Removal of 17β Estradiol and Fluoranthene by Nanofiltration and Ultrafiltration

With the recent emergence of endocrine disrupting compounds as an important potable drinking water and reclaimed wastewater quality issue, the removal of two estrogenic compounds (17β-estradiol and fluoranthene) by nanofiltration and ultrafiltration membranes was investigated. A less hydrophobic organic compound model species [parachlorobenzoic acid (PCBA)] was tested. 17β-estradiol (E2), fluoranthene, and PCBA were applied to the membrane in the presence and absence of natural organic matter (NOM). Both batch adsorption and dead-end stirred-cell filtration experiments indicated that adsorption is an important mechanism for transport/removal of relatively hydrophobic compounds, and is related to the octanol-water partition coefficient (KOW) values. All filtration measurements were performed approximately the same permeate flow rate in order to minimize artifacts from concentration polarization varied with different hydrodynamic operating conditions at the membrane interface. The percent removal by dead-end stirred-cell filtration ranged from 10 to >95% depending upon membrane pore size/hydrophobicity and presence/absence of NOM at an initial concentration ranging from 0.1 to 0.5 μM. Additional batch adsorption experiments with radio-label (3H) E2 at lower concentrations ranging 0.025 to 5 nM showed that E2 removal due to adsorption was independent of its initial concentration. Adsorption occurs both on the membrane surface and interior membrane pore surfaces. However, adsorption was insignificant for PCBA (log?KOW = 2.7), but removal presumably occurred due to electrostatic exclusion. Partition coefficients (log?K) of 0.44 to 4.86 measured in this study increased with log?KOW and membrane pore size.     

Effects of Bromide Ion and Natural Organic Matter Fractions on the Formation and Speciation of Chlorination By-Products

The impacts of bromide concentration and natural organic matter (NOM) characteristics on the formation and speciation of disinfection by-products (DBPs) in chlorinated NOM fractions were investigated. A total of 20 bulk water NOM fractions with a wide range of specific ultraviolet (UV) absorbance (SUVA254) values were obtained from a source water employing XAD-8 or XAD-4 resin adsorption in completely mixed batch reactors. SUVA was not a good predictor of DBP [trihalomethanes (THMs), haloacetic acids (HAAs), and adsorbable organic halogens (AOX)] formation and speciation. The destruction in the UV254 absorbance from chlorination did not correlate with DBP formation at any bromide level. NOM moieties which do not absorb UV light at 254?nm significantly contributed to DBP formation. Mass balance calculations on halogens using THMs, HAAs, and AOX data indicated that significant amounts of DBPs (>54% of AOX) other than THMs and HAAs were formed in NOM fractions with 60–110?μg/L bromide concentration. The relative occurrence of such other halogenated by-products decreased with increasing bromide concentrations up to 500?μg/L level. NOM in the studied water was more susceptible to the formation of brominated THM species as opposed to brominated HAAs. At constant dissolved organic carbon concentration, chlorine dose and pH, increasing bromide concentrations in NOM fractions increased the total concentrations of DBPs and resulted in a shift toward the formation of brominated species. Further, increasing bromide concentrations increased the spectrum of detected species (i.e., occurrence of all nine HAAs) and provided a competitive advantage to THM and HAA precursors in NOM over precursors of other DBPs.     

Modeling Uncoupled Solute Transport in Natural Organic Matter Size Fractionation by Ultrafiltration

Physicochemical separation of organic macrosolutes and colloidal particles is routinely required during the analysis of natural, waste, and process waters derived from aquatic and terrestrial environmental samples. This study was conducted to demonstrate the utility of a two-parameter nonlinear permeation coefficient model (PCM) in describing the uncoupled transport of solutes in dilute heterogeneous solutions subjected to batch ultrafiltration (UF). The PCM was used in conjunction with natural organic matter (NOM) permeate data for a natural water and six hydrophobic and hydrophilic subfractions to determine permeation coefficients p and NOM concentrations Cr0 with apparent molecular weight less than membrane specific cutoff values of moderately hydrophilic YC/YM series Amicon? UF membranes. Experimentally measured permeation coefficients p determined for the whole water were found to correlate well with composite permeation coefficients p? calculated using a mass-fraction weighted average of individual NOM subfraction permeation coefficient values. Correlation of experimentally measured and calculated permeation coefficient values (p and p?) indicated that the PCM can adequately describe uncoupled transport of chemically distinct solute fractions during batch UF of heterogeneous dilute solutions.     

Characterization of permeability and network structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels

Hydrogel membranes formed by interfacially photopolymerizing poly(ethylene glycol) (PEG) diacrylate precursor solution were prepared from PEG diacrylate of molecular weights (MW) ranging from 2000 (2K) to 20000 (20K) with concentrations ranging from 10% to 30% w/w. The effects of PEG diacrylate MW and concentration in the membrane precursor solution upon the diffusivities of vitamin B12, myoglobin, ovalbumin, albumin, and IgG were determined. Regardless of the concentration of the PEG diacrylate in the precursor solution, hydrogels prepared with PEG 2K, 4K, and 8K diacrylate were impermeable to proteins with a size equal to or larger than myoglobin (22 kDa), while hydrogels prepared with PEG 20K diacrylate were impermeable to proteins with a size equal to or larger than ovalbumin (45 kDa). Similarities between hydrogels formed from PEG 2K, 4K, and 8K diacrylates were also seen in calculations of the molecular weight between crosslinks and the mesh size, with values in the range of 150-750 g/mol and 15-35 A, respectively, depending on PEG diacrylate concentration. In contrast, hydrogels formed from PEG 20K diacrylate had molecular weight between crosslinks ranging from 1150 to 2000 g/mol and mesh sizes ranging from 45-70 A, with larger values being observed in membranes polymerized from more dilute PEG diacrylate precursor.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin-Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by alpha-thrombin (IC50 = 28 microg/ml). A single band of approximately 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation (Ic50 = 0.19 microg/ml) compared to bothrojaracin (IC50 = 0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Purification and properties of the F1F0 ATPase of Ilyobacter tartaricus, a sodium ion pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F1F0 ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the alpha, beta, gamma, epsilon, and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na(+)-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and delta subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na+ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na+ or Li+ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na+, Li+, or Hi+. Proton transport was specifically inhibited by Na+ or Li+ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na+ as the physiological coupling ion for ATP synthesis.     

Recovery of Metal Working Fluids Using Chelation-Ultrafiltration Process

Metal working (MW) fluids are used to control friction and temperature, improve workpiece surface quality, and reduce tool wear. The batch-life of the MW fluid investigated was controlled primarily by the concentration of metallic soaps (complexes between the oil emulsifier and Al∕Mg. Ethylenediaminetetraacetic acid, a strong metal chelator, was added to the MW fluid to break the oil-Al∕Mg complexes. The aqueous-phase ethylenediaminetetraacetic acid-Al∕Mg complexes were then separated from the oil phase by ultrafiltration (UF). Al levels were reduced between 39 and 49%, and Mg levels were reduced between 67 and 77%. Al transfer from the oil phase to the aqueous phase was slow and increased during UF concentration. Rinsing the concentrated MW fluid with deionized water decreased the ash content but had a lesser impact on Al∕Mg removal. Al mass balances produced errors of 2.8% for run 3 and 6.2% for run 4. The UF system (0.11-μm ceramic membrane) effectively separated the oil and aqueous phases. Permeate turbidity was generally <1 nephelometric turbidity unit and permeate flux ranged from 390 to 220 L∕m2-h (230 to 130 gal.∕ft2-day) depending on the degree of concentration.     

Disinfection By-Product Formation during Long-Term Water Storage in Alaska

In many areas of Northern and Western Alaska, small streams and shallow lakes serve as community raw water supplies. These water supplies freeze completely during winter. In order to supply drinking water during the 6–9 month winter, communities store water that was treated during summer. A chlorine residual is maintained in the stored water. Raw water sources derived from surface water may be heavily laden with dissolved organic matter. At utilities where organic matter escapes treatment, the potential for accumulation of disinfection by-products (DBPs) during storage is a significant health concern. The following study was performed to evaluate this potential threat. Water was collected from five operating utilities, four that normally store water for 6–9 months and one that produces drinking water year-round. Raw, filtered (i.e., unchlorinated) and “finished” (i.e., filtered and chlorinated) water samples were collected during the summer pumping season and stored in the laboratory for 8 months. In order to mimic practice in the field, the chlorine residual was maintained in the finished water for the full storage period. While the concentration of DBPs in the finished water varied over the study period, there was not a statistically significant trend from the third to the eighth month of storage. The observed DBP values were strongly a function of the type of treatment system used. Those systems passing more organic matter had higher DBP values throughout the storage period. The ultraviolet absorbance at 254 nanometers ?start(UV254)end? decreased continuously in the finished water coincident with chlorine consumption. ?startUV254end?, often used as a surrogate for DBPs, remained constant during the entire storage periodin raw and filtered water samples. Filtered water that was stored prior to chlorination accumulated fewer DBPs than finished water that was continuously chlorinated during the storage period. This result suggests that storing filtered water instead of finished water for long periods would limit DBP exposure to consumers. This conclusion was based on a comparison of DBP formation potentials (i.e., raw and filtered water) to DBPs (i.e., finished water). It is important to note that DBP formation potentials are based on a ?start24?hend?chlorine contact time. If long term storage were provided for filtered water, a smaller volume of secondary storage would still be needed to provide contact time for disinfection.     

A locus coding for putative non-ribosomal peptide/polyketide synthase functions is mutated in a swarming-defective Proteus mirabilis strain

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K = 10(10)M-1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of 3H-folate labelled protein (> = 130 and 100 kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.     

Human amyloid precursor-like protein 1--cDNA cloning, ectopic expression in COS-7 cells and identification of soluble forms in the cerebrospinal fluid

Amyloid precursor-like protein 1 (APLP1) represents an integral membrane type 1 protein of unknown function which was originally cloned from a mouse cDNA library on the basis of sequence similarity with the Alzheimer's amyloid precursor protein (APP). Here we report on the molecular cloning and expression of the human APLP1 (hAPLP1). hAPLP1 consists of 650 amino acids, displays 89% identity on the amino acid level to its mouse homologue and has a calculated molecular mass of 72 kDa. hAPLP1 synthesized in a cell-free system displays an apparent molecular mass of approximately 80 kDa in SDS-containing gels and becomes N-glycosylated when the in vitro translation is performed in the presence of microsomes. The hAPLP1 cDNA was also expressed ectopically in COS-7 cells and the protein expression was analyzed by immunoprecipitation and western blotting. We have demonstrated that hAPLP1 represents a novel glycoprotein which carries both N- and O-linked glycans. Moreover, hAPLP1 undergoes limited proteolysis which results in the secretion of the carboxy-terminal truncated molecule into the cells conditioned medium. Examination of cells transfected with hAPLP1 cDNA by confocal laser microscopy reveals an intense perinuclear and Golgi staining, a pattern resembling the subcellular distribution of APP. Using a novel hAPLP1-specific antiserum, we identified soluble hAPLP1 in the human cerebrospinal fluid, which suggests that secretion of hAPLP1 from brain cells also takes place in vivo.     

Isolation of a novel Kunitz family protease inhibitor in association with Tethya hemolysin from the sponge Tethya ingalli

Aqueous extracts from the New Zealand sponge Tethya ingalli (Hadromerida) displayed potent cytotoxicity in the NCI's 60-cell-line human tumor panel. Fractionation of the extract by ammonium sulfate precipitation, gel filtration, ultrafiltration, and both hydrophobic interaction and reversed-phase chromatography resulted in the isolation of two biologically active proteins. The first protein, Tethya protease inhibitor (TPI), which was purified to homogeneity, inhibited trypsin with an EC50 of 65 nM. TPI had a molecular mass of 11,431 Da, and an isoelectric point of 8.2. A partial N-terminal amino acid sequence determined for TPI showed significant homology with protease inhibitors of the Kunitz family. The second isolated protein displayed potent cytotoxicity, with pronounced selectivity for certain tumor cell lines (e.g., ovarian, renal, CNS, and breast). The latter protein, which had an apparent molecular weight of 21 kDa (SDS-PAGE), also lysed human red blood cells (EC50 of 39 nM) and was similar to a hemolysin previously isolated from the sponge Tethya lycinurium.     

Effect of proton pump inhibitor, omeprazole, on expression of rat parietal cell specific carbohydrate antigen, type 2 chain N-acetyllactosamine

The effect of a proton pump inhibitor, omeprazole, on the rat gastric mucosal expression of carbohydrate antigens was studied. Type 2 chain N-acetyllactosamine was detected specifically on the apicocanalicular cell membranes of parietal cells. Pretreatment of rats with omeprazole profoundly suppressed the antigen expression, which followed the inhibition of gastric acid secretion. When omeprazole was discontinued, the antigen was reexpressed, which preceded the restoration of acid secretion. The antigen-negative tissues became antigen-positive when they were desialylated. Gastric membrane vesicles from the normal and omeprazole-treated rats were antigen-positive and -negative, respectively. SDS-PAGE revealed that a glycoprotein with an apparent molecular weight of 64-78 kDa carried type 2 chain N-acetyllactosamine. In the omeprazole-treated rats, the same molecular weight glycoprotein was positively immunostained only after desialylation. We concluded that: (1) the expression of type 2 chain N-acetyllactosamine was closely correlated with gastric acid secretion, and (2) the inhibition of acid secretion was accompanied by the sialylation of the parietal cell membrane glycoprotein.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC)

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Airway neural responses to kinins: tachyphylaxis and role of receptor subtypes

Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced.     

Exposure estimates to disinfection by-products of chlorinated drinking water

Exposure to disinfection by-products (DBPs) of drinking water is multiroute and occurs in households serviced by municipal water treatment facilities that disinfect the water as a necessary step to halt the spread of waterborne infectious diseases. Biomarkers of the two most abundant groups of DBPs of chlorination, exhaled breath levels of trihalomethanes (THMs) and urinary levels of two haloacetic acids, were compared to exposure estimates calculated from in-home tap water concentrations and responses to a questionnaire related to water usage. Background THM breath concentrations were uniformly low. Strong relationships were identified between the THM breath concentrations collected after a shower and both the THM water concentration and the THM exposure from a shower, after adjusting for the postshower delay time in collecting the breath sample. Urinary haloacetic acid excretion rates were not correlated to water concentrations. Urinary trichloroacetic acid excretion rates were correlated with ingestion exposure, and that correlation was stronger in a subset of individuals who consumed beverages primarily within their home where the concentration measurements were made. No correlation was observed between an average 48-hr exposure estimate and the urinary dichloroacetic acid excretion rate, presumably because of its short biological half-life. Valid biomarkers were identified for DBP exposures, but the time between the exposure and sample collection should be considered to account for different metabolic rates among the DBPs. Further, using water concentration as an exposure estimate can introduce misclassification of exposure for DBPs whose primary route is ingestion due to the great variability in the amount of water ingested across a population.     

Cloning of the gene encoding the Actinobacillus actinomycetemcomitans serotype b OmpA-like outer membrane protein

The gene encoding an outer membrane protein A (OmpA)-like, heat-modifiable Omp of Actinobacillus actinomycetemcomitans ATCC 43718 (strain Y4, serotype b) was cloned by a PCR cloning procedure. DNA sequence analysis revealed that the gene encodes a protein of 346 amino acid residues with a molecular mass of 36.9 kDa. The protein expressed by the cloned gene reacted with a monoclonal antibody to the previously described 29-kDa Omp (Omp29) of strain Y4. This monoclonal antibody reacted specifically with Omp29 of A. actinomycetemcomitans (serotype b), but not with any Omp of Escherichia coli, including OmpA. This protein exhibited characteristic heat modifiability on sodium dodecyl sulfate-polyacrylamide gels, showing an apparent molecular mass of 29 kDa when unheated and a mass of 34 kDa when heated. The N-terminal amino acid sequence of the protein expressed in E. coli perfectly matched those deduced from the purified Omp29 of strain Y4. The deduced amino acid sequence of the gene coding for Omp29 from serotype b matched completely (except for valine at position 321) that of a recently reported omp34 gene described for A. actinomycetemcomitans serotype c (NCTC 9710). Because of the conserved nature of the gene within these serotypes, we designated the gene described herein from serotype b as omp34.     

Proteolysis of the carboxyl-terminal GPI signal independent of GPI modification as a mechanism for selective protein secretion

Variable amounts of soluble forms of a variety of glycosyl-phosphatidylinositol (GPI)-anchored proteins occur extracellularly, but the molecular mechanisms governing their release are not entirely clear. When the GPI-anchored folate receptor (FR) type beta was expressed transiently in human 293 fibroblasts, there was a roughly equal distribution of [3H]folic acid binding protein between the cell surface and the medium after 24 h over a wide range of expression levels of FR-beta. The difference in apparent molecular masses between the soluble FR-beta and the PI-PLC-treated membrane protein indicated that the former was not released from the membrane by the action of phospholipase. Brefeldin A inhibited the release of soluble FR-beta from both the transfected 293 cells and stable recombinant CHO (CHO-FR-beta) cells while pre-existing levels of cell surface FR were unaltered suggesting the absence of a precursor-product relationship between the membrane-associated FR-beta and the soluble protein in the medium. [35S]Cysteine pulse-chase analysis was consistent with this finding. Interchanging of carboxyl-terminal peptides between FR-beta and FR-alpha revealed that the nature of the processed signal for GPI modification was responsible for the quantitative membrane anchoring of FR-alpha and the production of soluble FR-beta. When total cell lysates were analyzed by Western blot, a diffuse band of apparent 41 kDa and three additional sharp bands of apparent 35, 33, and 29.3 kDa were seen. The 41 kDa band was identified as the PI-PLC sensitive cell surface receptor. Several mutant constructs of FR-beta, in which the carboxyl-terminal signal for GPI modification was either disrupted or deleted only gave the three lower bands. The three sharp bands from the wild-type and the mutant forms of FR-beta were identified as nonglycosylated (29.3 kDa) or glycosylated polypeptides in which the carboxyl-terminal peptide was at least partially proteolyzed without GPI modification. All of the mutations in the GPI signal resulted in the recovery of [3H]folic acid binding protein in the media which, similar to the wild-type FR recovered from the media, were converted to the 29.3 kDa band by N-glycanase. The results from this study indicate that a carboxyl-terminal peptide in FR-beta is efficiently proteolyzed intracellularly by a pathway that is independent of GPI signal recognition resulting in proper protein folding and secretion. Such carboxyl-terminal sequences could represent a simple adaptation for proteins whose physiologic functions reside both at the cell surface and in extracellular fluids, allowing their selective and tissue-specific release.     

Soluble Microbial Products in ABR Treating Low-Strength Wastewater

The chemical composition, molecular weight (MW) distribution, and biodegradability (both aerobic and anaerobic) of soluble microbial products (SMPs) in an anaerobic baffled reactor (ABR) treating low-strength wastewater were investigated. The effect of various process parameters on the production of SMPs was also examined. Results indicated that high MW (>300 kDa) compounds were produced in the middle compartments of the reactor and formed 22% of the effluent chemical oxygen demand (COD). This fraction was found to be 86% degradable under aerobic conditions but only 4% under anaerobic conditions. Low MW (<1 kDa) material represented the highest portion (36%) of the effluent COD and was mainly found in the first compartment of the ABR and in the effluent. This fraction was more easily degraded under anaerobic conditions (33%) than aerobic conditions (17%). Analysis of a hydrolyzed sample of the high MW fraction revealed the presence of several sugars and volatile fatty acids. Therefore, it was concluded that the high MW material contains heteropolysaccharides. Nuclear magnetic resonance analysis of the low MW fraction revealed the possible presence of alcohol, carboxylate, and aromatic chemical groups. SMP production increased with increasing hydraulic retention time (HRT), probably due to enhanced biomass decay at high HRTs, and also increased with decreasing temperature, probably due to increased stress on the biomass and a reduced metabolism of the SMP at low temperatures. Finally, SMP production in an ABR containing higher levels of initial biomass concentration was greater than for an ABR operating at the same conditions but with lower levels of initial biomass.