The use of a new mobile phase,with no multivalent cation binding properties,to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment

The fingerprints of extracellular polymeric substances (EPS) extracted from different types of biomass used for wastewater treatment (i.e., activated sludge, filamentous activated sludge, anaerobic granular sludge, anaerobic flocculated sludge) were studied by size exclusion chromatography (SEC) with Amersham Biosciences Superdex 200 10/300 GL column with a theoretical resolving range of 10–600 kDa. A new mobile phase, which does not display binding properties for multivalent cations, was previously optimized. This mobile phase contained 75 mM Hepes buffer at pH 7 with 15% acetonitrile (v/v) and was selected to minimize ionic and hydrophobic interactions between the molecules that make up the EPS and the column packing.When EPS extracted from similar sludges is analyzed using different mobile phases, the number of chromatographic peaks obtained is quite similar, and differences are mainly observed in the relative absorbance of the chromatographic peaks. However, very different chromatograms (number and relative absorbance of chromatographic peaks) are obtained for EPS extracted from different types of sludges. Furthermore, when dysfunctions, such as filamentous bulking in the activated sludge, occur in a bioreactor, they also induce strong variations in chromatographic profiles.     

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g⁻¹ of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.     

Sorption of Cd(II) and Pb(II) by exopolymeric substances (EPS) extracted from activated sludges and pure bacterial strains: Modeling of the metal/ligand ratio effect and role of the mineral fraction

The present study deals with the sorption of Cd(II) and Pb(II) by exopolymeric substances (EPS) extracted from activated sludges or pure bacterial strains. The percentage of sorbed metal increases with the concentration of the EPS–water solution. Pb(II) always presents a higher affinity than Cd(II) for EPS. For the EPS extracted from pure bacterial strains, only one global binding constant from a simple equilibrium sorption model, may be used to assess the effect of microbial products such as EPS on Cd(II) and Pb(II) speciation or mobility in the environment. However, for EPS extracted from activated sludges, the wide variation of the global binding constants determined for Cd(II) and Pb(II) do not permit such a simple approach. The differences in sorption to metals between the two types of EPS (bacterial, activated sludges) could be explained by the differences in EPS composition: organic macromolecules, as well as the nature of the mineral fraction.     

Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods

The efficiency of eight extracellular polymeric substances (EPS) extraction methods was compared on two different activated sludges. Three chemical methods (EDTA, formaldehyde + NaOH, glutaraldehyde), four physical methods (sonication, cation exchange resin, sonication + cation exchange resin, heating) and a control method (centrifugation alone) were tested.EPS quantities extracted were more greater for chemical methods than those for physical methods. For the chemical methods used EPS contamination due to extracting reagents was pointed out by infra-red analysis. The EPS extracted by physical methods can show a different qualitative composition with protein and carbohydrate as predominant compounds. This study therefore underlines that the choice of EPS extraction method should not only be limited to extraction yield and nucleic acid content but should also consider that the EPS solution may be contaminated by extracting reagents and/or be greatly modified by the extraction protocol.     

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.     

Hydrophobic features of EPS extracted from anaerobic granular sludge: an investigation based on DAX-8 resin fractionation and size exclusion chromatography

The hydrophobic fractionation of extracellular polymeric substances (EPS) extracted from anaerobic granular sludge was performed on the DAX-8 resin (two elution pH conditions, i.e., pH 2 and pH 5 were tested). The impact of seven different EPS extraction methods on EPS hydrophobicity features was assessed. The results showed that the extraction methods and bulk solution pH influenced dramatically the biochemical composition of the EPS, and in turn, the hydrophobicity determined. Besides, EPS extracting reagents i.e., formaldehyde, ethanol, sodium dodecyl sulfate (SDS), and Tween 20 not only introduced extra carbon content in the total organic carbon (TOC) measurement but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of untreated and pH-adjusted EPS samples, more complete EPS aMW information was preserved at pH 5. Thus, elution at pH 5 was preferred in this study for the qualitative analysis of EPS hydrophobic features. The hydrophobic fraction of EPS retained by the resin at pH 5 was ascribed to a wide aMW range, ranging from >440 to 0.3 kDa. Within this range, EPS molecules ranging from 175 to 31 kDa were mostly retained by the DAX-8 resin, which indicates that these EPS molecules are highly hydrophobic.

     

Effect of sulfate and iron on physico-chemical characteristics of anaerobic granular sludge

This research investigated the effect of the substrate composition (no substrate, glucose, glucose + sulfate or glucose + sulfate + iron) on the physico-chemical characteristics of two different anaerobic granular sludges as a function of time. The sludges were fed batch wise (pH 7, 30 °C) at an organic loading rate of 1.2 g COD l⁻¹ d⁻¹ (0.04 g COD g VSS⁻¹ day⁻¹) for 30 days. The presence of sulfate (COD/sulfate ratio = 1) in the feed of glucose fed anaerobic sludges did not change the physico-chemical characteristics throughout the incubation. In contrast, the presence of iron in the feed (in addition to glucose and sulfate, COD/iron ratio = 1) reduced the protein and carbohydrate content in the SMP and EPS with about 50% after 30 days incubation compared to the other feeding conditions. The sludge grown on glucose + sulfate + iron contained much more iron (+300–500%) and sulfur (+200–350%) than the other incubated sludges both after 14 and 30 days. The higher mineral content (lower VSS content) and the decrease of the EPS content contributed to the disintegration of iron fed granules, as shown by their lower size particles. However, the iron fed sludge displayed a higher granule strength than the other incubated sludges. Although an appreciable variation in the granule strength was noticed between the sludges investigated, it was not possible to relate these differences to their inorganic composition, the chemical composition of the extracted polymers or to the physical characteristics investigated.     

EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR)

Membrane bioreactors (MBR) technology for wastewater offers many advantages over conventional technologies such as high effluent quality, less footprint and others. The main disadvantage of membrane bioreactors (MBR) is related to membrane fouling, which is mainly caused by extracellular polymeric substance (EPS) and soluble microbial products (SMP). This research studied EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR). The SAMBR was operated under two organic loading rates (OLR) (0.79 and 1.56 kg/m³ d) and was fed with synthetic wastewater with glucose as the carbon source. The results showed percentages of chemical oxygen demand (COD) removal above 95% and the highest COD removal rates were observed at the bottom of the reactor (>83%) for both OLR. The EPS showed a stratification with highest quantities in the supernatant. For the SMP the highest concentration was in the bottom of SAMBR where utilization predominated associated products whereas in the SAMBR supernatant predominated biomass associated products. The OLR change led to a significant increase in SMP accumulation but not in EPS. These facts showed that EPS and SMP dynamic in the SAMBR seemed to be mainly influenced by biological activity, total suspended solids concentration and substrate composition.     

Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography-mass spectrometry (LC-MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS-PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60-70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.     

The production-influencing factors of extracellular polysaccharide (EPS) from a strain of lactic acid bacteria and EPS extraction

The influencing factors of extracellular polysaccharide (EPS) produced from a strain of lactic acid bacteria (LAB L15) were studied by using the phenol-H₂SO₄ method. It was demonstrated that the strain produced EPS at the most amount when it was incubated for 40–48 h and when the pH value was 4 under 30°C. Glucose was the most suitable carbon source for LAB-producing EPS. The rough EPS was obtained from L15 culture after centrifugation, dialysis, deprotein, decoloration, and ethanol-precipitation. The sample was at least composed of two polysaccharides that were completely different in molecular weight and the amount. The purified EPS was passed through the SephadexG-200 column and it showed that it was a sample purified by thin layer chromatography. __________ Translated from Microbiology, 2005, 32(4): 85–90 [译自: 微生物学通报, 2005, 32(4): 85–90]     

Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge

This study extracted the soluble microbial products and loosely bound and tightly bound extracellular polymeric substances (EPS) from suspended sludge from a membrane bioreactor, original and aerobically/anaerobically digested, and compared their fouling potentials on a microfiltration membrane. The resistance of cake layer accounts for 95–98% of the total filtration resistances when filtering the whole sludges, with anaerobically digested sludge presenting the highest resistance among the three tested sludges. The tightly bound EPS has the highest potential to foul the membrane; however, the loosely bound EPS contribute most of the filtration resistances of the whole sludges. The foulants corresponding to the irreversible fouling have chemical fingerprints similar to those from loosely bound EPS, which have a greater predilection to proteins and humic substances than to polysaccharides.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

Characteristics of extracellular polymeric substances (EPS) fractions from excess sludges and their effects on bioflocculability

Extracellular polymeric substances (EPS) of biological origin are ubiquitous in excess sludges and can be applied as an underlying bioflocculant, owing to their high content of macromolecules and cations. However, low flocculating activity limits the feasibility of their practical applications. This study provides a novel EPS fractionation approach to improve their flocculability by extracting an active EPS fraction and removing the others with low flocculability. The results showed that for two excess sludges (called sludge A and sludge B), the tightly bound EPS (TB-EPS) fraction possessed a high flocculating rate to kaolin suspension compared with the other EPS fractions [i.e., supernatant, slime, and loosely bound EPS (LB-EPS) fraction] (>54.1 ± 1.4% vs <7.8 ± 1.6%). High bioflocculability of TB-EPS fraction could be attributable to high contents of macromolecules (330–1200 kDa) and trivalent cations (Fe³⁺ and Al³⁺). Further investigation reveals that the TB-EPS fraction caused aggregation of particles by bridging and sweep flocculation.     

Solution properties of an exopolysaccharide from a Pseudomonas strain obtained using glycerol as sole carbon source

We report the solution properties of a new exopolysaccharide (EPS) obtained from a Pseudomonas strain fed with glycerol as the sole source of carbon. This high molecular mass (3 × 10⁶ g mol⁻¹) biopolymer is essentially made of galactose monomers with pyruvate and succinate groups imparting a polyelectrolyte character. The Smidsrod parameter B computed from the ionic strength dependence of the intrinsic viscosity indicates that the EPS backbone is rather flexible. In salt free aqueous solutions, the zero shear viscosity scaling with concentration follows a typical polyelectrolyte behavior in bad solvent, whereas at high ionic strength the rheological response is reminiscent from neutral polymers. Light scattering data indicate that the EPS adopts a globular conformation as a result of hydrophobic interactions. EPS solutions are stable within 4 days as particle sizing does not indicate EPS aggregation. Both globular conformation and stability against precipitation from solution are attributed to the low charge density of the polyelectrolyte.     

Extraction of extracellular polymeric substances (EPS) of sludges

The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1-1.2% of DNA in the sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For each gram of volatile solids, formaldehyde-NaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge (62% in the EPS extracted by formaldehyde-NaOH), whereas protein was predominant in the methanogenic sludge (41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated from confocal laser scanning microscopic observations, formaldehyde-NaOH extracted only a limited portion of EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm × 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)—acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Fractionation of phosphorus and trace elements species in soybean flour and common white bean seeds by size exclusion chromatography-inductively coupled plasma mass spectrometry

Soluble species of phosphorus, sulfur, selenium and eight metals (Mn, Fe, Co, Ni, Cu, Zn, Mo and Cd) in soybean flour and common white bean seeds were investigated by size exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). Samples were extracted by 0.02 mol l(-1) Tris-HCI buffer solution (pH 7.5). Fractionation of sample extracts by preparative scale SEC was accomplished using a Fractogel EMD BioSEC column (600 x 16 mm) and 0.02 mol l(-1) Tris-HCl buffer solution (pH 7.5) as mobile phase (flow rate: 2 ml min(-1)). A 2-ml sample was injected. Contents of elements in chromatographic fractions were determined by AAS, ICP-AES and ICP-MS. The elution profiles of P, Fe, Co, Ni, Cu, Zn and Mo in both samples were similar. Main species of Co, Ni, Cu, Zn and Mo were found in the low molecular weight region (2-5 kDa), whereas Fe is predominantly bound to high molecular weight compounds (180 kDa). The dominant phosphorus fraction was detected in the medium molecular weight region (10-30 kDa) and the other fraction in the low molecular weight region. Isotachophoretic analysis of chromatographic fractions revealed that the main phosphorus compound in the medium molecular weight region is phytic acid. SEC on Superdex 75 and Superdex Peptide columns (300 x 10 mm) was performed in on-line hyphenation with ICP-MS. The same mobile phase was used with a flow rate of 0.5 ml min(-1); volume of injected sample was 200 microl. Element specific chromatograms were obtained by continuous nebulization of effluent into ICP-mass spectrometer measuring intensities of 47(PO)+ and 48(SO)+ oxide ions and 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se, 95Mo and 114Cd nuclides. Chromatographic profiles of elements are generally analogous to those obtained with a Fractogel column, but better chromatographic resolution of separated species was achieved so that slight differences between samples were revealed. Estimated molecular weights of major phosphorus species in soybean flour and common white bean seed extracts are 6 and 3.6 kDa, respectively, whereas those of minor phosphorus species in both samples are 0.7 kDa. Traces of phosphorus were also detected in the high molecular weight region (130 kDa). Chromatograms of P, Ni, Cu, Zn and Mo compounds in both extracts are similar but not identical. Molecular weights of major Cu and Zn species are approximately 1 and 0.4 kDa for soybean flour and white bean seeds, respectively. In cases of Mn, Fe, Co and Se, the element profiles of soybean flour and white bean seed extracts are significantly different.     

Dynamic behavior of adsorber membranes for protein recovery

In recent years there has been a considerable interest in developing membrane chromatography systems that function as a short, wide chromatographic column in which the adsorptive packing consists of one or more microporous membranes. This study reports the use of new adsorber membranes prepared by the incorporation of various types of ion exchange resins into an EVAL porous membrane for protein recovery. The obtained heterogeneous matrixes composed of solid particles surrounded by the polymeric film possess a good accessibility for the protein to the adsorptive sites. Furthermore, small particles can be embedded into porous polymeric structures without the disadvantages of classical chromatographic columns (high pressure drop, fouling and plugging sensitivity, low flow rate), but with the advantages of membrane technology (easy scale-up, low-pressure drop, high flow rate). The adsorptive membranes feature high static as well as dynamic protein adsorption capacities for operating flow rates ranging from 200 to 400 L h bar per m(2) and ionic strength of 20-200 mM. In a sequential desorption step by changing the pH and/or the ionic strength of the eluent, up to 90% protein recovery was obtained. Next to the separation, the mixed matrix adsorber membrane functions as a concentration medium since the protein can be concentrated up to 20-fold in the eluent. The adsorber membranes can be reused in multiple adsorption/desorption cycles with good adsorption performances.     

Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II)

Different chemical extractants (NaCl, EDTA, HCl and NaOH) and physical methods (ultrasonication and heating) were examined by their efficacies of extracting “attached” exopolymeric substances (EPS) secreted by marine bacterium Sagittula stellata (SS) and terrestrial bacterium Pseudomonas fluorescens Biovar II (PF). Extraction by 0.5 N HCl for 3 h was best for SS while extraction by 0.05 N NaCl for 3–5 h was regarded as optimal for PF. Improvements in EPS purification included a pre-diafiltration step to remove the broth material and reduce the solution volume, thus the usage of ethanol, and time. The EPS harvested at the optimal time and purified by the improved method were enriched in polysaccharides, with smaller amounts of proteins, thus having amphiphilic properties. Isoelectric focusing of ²³⁴Th or ²⁴⁰Pu labeled EPS showed both actinides were strongly bound to macromolecules with low pI, similar to reported marine or soil colloidal natural organic matter (NOM).