Determination of the permeability and porosity of anaerobic sludge granules by size exclusion chromatography

Summary A new application of size-exclusion chromatography is described for assessment of the permeability and internal pore distribution of anaerobic sludge granules. The fractionation range and adsorption characteristics were investigated for a series of standard proteins and dextrans. To determine possible adsorption of solutes and stability of the sludges, the pH and salt concentration of the mobile phase were varied. Good results were obtained using dextrans as solutes and tap water as the mobile phase. To inhibit the sludge activity without affecting the granule characteristics the experimental arrangement was operated at 4°C. Three granular sludge types were investigated. The permeability of the granular sludges varied from 7% to 96%. The exclusion limit expressed as molecular mass also showed large differences. For two sludges, molecules greater than 80 000 Da cannot penetrate the pores; for one sludge the exclusion limit is 1300 Da. Experiments using acetic acid as an indicator of permeability gave corresponding results.Offprint requests to: P. A. Alphenaar     

On the use of size exclusion chromatography for the resolution of mixed amyloid aggregate distributions: I. equilibrium partition models

In this study, we investigated the theoretical potential of size exclusion chromatography (SEC) for resolving mixtures of protein aggregates (of various sizes and shapes) produced in the generation of amyloid fibrils. We present our findings in the form of an equilibrium partition model. We first review the general characteristics of SEC and discuss the physicochemical features affecting solute transport and partition. We then develop new methods for estimating the transport and partition coefficients of protein aggregates on the basis of their molecular dimensions and the SEC column properties. We detail how these calculated properties can be used to estimate the likely resolving power of an SEC column. Model predictions were found to be in general agreement with experimental data gained from the measurement of the elution profile of sheared amyloid fibrils prepared from bovine insulin and passed through a Superose 6 precision SEC column. Our formalism should provide a basic appreciation of the competing factors at work and allow an informed choice to be made for optimal selection of SEC column medium to separate a desired size range of aggregate.     

Relationship between size and mass transfer resistance in aerobic granules

AIMS: To investigate the size effect of aerobic granules on mass transfer efficiency by introducing the effective factor and the modified Thiele modulus. METHODS AND RESULTS: Batch experiments of aerobic granules with different sizes were conducted to study the size effect of granules on mass transfer resistance. Results showed that both specific substrate removal and biomass growth rates were size dependent, i.e. reduced rates were observed at big sizes. It was found that the diffusion resistance described by the effective factor and the Thiele modulus increased with the increase of the size of aerobic granules. CONCLUSIONS: The effective factor should be controlled at values higher than 0.44 and the Thiele modulus lower than 1.05 for efficient mass transfer in aerobic granules. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the coupled effective factor and Thiele modulus, an operation guidance including granule radius, kinetics of biomass and environmental conditions could be proposed for stable aerobic granulation.     

Solute exclusion from cellulose in packed columns: process modeling and analysis

In this investigation, process modeling and analysis were used to explore the behavior of solute exclusion from cellulose in packed columns. The study focused on modeling the effects of dispersion, mass transport, and pore diffusion. Three mathematical models were used to predict the behavior of the columns: an equilibrium model, a mass transfer model, and a combined mass transfer and pore diffusion model. Computer implementations of these models were tested against experimental conditions where cellulose particle size and solution velocity were used to either amplify or minimize dispersion or skewness in the elution curves. For small cellulose particles (200-300 mesh), all three models accurately predicted the shape of the elution curve and the particle porosity. For larger particles (45-60 mesh), the mass transfer model and the combined mass and pore diffusion model best represented the behavior of the column. At high solution velocities (0.63 cm(3) min(-1)) and large particles, only the combined mass transfer and pore diffusion model accurately represent the column behavior. Sensitivity analysis revealed that the mass transfer coefficient had little effect on the elution curves for the range of values (10(-6)-10(-3) cm s(-1)) calculated from the experimental data. The combined mass transfer and pore diffusion model presented in this article can be used to design solute exclusion measurement experiments for the larger cellulose particles found in a commercial cellulose-to-ethanol plant.     

Diffusivity of oxygen in aerobic granules

This work for the first time estimated apparent oxygen diffusivity (D(app)) of two types of aerobic granules, acetate-fed and phenol-fed, by probing the dissolved oxygen (DO) level at the granule center with a sudden change in the DO of the bulk liquid. With a high enough flow velocity across the granule to minimize the effects of external mass transfer resistance, the diffusivity coefficients of the two types of granules were estimated with reference to a one-dimensional diffusion model. The carbon source has a considerable effect on the granule diameter (d) and the oxygen diffusivity. The diffusivity coefficients were noted 1.24-2.28 x 10(-9) m2/s of 1.28-2.50 mm acetate-fed granules, and 2.50-7.65 x 10(-10) m2/s of 0.42-0.78 mm phenol-fed granules. Oxygen diffusivity declined with decreasing granule diameter, in particular, the diffusivity of acetate-fed granules is proportional to the size, whereas the diffusivity of phenol-fed granules is proportional to the square of granule diameter. The existence of large pores in granule, evidenced by FISH-CLSM imaging, was proposed to correspond to the noted size-dependent oxygen diffusivity. The phenol-fed granules exhibited a higher excellular polymer (ECP) content than the acetate-fed granules, hence yielding a lower oxygen diffusivity.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands. A quantitative immunocytochemical approach

Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/micron2) was almost 3 times as high as that of ACTH. In the mature granules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules' antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

The granule size distribution in an anammox-based granular sludge reactor affects the conversion--implications for modeling

Mathematical models are useful tools to optimize the performance of granular sludge reactors. In these models, typically a uniform granule size is assumed for the whole reactor, even though in reality the granules follow a size distribution and the granule size as such affects the process performance. This study assesses the effect of the granule size distribution on the performance of a granular sludge reactor in which autotrophic nitrogen removal is realized through one-stage partial nitritation-anammox. A comparison is made between different approaches to deal with particle size distributions in one-dimensional biofilm models, from the use of a single characteristic diameter to applying a multiple compartment model. The results show a clear impact on the conversion efficiency of the way in which particle size distribution is modeled, resulting from the effect of the granule size on the competition between nitrite oxidizing and anammox bacteria and from the interaction between granules of different sizes in terms of the exchange of solutes. Whereas the use of a uniform granule size is sufficient in case only the overall reactor behavior needs to be assessed, taking into account the detailed granule size distribution is required to study the solute exchange between particles of different sizes. For the latter purpose, the application of the widespread software package Aquasim is limited and the development of dedicated software applications is required.     

Preparation and characterization of soluble starches having different molecular sizes and composition,by acid hydrolysis in different alcohols

Potato and waxy-maize starches were separately modified for 1 h at 65° with 0.36% hydrochloric acid in methanol, ethanol, 2-propanol, and 1-butanol. All of the modified starches were readily soluble in hot water, to give crystal-clear solutions up to a concentration of at least 20% (w/v). The modified granules were studied by light-microscopy and iodine-iodide staining. All of the modified starches retained their granule appearance, although with various degrees of damage that progressively increased from methanol to 1-butanol. Both hydrolysis and alcoholysis occurred, but to different extents in the different alcohols. The highest proportion of alcoholysis occurred in methanol where 50% of the resulting molecules were glycosides, the lowest in 1-butanol where 6% were glycosides. The number-average molecular weights of the modified starches also progressively decreased from 126,670 for the methanol-modified waxy-maize starch to 4,750 for the 1-butanol-modified potato starch. The methanol- and ethanol-modified potato starches were fractionated into amylose and amylopectin components. The 2-propanol- and 1-butanol-modified potato starches gave only an amylopectin component. The amylose components were characterized by gel-permeation chromatography on Bio-Gel A-5m, and the amylopectin components, on Bio-Gels A-150m and A-0.5m. The molecular sizes of the amylose and amylopectin components progressively decreased from methanol- to 1-butanol-modified starches. Furthermore, the polymodal composition of the amylopectin component was decreased to give a more homogeneous product. Waxy-maize starch was modified in methanol and 2-propanol and gave products that were of lower molecular size and more homogeneous than the polymodal native starch. It is shown that the differential effect of the different alcohols on the modification of the starch granules is produced by effecting different concentrations of acid inside the granule, where hydrolysis occurs in the 10–12% of water contained in the granule. It is postulated that 2-propanol and 1-butanol dissolve the double-helical, crystalline regions in the starch granule to give different types of products under otherwise identical conditions of modification.     

The influence of substrate transport limitation on porosity and methanogenic activity of anaerobic sludge granules

The relationship between porosity, diameter and methanogenic activity of anaerobic granules has been investigated. Experiments with different granular sludges revealed that substrate transport limitations increase with the diameter of the granules. As a consequence, autolysis can occur in the core of the granule, producing hallow granules. The porosity measurements revealed that the hollow centre is not available for substrate transport. Possibly as an effect of bacterial lysis, the porosity decreases in the more interior layers of the granules. This results in a inactive inner part of the large granules, which is not involved in the treatment process; the specific methanogenic activity decreases with granule size. No marked difference in substrate affinity is observed between granules of different sizes, which probably indicates that for large granules only the exterior is biological active. Correspondence to: G. Lettinga     

Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells

Using patch-clamp techniques, we have followed the attributes of the secretory granules of peritoneal mast cells obtained from rats of different ages. The granule attributes were determined by following the step increases in the cell surface membrane area caused by the exocytosis of the granules in GTP gamma S stimulated mast cells. Our data show that the amount of granule membrane available for exocytosis depends exponentially on the weight (age) of the donor rat, reaching a maximum at approximately 300 g. The data are consistent with an exponential growth in the number of granules contained by mast cells of maturing animals. Histograms of the sizes of the step increases in surface area caused by exocytosis of the granules showed at least four equally spaced peaks of similar variance where the position of the first peak and the spacing between peaks averaged 1.3 +/- 0.4 micron2. In all cells recorded, no more than seven peaks could be found, the higher order peaks having a lower probability of occurrence. The distribution of granule sizes did not change measurably between young and adult animals. This study suggests that at least two separate steps may determine the size of a secretory granule: granule to granule fusion that may account for the subunit composition of granule sizes and traffic of microvesicles through the maturing granules that may account for the variance observed in the granule sizes. This study also demonstrates a novel way to study granulo-genesis in living cells.     

Stepwise maturation of lytic granules during differentiation and activation of human CD8+ T lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1(+) lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands

Summary Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/m²) was almost 3 times as high as that of ACTH. In the mature igranules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

Human beta-adrenergic receptors. Simultaneous purification of beta 1- and beta 2-adrenergic-receptor peptides.

Monoclonal antibodies to insulin secretory granule membranes were obtained following immunization of mice with granule membranes purified from a rat transplantable insulinoma. The specificities of the antibodies were investigated by using binding assays with different insulinoma subcellular fractions, by indirect immunofluorescence studies with intact and permeabilized cells, and by immunoblotting of granule membrane proteins fractionated by SDS/polyacrylamide-gel electrophoresis. Fifty-six antibodies were characterized initially, and 21 representative cell lines were cloned. The antibodies fell into four categories: (1) binding preferentially to secretory granules, and reacting with a component of approx. 80,000 Da on immunoblots (antigen designated SGM 80); (2) binding preferentially to secretory granules, and reacting with components of approx. 110,000 and 50,000 Da on immunoblots (antigen designated SGM 110); (3) binding preferentially to secretory granules but unreactive on immunoblots; (4) binding to membrane antigen(s) with a widespread intracellular distribution which included granules and plasma membranes. The antigens SGM 80 and SGM 110 were studied in more detail and both were shown to be integral membrane glycoproteins with antigenic determinants located on the internal face of the secretory granule membrane. These antigens were also present in normal rat islets of Langerhans and similar components were detected by immunoblotting in secretory granules from anterior pituitary and adrenal medulla. Proteins which were immunologically related to SGM 80 and SGM 110, but distinct in molecular size, were also identified in liver. It is concluded that secretory granules contain specific components which are restricted in subcellular location but widespread in tissue distribution. The antibodies obtained will be valuable reagents in the further investigation of the biogenesis and turnover of insulin secretory granules.     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.     

Determination of molecular mass of the aroid alternative oxidase by radiation-inactivation analysis.

The functional molecular mass of the cyanide-resistant salicylhydroxamate-sensitive duroquinol oxidase activity from Sympocarpus foetidus (skunk cabbage) and Sauromatum guttatum spadix mitochondria was determined by radiation-inactivation analysis. The functional molecular mass for the oxidase activity was found to be 26,700 Da for skunk cabbage and 29,700 Da for Sauromatum guttatum mitochondria frozen at -70 degrees C. Irradiation of dried mitochondrial samples resulted in a larger target size of 38,000 Da, and in some cases, a stimulation of activity at low dose of radiation. The functional molecular mass of cytochrome c oxidase activity from skunk-cabbage and bovine heart mitochondria was also investigated. Dried and frozen mitochondrial samples from both species yielded similar target sizes, in the range 70,900-73,400 Da. Purified bovine heart cytochrome c oxidase was also irradiated, and yielded a functional molecular mass of 66,400 Da. The target size of cytochrome c oxidase agrees with literature values insofar as the target size is considerably smaller than the molecular mass of the entire complex.     

Zymogen granule size in pancreas of nursing rats

Dramatic depression in granule volume density and size was measured in acinar cells of postnatal rat pancreas following the initiation of feeding. Volume density decreased about threefold from 45% at birth to 16% 2 days thereafter. Mean granule diameter decreased from 1.50 micron to 0.80 micron, an 85% decrease in corresponding granule volume. At the same time, numerical density approximately doubled. At 2 days after birth, cells with smaller granules had lower volume densities, and differences in mean granule volume between cells accounted for most of the differences in volume density. Although the distribution of granule diameter in newborns was lognormal, the distribution at 2 days was heavily skewed to larger sizes. This was the result of skewed distributions within individual cells and not an artifact of sampling. The results corroborate the central role of granule volume in determining changes in the volume density of zymogen granules in the pancreas and suggest that zymogen granules can act as capacitors that can change size as a function of the enzyme contained within.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Solute exclusion from cellulose in packed columns: experimental investigation and pore volume measurements

A packed column approach was used in this investigation to determine pore volume and surface area distributions of several celluloses. Specific surface areas for Avicel PH 102, Solka Floc BW 300, and two size fractions of corn cobs that have been pretreated to remove lignin and hemicellulose were measured using this technique. In addition to measuring pore volume and specific surface area, the molecular diameters of several PEGs (polyethylene glycol) were estimated using viscosity measurements. Also, the influence of cellulose particle size, molecular diameter of PEGs, and PEG solution velocity on dispersion and tailing were investigated. Molecular diameter estimates from this investigation were 30%-35% lower than those reported in the literature. This discrepancy is due to earlier investigators using an inappropriate relationship for estimating molecular diameter from viscosity measurements. The precision of the column approach to solute exclusion was higher than that obtained by investigators using a batch approach. Dispersion increased with increasing particle size. Tailing of the elution curve was increased with increasing solute molecular diameter and elution rate. For a cellulase with a molecular diameter of 5.1 nm, estimated specific surface area ranged from 7.2 to 10.5 m(2) g(-1).     

A novel method to measure self-association of small amphipathic molecules: temperature profiling in reversed-phase chromatography

Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the self-association of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5-80 degrees C during gradient elution reversed-phase high-performance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution.