Chemical pretreatment of combined sewer overflows for improved UV disinfection.

The aim of this research was to better understand chemical pre-treatment of combined sewer overflows (CSOs) for subsequent ultraviolet (UV) disinfection. Approximately 200 jar tests were completed. Alum (Al2(S04)3·12H2O) resulted in a higher UV light transmission (UVT), and equivalent total suspended solids (TSS) removal, than ferric chloride (FeCl3). An alum dose of 20 mg/L increased the UVT of the raw CSO from 30 to 60% after settling. The addition of 100 mg/L of alum maximized UVT reaching approximately 85%. Flocculation did not increase UVT. However, it did improve the removal of TSS. Cationic polymers worked quickly compared with metal coagulants, but only reached a UVT of 60%. A high positive charge density on the polymer improved the removal of turbidity when compared with low charge, but did not affect UVT. If the goal is to maximise UVT, a very high alum dose may be preferred. If the goal is to minimize coagulant dose with moderate UV performance, cationic polymer at approximately 3 mg/L is recommended.

UV disinfection of wastewater flocs: the effect of secondary treatment conditions.

Activated sludge flocs that are carried to the final effluent can significantly decrease the effectiveness of ultraviolet (UV) disinfection of wastewater. This effect is detected in a typical UV dose-response curve, where at higher UV doses there is a decrease in the inactivation rate (tailing). In this study, the effect of activated sludge process conditions on the UV inactivation kinetics of flocs was investigated. The conditions compared were nitrifying vs. non-nitrifying vs. an enhanced biological nutrient removal-University of Cape Town (BNR-UCT) system. The results showed that the flocs generated in the BNR-UCT process were easier to disinfect. The final effluent from the BNR-UCT process also showed improved kinetics of inactivation and reached higher levels of disinfection. The nitrifying system's final effluent had a lower number of initial fecal coliforms, which contributed to reaching higher disinfection levels compared to the non-nitrifying system.

Hydrodynamic treatment of wastewater effluent flocs for improved disinfection.

Hydrodynamic forces generated by an orifice plate under low pressure were examined as a means of disrupting flocs, in order to improve disinfection of treated wastewater effluents. Changes in cavitation conditions were found to have little impact on the extent of particle breakage in this experimental setup. The rate of strain (flow rate divided by the hole radius cubed), however, was found to be the best predictor of floc breakage. Floc breakage was not affected by changes in floc concentration, but was very sensitive to differences between flocs collected from different sources. Larger flocs (90 to 106 microm) were broken apart to a greater extent than smaller ones (53 to 63 microm). Hydrodynamic treatment decreased the viability of bacteria associated with large flocs, and also increased the ultraviolet dose response by up to one log unit (i.e., a factor of ten). Subjecting final effluent wastewaters to hydrodynamic treatment, therefore, provides a treatment strategy for conditions in which the presence of flocs limits the level of disinfection that can be achieved.

An assessment of oxygen transfer efficiency in a gas permeable hollow fibre membrane biological reactor.

The clean water oxygen transfer efficiency (OTE) of a full scale non-porous hollow fibre gas permeable (GP) membrane (surface area of 500 m(2)) was evaluated at inlet air pressures of 1.2, 1.4, and 1.8 atm using two established testing methods. To form a basis of comparison with traditional aeration technologies, additional testing was done with conventional aerators (fine bubble and coarse bubble diffusers) replacing the GP membrane. OTE can be established based on the re-aeration of deoxygenated water or by monitoring the catalytic oxidation of a sodium sulphite (Na(2)SO(3)) solution. In this study, OTE values determined by sulphite oxidation (SOTE(S)) were consistently higher than those established during re-aeration (SOTE(R)) suggesting that the chemical reaction was enhancing the mass transfer. The chemical reaction was sufficiently fast in the case of the GP membrane, that the gas phase limited the mass transfer. The GP membrane operating at 1.2 atm had a SOTE(S) of 70.6% and a SOTER of 52.2%. SOTE(R) for the coarse bubble and fine bubble diffusers were 3.8% and 23.6%, respectively. This is comparable to the manufacturer's values, corrected for depth of 3.4% and 18.3%, respectively. Particularly, the derived OTE values were used to evaluate differences in energy consumption for a conventional treatment plant achieving carbon removal and nitrification. This analysis highlights the potential energy efficiency of GP membranes, which could be considered for the design of the membrane modules.

Library-dependent and library-independent microbial source tracking to identify spatial variation in faecal contamination sources along a Lake Ontario beach (Ontario, Canada).

Multiple microbial source tracking methods were applied to investigate spatial variation in faecal pollution sources impacting a 1.7 km freshwater beach on Lake Ontario (Canada). The highest E. coli concentrations measured in the study area were from interstitial sand pore water at Sunnyside Beach, reaching 2.6 x 10(6) CFU/100 ml. These E. coli concentrations exceeded those in the nearby Humber River and Black Creek, which are impacted by combined sewer overflows containing municipal wastewater and by stormwater conveying washoff from the urban area. Library-independent Bacteroidales HF183 analyses identified the more frequent occurrence of municipal wastewater contamination in the Humber River and at a Sunnyside Beach location closest to the mouth of the river. Library-dependent E. coli antibiotic resistance and rep-PCR DNA fingerprinting analyses identified the more frequent occurrence of bird faecal contamination at Sunnyside Beach locations away from the river mouth. These microbial source tracking results raise caution about managing beaches with multiple sources of contamination as a single entity without considering spatial variability in faecal pollution sources and the need for more localized beach management practices.

Laboratory pilot scale study for H2S removal from biogas in an anoxic biotrickling filter.

The purpose of this laboratory pilot scale study at the Wastewater Technology Centre (WTC), Environment Canada, Burlington, ON was to investigate the anaerobic biological removal of H2S from biogas under real-time operating conditions. Biogas produced in a 538 litre pilot anaerobic digester was continuously fed into a 12 litre biotrickling filter containing plastic fibres as packing bed media. The process was monitored for several months. The biogas flowrate and H2S concentration ranged between 10 to 70 L/h and 1,000 to 4,000 ppmv respectively over the course of the test period. Nitrate-rich wastewater from a pilot scale sequencing batch reactor effluent was used as the nutritive solution for the biotrickling filter. The paper presents the influence of several operational parameters such as biogas flowrate, hydrogen sulphide concentration and composition of nutrient solution on process performance. To date, our results show H2S removal rates up to 100% without adverse effects on the methane concentration of the biogas. No system deterioration was observed over long term operation. This non-conventional technology is very promising and could be considered for full scale applications.

Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis.

A full-length cDNA clone for human thyroid peroxidase (TPO) inserted into the mammalian cell expression vector pECE was stably transfected into Chinese hamster ovary (CHO) cells. Clones were assayed for human TPO mRNA, TPO protein, and TPO enzymatic activity. One subclone, expressing the highest TPO enzymatic activity, was used in further studies. FACS analysis of these cells preincubated in Hashimoto's serum revealed approximately 100-fold greater fluorescence compared with controls, indicating that recombinant TPO is expressed on the cell surface. Particulate antigen was extracted from these cells and studied by Western blot analysis using a panel of Hashimoto's sera of known antimicrosomal antibody (anti-MSA) titer. Under nonreducing conditions a broad, immunoreactive band of approximately 200 kD was observed, as well as a doublet of approximately 110 kD. All of the 36 Hashimoto's sera tested reacted with these bands, most in proportion to their anti-MSA titer. Six normal sera tested against this antigen(s) were nonreactive, as were the Hashimoto's sera tested against nontransfected CHO cells. Western blots under reducing conditions revealed a considerably diminished signal, with some of the sera of lower anti-MSA titer becoming negative, the loss of the 200-kD broad band, and the apparent conversion of the 110-kD doublet into a single band. Preincubation of cells in tunicamycin revealed no decrease in TPO immunoreactivity. In conclusion, we expressed enzymatically active human TPO in nonthyroidal eukaryotic cells. Our data prove that functionally active TPO is a major component of the thyroid microsomal antigen.

Isolation of a complementary DNA clone for thyroid microsomal antigen. Homology with the gene for thyroid peroxidase.

The thyroid microsomal antigen (MSA) in autoimmune thyroid disease is a protein of approximately 107 kD. We screened a human thyroid cDNA library constructed in the expression vector lambda gt11 with anti-107-kD monoclonal antibodies. Of five clones obtained, the recombinant beta-galactosidase fusion protein from one clone (PM-5) was confirmed to react with the monoclonal antiserum. The complementary DNA (cDNA) insert from PM-5 (0.8 kb) was used as a probe on Northern blot analysis to estimate the size of the mRNA coding for the MSA. The 2.9-kb messenger RNA (mRNA) species observed was the same size as that coding for human thyroid peroxidase (TPO). The probe did not bind to human liver mRNA, indicating the thyroid-specific nature of the PM-5-related mRNA. The nucleotide sequence of PM-5 (842 bp) was determined and consisted of a single open reading frame. Comparison of the nucleotide sequence of PM-5 with that presently available for pig TPO indicates 84% homology. In conclusion, a cDNA clone representing part of the microsomal antigen has been isolated. Sequence homology with porcine TPO, as well as identity in the size of the mRNA species for both the microsomal antigen and TPO, indicate that the microsomal antigen is, at least in part, TPO.

Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.

Experience with the antibiotic resistance analysis and DNA fingerprinting in tracking faecal pollution at two lake beaches.

Posting or closing of swimming beaches because of faecal contamination is a widespread problem reported in many locations. In a risk-based approach to this problem, the risk to swimmers' health is assessed by field monitoring of indicator bacteria and the associated risks are managed by source controls and other remedial measures. In risk assessment, great advances have been made in recent years with the introduction of microbial source tracking (MST) techniques. Two such techniques, antibiotic resistance analysis and DNA fingerprinting, were applied in a study of causes of faecal contamination at two lake beaches in Toronto, Ontario. Both methods identified bird faeces as the dominant sources of E. coli. Coping with this type of pollution presents a major environmental challenge.

An abnormal splice donor site in one allele of the thyroid peroxidase gene in FRTL5 rat thyroid cells introduces a premature stop codon: association with the absence of functional enzymatic activity.

Low stringency screening of an FRTL5 cDNA library with a human thyroid peroxidase (TPO) cDNA probe yielded two different types of TPO cDNA clones. One type contained the full-length structural gene, but there was an A at the first nucleotide of an intronic splice donor site that leads to alternate splicing, with the retention of part of an intron. This 54-basepair retained intron fragment contains a premature inframe stop codon that would truncate the protein at its carboxyl-terminus by 71% with the loss of enzymatic activity. The second type of TPO cDNA does not contain the retained intron fragment and premature stop codon, but it is not full-length and lacks 580-680 basepairs at its 5' end. However, Northern blot analysis reveals only full-length copies (3.2 kilobases) of TPO mRNA in FRTL5 cells, and this 5' truncation is, therefore, an artifact of library construction. The relative proportions of the two types of TPO mRNA in FRTL5 cells was determined by the polymerase chain reaction, using as template single stranded cDNA generated by reverse transcription of FRTL5 mRNA. Slightly more than half of the TPO mRNA in the FRTL5 cells represented the form with the abnormal splice donor site. The relative proportions of the two TPO mRNA forms was not influenced by TSH stimulation of the FRTL5 cells, and the proportion remained unaltered even after the FRTL5 cells were subcloned by limiting dilution. At the genomic level, we used allele-specific oligonucleotides for the mutant and normal forms of TPO, and found FRTL5 cells to have both normal and abnormal TPO alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain.

The most widely held model for the human TSH receptor is of holoreceptor of 80 kDa with two subunits of approximately 50 and 30 kDa linked by disulfide bridges, with the former subunit containing the major hormone-binding site. We reexamined this model by covalently cross-linking radiolabeled TSH to the recombinant human TSH receptor stably expressed in Chinese hamster ovary (CHO) cells. When cross-linking was performed after the preparation of CHO membranes, analysis of hormone-receptor complexes under reducing and nonreducing conditions provided results supporting the two-subunit TSH receptor model. In contrast, however, cross-linking of TSH to the TSH receptor in intact CHO cells before membrane preparation revealed, even under reducing conditions, an approximately 100-kDa receptor as well as an approximately 54-kDa hormone-binding subunit. The approximately 100-kDa holoreceptor size is consistent with the size of the TSH receptor, as predicted from its derived amino acid sequence. The proportions of the approximately 100-kDa TSH receptor and the 54-kDa fragment varied in different experiments, suggesting the occurrence of proteolytic cleavage. Cross-linking of radiolabeled TSH to intact cells expressing a mutant TSH receptor (TSHR-D1) lacking amino acids 317-366 localized the proteolytic cleavage site to just up-stream of amino acid residue 317. In summary, the present data obtained by cross-linking TSH to recombinant human TSH receptors in intact cells provides evidence that the receptor exists in vivo as an approximately 100-kDa glycoprotein with a single polypeptide chain with intramolecular disulfide bridges.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase.

Five overlapping cDNA clones representing the entire mRNA for human thyroid peroxidase (TPO) have been isolated from a human Graves' thyroid cDNA library. The cDNA sequence has been determined. Human TPO cDNA contains 3060 bases from the start of transcription to the beginning of the poly (A) tail at the 3'-end. The derived amino acid sequence of human TPO consists of 933 amino acids with a mol wt of 102,937. The derived amino acid sequence contains five potential glycosylation sites (Asn-X-Ser/Thr), a probable transmembrane signal peptide sequence at the amino terminus, and a hydrophobic putative membrane-spanning region beginning 85 amino acid residues from the carboxyl terminal end. Comparison of the human TPO amino acid sequence to that of pig TPO shows strong homology extending from the amino terminus to within 44 amino acid residues of the carboxyl-terminus.

Distribution of estrogens, 17beta-estradiol and estrone, in Canadian municipal wastewater treatment plants.

The distribution of female hormones, 17beta-estradiol and estrone, was determined in effluents of 18 selected municipal treatment plants across Canada. Replicate 24-h composite samples were collected from the influent and final effluent of each treatment plant, and the removal efficiency compared to the operational characteristics of the plants. In conventional activated sludge and lagoon treatment systems, the mean concentrations of 17beta-estradiol and estrone in influent were 15.6 ng/l (range 2.4-26 ng/l) and 49 ng/l (19-78 ng/l). In final effluents, the mean concentrations of both 17beta-estradiol and estrone were reduced to 1.8 ng/l (0.2-14.7 ng/l) and 17 ng/l (1-96 ng/l), respectively. 17beta-estradiol was removed effectively, >75% and as high as 98%, in most of the conventional mechanical treatment systems with secondary treatment. The removal of estrone was much more complex with removal varying from 98% to situations where the concentrations in the effluent were elevated above that detected in the influent. The estrogenicity, measured using a transfected estrogen receptor in yeast (YES) assay, was also variable, ranging from high removal to elevations of estrogenicity in final effluent. Although the apparent removals were not statistically correlated with either hydraulic (HRT) or solid (SRT) retention times, plants or lagoons with high SRT were very effective at reducing the levels of hormones. Well-operated plants that achieved nitrification also tended to have higher removal of hormones than those that did not nitrify. Laboratory aerobic reactor experiments confirmed the rapid removal of 17beta-estradiol, estrone, and estrogenicity when exposed to sewage slurries.

High-rate stormwater clarification with polymeric flocculant addition.

Treatment of urban stormwater by clarification, with flocculant addition, was studied in Toronto, Canada using a pilot-scale clarifier with removable lamellar plates. Almost 90 stormwater runoff events were characterised at the study site and found fairly polluted. The previous research phase indicated good treatability of this stormwater by lamellar clarification with flocculant addition (total suspended solids, TSS, removal of 84%, at a surface load of 15 m/h), but there were concerns about cleaning plates after storm events. With the aid of numerical modelling, hydraulic improvements to the clarifier inlet zone were retrofitted in 2004 and permitted the removal of the lamellar pack without a loss in treatment efficiency. In the modified clarifier, a cationic polymeric flocculant dosage of 4 mg/L with conventional clarification provided a TSS removal of 77%, at surface loads up to 43 m/h. The use of the polymer did not increase the acute toxicity of the treated effluent. The clarifier sludge was severely polluted by several heavy metals and would require special disposal. The treatment process tested could be well applied in projects requiring intensive stormwater treatment at compact sites.

Bovine thyrotropin has a specific bioactivity 5- to 10-fold that of previous estimates for highly purified hormone.

Bovine TSH (bTSH) was subjected to reverse phase HPLC using a Synchropak RP-P C18 column. Elution was with a linear gradient of 0-50% 1-propranol in 0.1% trifluoroacetic acid. Individual fractions were assayed for TSH bioactivity by the cultured thyroid cell cAMP response assay. Optimum separation of biologically active TSH was obtained at a mobile phase pH of 5. Under these conditions, bTSH purified by conventional chromatographic techniques to 25 U/mg protein was purified further to approximately 200 U/mg protein. These data indicate that bioactivity is only present in a relatively small fraction of the molecules in a highly-purified TSH preparation. The implications of these findings are discussed.

Human thyroid peroxidase-myeloperoxidase chimeric molecules: tools for the study of antigen recognition by thyroid peroxidase autoantibodies.

We constructed seven chimeric molecules in which sequential segments in the cDNA for thyroid peroxidase (TPO) were replaced with the homologous regions of myeloperoxidase (MPO) cDNA. The sizes of the translated cDNA segments A through G ranged from 23-175 amino acid residues in length. The TPO-MPO cDNA chimeras, inserted into an eukaryotic expression vector, were stably transfected into Chinese hamster ovary cells. Protein expression was examined by immunoblotting under reduced/denaturing conditions with a murine monoclonal antibody to denatured wild-type TPO. Expression (at a low level) was confirmed for TPO-MPO chimeras A, B, F, and G. The amino acid substitutions in TPO-MPO-C eliminate the monoclonal antibody epitope, and this chimera, therefore, provides a negative control. TPO-MPO-D and TPO-MPO-E did not generate detectable levels of protein. To study TPO autoantibody interaction with native protein, we performed fluorescence-activated cell sorter analysis using intact Chinese hamster ovary cells stably transfected with the wild-type and TPO-MPO chimeric cDNAs. Of the chimeras, only cells transfected with TPO-MPO-A (N-terminal 146 amino acids of MPO substituted for the N-terminal 121 amino acids of TPO) were recognized by TPO autoantibodies, although to a lesser degree than cells expressing wild-type TPO. In conclusion, the present data indicate that TPO autoantibodies can interact with TPO molecules in which the amino-terminus is replaced with the homologous MPO prosequence region, not normally present in mature MPO. Our study provides a foundation for designing future TPO mutants that may be of value for characterizing disease-associated B-cell epitopes in autoimmune thyroid disease.

Immunoprecipitation of radiolabeled human thyroid cell proteins by serum from patients with autoimmune thyroid disease.

Characterization of the antigen to thyroid-stimulating immunoglobulin (TSI), presumably the TSH receptor, remains elusive. We, therefore, attempted immunoprecipitation of the TSI antigen using cultured human thyroid cells radiolabeled to high specific activity by [35S]methionine (all proteins labeled) or by 125I and lactoperoxidase (primarily cell surface proteins labeled). Both techniques produced similar results. Crude membrane preparations from the labeled cells were solubilized in buffer containing 1% Triton and then incubated with TSI-containing or control serum. Immunoglobulin was precipitated with goat antihuman immunoglobulin and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 30 proteins were immunoprecipitated by both normal and control sera. However, only 1 protein (mol wt, approximately 320,000) was preferentially selected by TSI-containing serum (6 of 6 tested). This protein was also immunoprecipitated by the majority of sera from patients with Hashimoto's thyroiditis. The 320,000 mol wt protein constituted only a minute amount of total radiolabeled membrane protein, because it was not apparent on electrophoresis of the membrane extract before immunoprecipitation. It corresponded (in migration) to a human thyroglobulin (Tg) standard. Tg also inhibited immunoprecipitation of the 320,000 mol wt band. The anti-Tg antibody titer correlated significantly with the degree of immunoprecipitation of the 320,000 mol wt band. No specific immunoprecipitation of any protein was observed when radiolabeled guinea pig membrane proteins were used. These data provide insight into the difficulties involved in the identification of the TSH receptor with serum containing TSI.

Characterization, by molecular cloning, of smaller forms of thyroid peroxidase messenger ribonucleic acid in human thyroid cells as alternatively spliced transcripts.

The mRNA for human thyroid peroxidase (hTPO) is 3.1 kilobases (kb) in size, coding for a protein of 933 amino acids. However, there is controversy as to whether other hTPO mRNA transcripts exist in thyroid cells. There is one report of the existence of 2.1- and 1.7-kb transcripts (hTPO mRNA species I and II), representing up to half of the hTPO mRNA in TSH-stimulated human thyroid cells. On the other hand, numerous other studies have only observed 3.1-kb hTPO mRNA transcripts. The nature of these putative 2.1- and 1.7-kb mRNA transcripts, if present, is unknown. We now report the isolation and characterization of two smaller hTPO cDNA species, designated hTPO cDNA I and II. cDNA I and II are 1753 and 1044 basepairs (bp) in size, with open reading frames of only 225 and 174 amino acids, respectively. Comparison of the nucleotide sequences of cDNA I and II with available hTPO genomic sequence reveals that cDNA I consists of exons 1-6 (654 bp) and the 5'-end of intron 6 (1099 bp); cDNA II contains exons 1-5 (486 bp) and an unidentified DNA tract of 558 bp further down-stream, presumably an intron. Confirmation that cDNA I and II correspond to mRNA transcripts I and II, respectively, was provided by Northern blot analysis with DNA probes specific for cDNA I and II. hTPO mRNA transcripts I and II are present in TSH-stimulated and TSH-deprived human thyroid cells in culture as well as in intact thyroid tissue. In summary, the present data 1) demonstrate directly that hTPO mRNA transcripts I and II exist in human thyroid cells, 2) explain the discrepant data in the literature regarding their existence, 3) elucidate their molecular structure, 4) indicate that they are generated by alternative splicing, and 5) demonstrate that hTPO mRNA I and II exist in the thyroid gland in vivo as well as in thyroid cells in culture.

Evidence for the highly conformational nature of the epitope(s) on human thyroid peroxidase that are recognized by sera from patients with Hashimoto's thyroiditis.

To define the epitope(s) on human thyroid peroxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease, we constructed and screened a human TPO cDNA sublibrary containing 3.8 million random fragments of human TPO cDNA, each 200-500 basepairs in length. These fragments would code for TPO polypeptides of 66-166 amino acid residues. The validity of this approach was first tested with a murine monoclonal antibody against the denatured human thyroid microsomal antigen (TPO). Analysis of the nucleotide sequence of 14 clones selected from this library enabled molecular identification of the epitope recognized by this monoclonal antibody. In contrast to the data obtained with the monoclonal antibody, sera from patients with Hashimoto's thyroiditis containing polyclonal antimicrosomal/TPO antibodies did not recognize the TPO protein fragments generated by this library. These results differ from previous data obtained with recombinant human TPO fragments generated as bacterial fusion proteins. Our data suggest that, contrary to previous concepts, the natural B-cell epitope(s) on human TPO may be highly conformational (requiring a complex 3-dimensional structure) or may be discontinuous (formed by distant regions of the linear polypeptide chain being brought into apposition by protein folding).