compared with +/?. While naltrexone reduced the levels in ob/ob pituitary towards normal, no effect on β-endorphin levels in pituitary of lean mice was obtained. In vitro studies of effects of the opiate antagonists, naloxone, on insulin secretion by isolated islets provided additional evidence of resistance of lean mice to naloxone relative to ob/ob. (IRI secretion fell only in naloxone treated ob/ob islets.) These observations support the contention that this form of genetic obesity is characterized by elevated endogenous opiate levels and an increased sensitivity to opiate antagonists such as naltrexone or naloxone.     

Dietary Restraint and Control Over “Wanting” Following Consumption of “Forbidden” Food

Eating behavior can be influenced by the rewarding value of food, i.e., “liking” and “wanting.” The objective of this study was to assess in normal‐weight dietary restrained (NR) vs. unrestrained (NU) eaters how rewarding value of food is affected by satiety, and by eating a nonhealthy perceived, dessert‐specific food vs. a healthy perceived, neutral food (chocolate mousse vs. cottage cheese). Subjects (24NR age = 25.0 ± 8.2 years, BMI = 22.3 ± 2.1 kg/m²; 26NU age = 24.8 ± 8.0 years, BMI = 22.1 ± 1.7 kg/m²) came to the university twice, fasted (randomized crossover design). Per test‐session “liking” and “wanting” for 72 items divided in six categories (bread, filling, drinks, dessert, sweets, stationery (placebo)) was measured, before and after consumption of chocolate mousse/cottage cheese, matched for energy content (5.6 kJ/g) and individual daily energy requirements (10%). Chocolate mousse was liked more than cottage cheese (P < 0.05). After consumption of chocolate mousse or cottage cheese, appetite and “liking” vs. placebo were decreased in NR and NU (P < 0.03), whereas “wanting” was only decreased in NR vs. NU (P ≤ 0.01). In NR vs. NU “wanting” was specifically decreased after chocolate mousse vs. cottage cheese; this decrease concerned especially “wanting” for bread and filling (P < 0.05). To conclude, despite similar decreases in appetite and “liking” after a meal in NR and NU, NR decrease “wanting” in contrast to NU. NR decrease “wanting” specifically for a nonhealthy perceived, “delicious,” dessert‐specific food vs. a nutritional identical, yet healthy perceived, slightly less “delicious,” “neutral” food. A healthy perceived food may thus impose greater risk for control of energy intake in NR.     

“Action potentials” in NEUROSPORA CRASSA, a mycelial fungus

Occasional spontaneous “action potentiais” are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5–20 mV depolarized from the normal resting potential (near − 180 mV). The “action potentiais” are long-lasting, 1–2 min and at the peak reach a membrane potential near −40 mV. A 2− to 8−fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H⁺ or Cl^- and (b) periodic decreases in activity of the major electrogenic pump (H⁺) of the Neurospora membrane, coupled with a nonlinear (inverse sigmoid) current-voltage relationship.Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.     

Low-sulfur proteins from α-keratins. Interrelationships between their amino acid compositions, α-helix contents,and the supercontraction of the parent keratin

S-Carboxymethyl (SCM) kerateine preparations from a range of keratins were fractionated by acid precipitation into low-sulfur (SCMKA) and high-sulfur (SCMKB) fractions. Amiono acid analyses and optical rotatory dispersion measurements on the SCMKA fractions from different keratins indicated that the proportion of α-helical material (30–64%) increased with increasing leucine and glutamic acid contents and decreased with increasing SCM-cysteine and proline contents. It is shown that these variations in α-helix contents are due mainly to differences in the conformations of the principal components of the SCMKA fractions, although there is also a smaller contribution arising from contamination with varying amounts of non helical components. The thiol + disulfide contents for the parent keratins were measured and compared with the SCM -cysteine contents of the solubilized fractions. In general the SCM-cysteine contents of the SCMKA fractions increased with increasing thiol + disulfide content of the parent keratin, but these values were not directly proportional. The extent of total supercontraction in 8M LiBr solution was measured for the parent keratins and shown to bear no significant relationship to their disulfide contents. From the extents of first-stage and total supercontractions the disulfide contents of the contractile structures in the fibers were calculated according to a model described previously and compared with the analytical values for the SCM -cysteine contents of the SCMKA and SCMKB fraction. Data for both fractions were consistent with the model in that they were in each case related to the calculated values by a constant factor. However, because of simplifying assumptions in the model, it was not possible to identity positively one or other of these protein fractions with the contractile elements in the fiber.