Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges

The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1–7 kDa) and Superdex 20010/300GL (10–600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.     

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.     

Simultaneous analysis of disopyramide and quinidine in plasma by high-performance liquid chromatography

A new reversed-phase high-performance liquid chromatographic method allowing simultaneous measurement of plasma concentrations of disopyramide and quinidine is described. Disopyramide and quinidine were separated on a reversed-phase column using 0.05 M phosphate buffer (pH 3.0)—acetonitrile (73:27, v/v), as mobile phase and the peaks were monitored by UV absorbance at the wavelengths of 254 and 325 nm. The drugs were extracted from alkaline plasma with chloroform containing the internal standard. The organic phase was evaporated to dryness and the residue was redissolved in a small volume of the mobile phase before analysis by high-performance liquid chromatography. The method is convenient and reliable in routine monitoring of both drugs.     

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.     

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.     

Implementation of an HPLC polystyrene divinylbenzene column for separation of activated sludge exopolymers

A high-pressure size-exclusion chromatography procedure for separation of activated sludge exopolymers was investigated and implemented in order to achieve a documented and faster separation procedure than the conventional low-pressure size-exclusion chromatography methods previously suggested in studies of activated sludges from a traditional and an advanced from activated sludges from a traditional and an advanced activated sludge treatment plant performing biological nitrogen and phosphorus removal were used. For both types of exopolymers the separation was largely dependent on the mobile-phase. Using NaCl and ortho-phosphate in the molar proportion 10:1 it was shown that for a mobile-phase ionic strength of 0.011 and pH in the range 7.0–10.0 no irreversible column adsorption occurred. For a standard procedure a mobile-phase pH of 7.0 was selected in order to separate the exopolymers into the maximal number of peaks. Alterations in the mobile-phase, i.e. using a pH below 7.0 or a mobile-phase ionic strength above 0.011, changed the separation for both types of exopolymers and caused irreversible column adsorption. Similarly, using deionized water as the mobile-phase irreversible column adsorption was introduced and the separation was strongly affected. The method applicability for qualitative characterization of exopolymers was demonstrated. The method was found to be successful in showing differences and similarities between exopolymers from two different activated sludge treatment plants, showing degradation of exopolymer compounds due to exoenzymes in the exopolymers and showing that snow melting and subsequent high conductivity in the inlet to the waste-water treatment plant had an impact on the chromatographic fingerprint of the extracted exopolymers.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

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.     

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     

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.     

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.     

Monovalent cations and their influence on activated sludge floc chemistry, structure, and physical characteristics

Multivalent cations have been known to be important components of activated sludge floc structure due to their bridging ability of the negatively charged sites on the biopolymer network. Recently in batch systems it was found that excess concentration of monovalent cations led to the deterioration in settleability, dewaterability of sludges and effluent quality of the system. In this study, effect of influent monovalent cations (potassium and sodium) on activated sludge floc structure was investigated in semi-continuous reactors. Results revealed that the increase in concentration of both ions correlated to the general increase in total EPS concentration. The zeta potential values were affected by the cation type and dose in such a way that sludge from sodium reactors had always higher zeta potential values (higher negative charge) than the sludges from potassium reactors. Flocs from sodium reactors were more fragile and weak and the capillary suction time values of these sludges were higher compared to those from potassium reactors. The findings of this research conclude that the floc structure is significantly weakened with the increase of monovalent cations. Even though EPS is produced, it is unable to bind the floc components together. With this, the physical properties of sludge deteriorate for both cations.     

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cationexchange resin. In the EPS matrix a similar initial (2–3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.     

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.     

Determination of donepezil, an acetylcholinesterase inhibitor, in human plasma by high-performance liquid chromatography with ultraviolet absorbance detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of donepezil, a centrally and selectively acting acetyleholinesterase inhibitor, in human plasma. After sample alkalinization with 0.5 ml of NaOH (0.1 M), the test compound was extracted from I ml of plasma using isopropanol-hexane (3:97, v/v). The organic phase was back-extracted with 75 microl of HCl (0.1 M) and 50 microl of the acid solution was injected into a C18 STR ODS-II analytical column (5 microm, 150x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.02 M, pH 4.6), perchloric acid (6 M) and acetonitrile (59.5:0.5:40, v/v) and was delivered at a flow-rate of 1.0 ml/min at 40 degrees C. The peak was detected using a UV detector set at 315 nm, and the total time for a chromatographic separation was approximately 8 min. The method was validated for the concentration range 3-90 ng/ml. Mean recoveries were 89-98%. Intra- and inter-day relative standard deviations were less than 7.3 and 7.6%, respectively, at the concentrations ranging from 3 to 90 ng/ml. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.     

Determination of rifabutin in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of rifabutin in human plasma. Rifabutin and sulindac (internal standard) are extracted from human plasma using a C₈ Bond Elut extraction column. Methanol (1 ml) is used to elute the compounds. The methanol is dried down under nitrogen and reconstituted in 250 μl of mobile phase. Separation is achieved by HPLC on a Zorbax Rx C₈ column with a mobile phase composed of 0.05 M potassium dihydrogen phosphate and 0.05 M sodium acetate at pH 4.0-acetonitrile (53:47, v/v). Detection is by ultraviolet absorbance at 275 nm. The retention times of rifabutin and internal standard were approximately 10.8 and 6.9 min, respectively. The assay is linear over the concentration range of 5–600 ng/ml. The quantitation limit was 5 ng/ml. Both intra-day and inter-day accuracy and precision data showed good reproducibility.     

Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges

A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1.1 x 10(5) pfu g-1) and the lowest being that of Bacteroides fragilis phages (110 pfu g-1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others.     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.