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.     

A dynamic mathematical model for microbial removal of pyritic sulfur from coal

A dynamic mathematical model has been developed to describe microbial desulfurization of coal by Thiobacillus ferrooxidans. The model considers adsorption and desorption of cells on coal particles and microbial oxidation of pyritic sulfur on particle surfaces. The influence of certain parameters, such as microbial growth rate constants, adsorption-descrption constants, pulp density, coal particle size, initial cell and solid phase substrate concentration on the maximum rate of pyritic sulfur removal, have been elucidated. The maximum rate of pyritic sulfur removal was strongly dependent upon the number of attached cells per coal particle. At sufficiently high initial cell concentrations, the surfaces of coal particles are nearly saturated by the cells and the maximum leaching rate is limited either by total external surface area of coal particles or by the concentration of pyritic sulfur in the coal phase. The maximum volumetric rate of pyritic sulfur removal (mg S/h cm(3) mixture) increases with the pulp density of coal and reaches a saturation level at high pulp densities (e.g. 45%). The maximum rate also increases with decreasing particle diameter in a hyperbolic form. Increases in adsorption coefficient or decreases in the desorption coefficient also result in considerable improvements in this rate. The model can be applied to other systems consisting of suspended solid substrate particles in liquid medium with microbial oxidation occurring on the particle surfaces (e.g., bacterial ore leaching). The results obtained from this model are in good agreement with published experimental data on microbial desulfurization of coal and bacterial ore leaching.     

The use of solid lipid nanoparticles to target a lipophilic molecule to the liver after intravenous administration to mice

Taspine solid lipid nanoparticles (Ta-SLN) and taspine solid lipid nanoparticles modified by galactoside (Ta-G(2)SLN) were prepared by the film evaporation-extrusion method. The nanoparticles were spherical or near-spherical particles with smooth surface, small size and high encapsulation efficiency. Ta-G(2)SLN and Ta-SLN showed significant inhibition on 7721 cell growth. Intravenous injection of either Ta-SLN or Ta-G(2)SLN resulted in a higher plasma and liver concentration and a longer retention time in mice compared with the administration of Ta. These results suggested that SLN tended to be preferentially delivered to the liver and Ta-G(2)SLN may further enhance liver targeting.     

Modeling of heat and mass transfer for solid state fermentation process in tray bioreactor

A mathematical model is developed to simulate oxygen consumption, heat generation and cell growth in solid state fermentation (SSF). The fungal growth on the solid substrate particles results in the increase of the cell film thickness around the particles. The model incorporates this increase in the biofilm size which leads to decrease in the porosity of the substrate bed and diffusivity of oxygen in the bed. The model also takes into account the effect of steric hindrance limitations in SSF. The growth of cells around single particle and resulting expansion of biofilm around the particle is analyzed for simplified zero and first order oxygen consumption kinetics. Under conditions of zero order kinetics, the model predicts upper limit on cell density. The model simulations for packed bed of solid particles in tray bioreactor show distinct limitations on growth due to simultaneous heat and mass transport phenomena accompanying solid state fermentation process. The extent of limitation due to heat and/or mass transport phenomena is analyzed during different stages of fermentation. It is expected that the model will lead to better understanding of the transport processes in SSF, and therefore, will assist in optimal design of bioreactors for SSF.     

Spectrophotometric analysis as a complementary technique to aerobiology in the study of solid particles in the air

Hitherto, aerobiology has mainly focused on studying and analysing the biologic material found in the atmosphere (pollen grains and fungal spores), due to its allergologic and agricultural interest. However, the increase in respiratory tract problems caused by other solid particles suspended in the air has made studies including biologic and non-biologic material more frequent. This paper describes a simple technique based on spectrophotometric analysis. The results thus obtained can complement the outcome obtained by traditional aerobiologic techniques if the method is used properly and the equipment properly calibrated and an idea of the solid particles in the air can be obtained. The combination of both aerobiological and spectrophotometric analysis makes it possible to estimate the concentration of solid-particle material in the air, and we can also estimate the percentage of pollen grains and fungal spores among the general spectrum of particles. The results also show that a great part of the solid particulates originate from human activities. However, it was also observed that they emerge from natural sources as such as fungi and higher plants.     

Estimating cell concentration in the presence of suspended solids: a light scatter technique

A novel sensor was developed, based on light scatter, to estimate the cell concentration in the presence of suspended solids. The light scatter properties of cells in the presence of suspended solids were investigated. Two crucial observations were made: first, that the light scatter from cells is essentially a linear function of cell concentration and, second, that invariant regions are present in the light scatter spectrum of cell/solid substrate mixtures. Invariant regions are wavelength intervals of the light scatter spectrum in which the light scatter reading is independent of solid substrate concentration and only a function of cell concentration. The occurrence of invariant regions is the key behavior which allowed the quantification of cell concentration in the presence of suspended solids.An algorithm was developed for the estimation, from light scatter data, of cell concentration in the presence of solid substrate. The light scatter approach was validated by comparing cell concentrations estimated by this technique to those obtained from DNA and carbon dioxide evolution rate measurements during a series of fermentations. The model system used was Bacillus subtilis var sakainensis ATCC 21394 growing on fishmeal as the sole nitrogen source.A model was developed based on the interactions of scatter and absorbance. This model reflects the hypothesis that invariant regions are caused by changes in the absorbance of the solid substrate as a function of wavelength. (c) 1992 John Wiley & Sons, Inc.     

Arginine Inhibits Adsorption of Proteins on Polystyrene Surface

Nonspecific adsorption of protein on solid surfaces causes a reduction of concentration as well as enzyme inactivation during purification and storage. However, there are no versatile inhibitors of the adsorption between proteins and solid surfaces at low concentrations. Therefore, we examined additives for the prevention of protein adsorption on polystyrene particles (PS particles) as a commonly-used material for vessels such as disposable test tubes and microtubes. A protein solution was mixed with PS particles, and then adsorption of protein was monitored by the concentration and activity of protein in the supernatant after centrifugation. Five different proteins bound to PS particles through electrostatic, hydrophobic, and aromatic interactions, causing a decrease in protein concentration and loss of enzyme activity in the supernatant. Among the additives, including arginine hydrochloride (Arg), lysine hydrochloride, guanidine hydrochloride, NaCl, glycine, and glucose, Arg was most effective in preventing the binding of proteins to PS particles as well as activity loss. Moreover, even after the mixing of protein and PS particles, the addition of Arg caused desorption of the bound protein from PS particles. This study demonstrated a new function of Arg, which expands the potential for application of Arg to proteins.     

Electrical properties and interaction with silicon dioxide particles of Bacillus subtilis cells

It was shown that, depending on the quantity of phosphate in cultivation media, the surface layer of the Bacillus subtilis cell wall can mainly consist of either teichoic or teichuronic acids. It was found that the addition of silicon dioxide particles to the bacterial suspension induces an increase in the negative charge of the cell. This cell response to contact interaction with solid particles is observed in bacteria whose surface components are teichoic acids.     

Direct and Rapid Analysis of the Adhesion of Bacteria to Solid Surfaces: Interaction of Fluorescently Labeled Rhodococcus Strain GIN-1 (NCIMB 40340) Cells with Titanium-Rich Particles

A fluorimetric assay which enables direct and accurate analysis of the adhesion of bacteria to solid particles was developed. The assay is based on labeling of the bacteria with fluorescamine, which reacts with primary amino groups on the cell surface to yield a yellow fluorescence that is easily detectable by both fluorescence microscopy and spectrofluorimetry. As an example, fluorescent labeling of Rhodococcus strain GIN-1 (NCIMB 40340) cells enabled the detection and quantitative determination of their adsorption to TiO(inf2) and coal fly ash particles. Exposure of the cells to 10% acetone during the labeling reaction affected neither their viability nor their ability to adhere to these particles. Only a small fraction (^sim2%) of the total cell protein was labeled by fluorescamine upon staining of intact bacterial cells, which may indicate preferential labeling of certain proteins. Specificity studies carried out with the fluorescence assay confirmed previous findings that Rhodococcus strain GIN-1 cells possess high affinities for TiO(inf2), ZnO, and coal fly ash and low affinities for other metal oxides. In principle, the newly developed fluorimetric assay may be used for determination of cell adhesion to any solid matrix by either microscopic examination or epifluorescence measurements. In the present work, the adhesion of several other microorganisms to TiO(inf2) particles was tested as well, but their ability to adhere to these particles was significantly lower than that of Rhodococcus strain GIN-1 cells.     

Rheology of corn stover slurries at high solids concentrations--effects of saccharification and particle size

The rheological characteristics of untreated and dilute acid pretreated corn stover (CS) slurries at high solids concentrations were studied under continuous shear using plate-plate type measurements. Slurry rheological behavior was examined as a function of insoluble solids concentration (10-40%), extent of pretreatment (0-75% removal of xylan) and particle size (-20 and -80 mesh). Results show that CS slurries exhibit shear-thinning behavior describable using a Casson model. Further, results demonstrate that the apparent viscosity and yield stress increase with increasing solids concentration (which corresponds to a decrease in free water). Dilute acid pretreatment leads to lower viscosity and yield stresses at equivalent solids concentrations, as does smaller particle size. Taken together, these findings are consistent with the hypothesis that the availability of free water in the slurry plays a significant role in determining its rheological behavior. In particular, as the free water content of the slurry decreases, e.g., with increasing solids concentration, the greater interaction among particles likely increases the apparent viscosity and yield stress properties of the slurry. The results also suggest that the availability of free water, and thereby slurry rheological properties, depend on the chemical composition of the corn stover as well as its physical characteristics such as particle size and porosity. Hydrophilic polymers within the cell wall, such as xylan or pectin, or larger pores within bigger particles, facilitate sequestration of water in the solid phase resulting in decreased availability of free water. Thus, dilute acid pretreated slurries, which contain smaller size particles having significantly lower xylan content than slurries of untreated milled stover, exhibit much lower viscosities and yield stresses than untreated slurries containing large particles at similar solid concentrations.     

Distribution of the Receptor Sites for Sindbis Virus on the Surface of Chicken and BHK Cells

Sindbis virus was adsorbed to chicken cells or to BHK cells, and the distribution of virus over the surface of the cell was examined by electron microscopy of surface replicas. The distribution of virus particles on the cell was used to indicate the position of virus receptors at the cell surface. When purified Sindbis virus was adsorbed at 37 C to cells prefixed with glutaraldehyde, the virus particles were evenly distributed over the surface of most cells. There was a large variability from cell to cell, however, in the number of virus particles adsorbed, and regions with different concentrations of virus particles were sometimes observed on the same cell. The concentration of virus receptors observed varied from 20 to 160/mum(2) of cell surface, and, thus, the total number of virus receptors per chicken cell is on the order of 10(5). When virus was adsorbed to unfixed cells at 4 C, the virus particles were clustered into aggregates varying from a few particles to large crystalline arrays (the latter seen only in chicken cells). These conditions are apparently conducive to virus aggregation, and this, coupled with free lateral diffusion of the virus-receptor complex in the cell membrane at 4 C, leads to the observed clustering.     

Fibrillar pectin and contact cation exchange at the root surface

It is postulated that there is an extracellular apparatus on root surfaces which can remove nutrient cations from clay particles by contact ion exchange. This apparatus is composed of distinctive rhizoplane fibrils each of which is composed at least in part of pectin. A tuft of rhizoplane fibrils embedded proximally in a cell wall can give rise to a meshwork distally which makes extensive and intimate contacts with soil particles. The existence of these fibrils has revived interest in interpreting the series of events involved in movement of cations into roots directly from the solid phase of the soil, beginning with the contact cation exchange process. Accepted concepts from soil chemistry and solid state physics are employed to analyze possible roles of pectin fibrils in cation mobilization at and near the rhizoplane (root surface).     

Ultrastructural study of protein kinase C-betaII localization during the cell cycle of human glioma cells

Transmission electron microscopy and immunogold labeling were used to determine how PKC-betaII is localized at stages in the cell cycle of the human glioma cell line U-373MG. Results show that immunogold particles in both dimethylsulfoxide (DMSO) and calphostin C (0.5 microM)-treated cells were mainly located in the cytoplasm. The concentration of gold particles in the nucleus was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-betaII during the cell cycle. The concentration of gold particles in the DMSO-treated cells was constant until 8 h. Subsequently, cytoplasmic PKC-betaII oscillated with an increased at 10 h, a rapid decrease at 12 h, and a rise at 14 h. The concentration of the gold particles then gradually decreased. In contrast, immunogold labeling in calphostin C-treated cells increased gradually up to 10 h. Subsequently, the pattern of PKC-betaII labeling in calphostin C-treated cells recapitulated those of control cells as seen by the rapid decline of PKC-betaII labeling at 12 h and its re-accumulation at 14 h. Additionally, there was a rapid increase at 20 h. Western blots of PKC-betaII showed constant PKC-betaII immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-II immunoreactivity at 10 h and 14 h. This technique may represent intracellular immunoreactivity of PKC-betaII. The results from the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-betaII provokes a conformation change in PKC-betaII, preventing its activation and degradation.     

THE ADHESIVENESS OF LEUCOCYTES TO SOLID SURFACES

1. Methods are described for measuring the stickiness of cells to solid surfaces. 2. The effect of various factors such as temperature, serum concentration, sodium chloride concentration, etc., on the adhesiveness of leucocytes to solid surfaces and the phagocytosis of solid particles are compared, and certain marked differences pointed out. 3. On account of these differences it is concluded that forces of surface tension, though undoubtedly operative in determining the behavior of cells in contact with solid bodies, are not the only factors involved. 4. Ways in which changes in the structure or rigidity of the protoplasm might affect phagocytosis and adhesiveness in opposite directions are suggested.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : IV. VISCOSITY—Continued.

1. The proof is completed that the influence of electrolytes on the viscosity of suspensions of powdered particles of gelatin in water is similar to the influence of electrolytes on the viscosity of solutions of gelatin in water. 2. It has been suggested that the high viscosity of proteins is due to the existence of a different type of viscosity from that existing in crystalloids. It is shown that such an assumption is unnecessary and that the high viscosity of solutions of isoelectric gelatin can be accounted for quantitatively on the assumption that the relative volume of the gelatin in solution is comparatively high. 3. Since isoelectric gelatin is not ionized, the large volume cannot be due to a hydration of gelatin ions. It is suggested that this high volume of gelatin solutions is caused by the existence in the gelatin solution of submicroscopic pieces of solid gelatin occluding water, the relative quantity of which is regulated by the Donnan equilibrium. This would also explain why the influence of electrolytes on the viscosity of gelatin solutions is similar to the influence of electrolytes on the viscosity of suspensions of particles of gelatin. 4. This idea is supported by experiments on solutions and suspensions of casein chloride in which it is shown that their viscosity is chiefly due to the swelling of solid particles of casein, occluding quantities of water regulated by the Donnan equilibrium; and that the breaking up of these solid particles into smaller particles, no longer capable of swelling, diminishes the viscosity. 5. This leads to the idea that proteins form true solutions in water which in certain cases, however, contain, side by side with isolated ions and molecules, submicroscopic solid particles capable of occluding water whereby the relative volume and the viscosity of the solution is considerably increased. This accounts not only for the high order of magnitude of the viscosity of such protein solutions but also for the fact that the viscosity is influenced by electrolytes in a similar way as is the swelling of protein particles. 6. We therefore reach the conclusion that there are two sources for the viscosity of protein solutions; one due to the isolated protein ions and molecules, and the other to the submicroscopic solid particles contained in the solution. The viscosity due to the isolated molecules and ions of proteins we will call the general viscosity since it is of a similar low order of magnitude as that of crystalloids in solution; while the high viscosity due to the submicroscopic solid protein particles capable of occluding water and of swelling we will call the special viscosity of protein solutions. Under ordinary conditions of hydrogen ion concentration and temperature (and in not too high a concentration of the protein in solution) the general viscosity due to isolated ions and molecules prevails in solutions of crystalline egg albumin and in solutions of metal caseinates (where the metal is monovalent) while under the same conditions the second type of viscosity prevails in solutions of gelatin and in solutions of acid-salts of casein; and also in solutions of crystalline egg albumin at a pH below 1.0 and at higher temperatures. The special viscosity is higher in solutions of gelatin than of casein salts for the probable reason that the amount of water occluded by the submicroscopic solid gel particles in a gelatin solution is, as a rule, considerably higher than that occluded by the corresponding particles of casein.     

Effect of biologically produced sulfur on gas absorption in a biotechnological hydrogen sulfide removal process

Absorption of hydrogen sulfide in aqueous suspensions of biologically produced sulfur particles was studied in a batch stirred cell reactor, and in a continuous set-up, consisting of a lab-scale gas absorber column and a bioreactor. Presence of biosulfur particles was found to enhance the absorption rate of H(2)S gas in the mildly alkaline liquid. The mechanism for this enhancement was however found to depend on the type of particles used. In the gently stirred cell reactor only small hydrophilic particles were present (d(p) < 3 microm) and the enhancement of the H(2)S absorption rate can be explained from the heterogeneous reaction between dissolved H(2)S and solid elemental sulfur to polysulfide ions, S(x) (2-). Conditions favoring enhanced H(2)S absorption for these hydrophilic particles are: low liquid side mass transfer (k(L)), high sulfur content, and presence of polysulfide ions. In the set-up of gas absorber column and bioreactor, both small hydrophilic particles and larger, more hydrophobic particles were continuously produced (d(p) up to 20 microm). Here, observed enhancement could not be explained by the heterogeneous reaction between sulfide and sulfur, due to the relatively low specific particle surface area, high k(L), and low [S(x) (2-)]. A more likely explanation for enhancement here is the more hydrophobic behavior of the larger particles. A local increase of the hydrophobic sulfur particle concentration near the gas/liquid interface and specific adsorption of H(2)S at the particle surface can result in an increase in the H(2)S absorption rate.     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Biological properties of the phage-tail-like particles from Myxococcus coralloides D

Abstract Electron microscopy of preparations of the Myxocococcus coralloides D autolytic supernatants revealed the presence of phage-tail-like particles. The particles consisted of a core, a contractile sheath and a baseplate with fibers. The induction of the defective prophage occurred when the cultures reached the stationary phase, but the events occurring during induction did not lead to cell lysis. Particles nerver appeared during exponential growth. They were not observed when M. coralloides D grew on solid media, either. Purified samples of phage tails were able to inhibit most of the myxobacterial strains which were tested when the particles were in the extended state, but they were unable to multiply on the sensitive strains.     

Preferential attachment of membrane glycoproteins to the cytoskeleton at the leading edge of lamella

The active forward movement of cells is often associated with the rearward transport of particles over the surfaces of their lamellae. Unlike the rest of the lamella, we found that the leading edge (within 0.5 microns of the cell boundary) is specialized for rearward transport of membrane-bound particles, such as Con A-coated latex microspheres. Using a single-beam optical gradient trap (optical tweezers) to apply restraining forces to particles, we can capture, move and release particles at will. When first bound on the central lamellar surface, Con A-coated particles would diffuse randomly; when such bound particles were brought to the leading edge of the lamella with the optical tweezers, they were often transported rearward. As in our previous studies, particle transport occurred with a concurrent decrease in apparent diffusion coefficient, consistent with attachment to the cytoskeleton. For particles at the leading edge of the lamella, weak attachment to the cytoskeleton and transport occurred with a half-time of 3 s; equivalent particles elsewhere on the lamella showed no detectable attachment when monitored for several minutes. Particles held on the cell surface by the laser trap attached more strongly to the cytoskeleton with time. These particles could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/- 14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere on the lamella. Fluorescent succinylated Con A staining showed no corresponding concentration of general glycoproteins at the leading edge, but cytochalasin D-resistant filamentous actin was found at the leading edge. Our results have implications for cell motility: if the forces used for rearward particle transport were applied to a rigid substratum, cells would move forward. Such a mechanism would be most efficient if the leading edge of the cell contained preferential sites for attachment and transport.