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.

Impact of activated sludge configuration and operating conditions on in vitro and in vivo responses and trace organic compound removal.

This study tested municipal sewage effluents generated at the pilot scale using conventional activated sludge (CAS), nitrifying activated sludge (CAS-N) and biological nutrient removal (BNR) in terms of the removal of trace organic compounds (TrOCs) and final effluent quality as indicated by yeast estrogenicity screening (YES), short term zebrafish reproduction and fathead minnow life-cycle tests. Under cold weather conditions (extended SRTs), the BNR configuration reduced the concentrations of the largest number of TrOCs while under warm weather conditions (reduced SRTs) the CAS-N was most effective. By comparison, YES test results indicated statistically lower responses in the BNR effluent in the warm weather tests and no difference between the effluents of CAS-N and BNR in the cold weather tests. Short term tests with adult zebrafish revealed no impact of the BNR and CAS-N effluents on egg production. By contrast egg production and gene expression in the CAS-exposed zebrafish were substantially less than that of control exposures and were similar to that of exposures to ammonia at similar concentrations as the CAS exposures. In fathead minnow life-cycle tests, exposures to CAS effluent (70-50% v/v) resulted in considerable mortality, reduced growth and reduced egg production that was likely due to the elevated ammonia concentrations. The CAS-N effluent (100% v/v) also resulted in some mortality and reduced growth and egg production in the fathead minnows. By contrast, the BNR effluent (100% v/v) had no effect on mortality, growth or egg production. The results suggest that enhancements to wastewater treatment plants that are associated with improved nitrogen removal can result in enhanced removal of TrOCs and can reduce the harmful effects of the effluents on aquatic biota.

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.

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.

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.

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.

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.

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.

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.

Availability of oncogene activated production system for mass production of light chain of human antibody in CHO cells.

We previously established a ras-oncogene amplified Chinesehamster ovary (CHO) cell line, named ras clone I, as anuniversal host cell line for oncogene activated production(OAP) system to mass-produce recombinant protein by activationof the cytomegalovirus immediate early (CMV) promoter with ras protein. The lambda light chain(C5lambda) of human monoclonal antibody HB4C5 is expected tobe potentially useful for lung cancer targeting. We generated aC5lambda hyper-producing cell line by transfecting ras cloneI with the C5lambda gene expression plasmid regulated by theCMV promoter, of which productivity was 5.3 times greater thanthe hyper productive CHO cell line generated by using conventional CHO cells. Introduction of the adenovirus E1A geneinto the hyper-producing cell line derived from ras clone I resulted in further 9.5 times enhancement of the productivity,suggesting the synergistic effect of E1A and ras oncogenes on the recombinant protein production driven by the CMV promoter. In addition, intracellular accumulation of C5lambda andupregulation of BiP was found in hyper-producing cell lineswhich were introduced E1A and ras oncogene. This resultsuggests that excessive intracellular accumulation ofC5lambda protein, which might be caused by that the amount of produced C5lambda in ER is beyond the ability of CHO cells to secrete, might signal the BiP promoter. Our data imply that ras clone I is available as a general host cell for establishing the recombinant protein hyper-producing CHOcells by the OAP system, and suggest that further mass production of recombinant proteins in the OAP system can be possible by clarifying the accurate role of upregulated BiP protein.

Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes.

To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated 'primed' ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO(2) concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO(2) condition, only the slight effect can be seen. However when cultured in 8% CO(2) condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 mug/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition.

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells.

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO - Chinese hamster ovary; hCMV promoter - immediate early gene promoter of human cytomegalovirus; hIL-6 - human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase - phosphoinositide 3 kinase; PKA - cAMP dependent protein kinase; PKC - protein kinase C.

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.

Autoantibodies in the sera of patients with autoimmune thyroid disease recognize a secreted form of human thyroid peroxidase generated in a baculovirus system.

We used a baculovirus vector to express the cDNA for a truncated (amino acid residues 1-848), secreted form of thyroid peroxidase (TPO) in Sf9 insect cells. Immunoreactive TPO was detected in pooled conditioned media from 10 clones using polyclonal TPO autoantibodies in the sera of patients with autoimmune thyroid disease. As a further test of TPO immunogenicity, the pooled media completely inhibited autoantibody binding to antigen. We used an ELISA to compare autoantibody reactivity to insect cell-derived TPO and TPO antigen produced by Chinese hamster ovary (CHO) cells, the standard form of antigen in present use. In a study of 22 TPO antibody-negative sera and 24 sera with different TPO autoantibody potencies, there was a highly significant correlation (r = 0.977; p < 0.001) in OD values obtained with TPO from the two different sources. The highest producing baculovirus clone generated 8.5 micrograms TPO/ml of conditioned medium, nearly 10-fold higher than previously achieved with stably transfected Chinese hamster ovary cells. Baculovirus-derived, soluble TPO therefore is an excellent source of recombinant TPO in further studies to examine the precise B cell epitopes for human autoantibodies.

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.

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)

Determination at the molecular level of a B-cell epitope on thyroid peroxidase likely to be associated with autoimmune thyroid disease.

In a panel of 13 mouse monoclonal antibodies generated against native (nondenatured) human thyroid peroxidase (TPO), only 1 (monoclonal antibody 47) recognized TPO protein fragments expressed in a human TPO cDNA sublibrary. Determination of the nucleotide sequences of 18 clones recognized by monoclonal antibody 47 localized its epitope to 9 amino acids (residues 713-721) in the human TPO protein. On Western blot analysis, only TPO monoclonal antibody 47 recognized the 933-amino acid TPO molecule after denaturation and reduction of the latter, supporting the concept that the major part of the epitope is represented by a continuous portion of the TPO sequence. The binding of TPO monoclonal antibody 47 to native TPO is inhibited by immunoglobulin G in the serum of patients with autoimmune thyroid disease. The epitope for monoclonal antibody 47 defined in the present study is, therefore, part of or in the vicinity of an epitope for autoimmune thyroid disease-associated TPO antibodies.

A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

Overexpression of an immunologically-intact, secreted form of human thyroid peroxidase in eukaryotic cells.

Recombinant human thyroid peroxidase (hTPO) has been expressed in eukaryotic cells as both the membrane-associated enzyme and as a secreted protein. We now report overexpression of the secreted form of recombinant hTPO in eukaryotic cells. For hTPO gene amplification we used a vector containing the mouse dihydrofolate reductase (DHFR) gene. Stably transfected Chinese hamster ovary (CHO) cells were grown in the presence of increasing concentrations of methotrexate (MTX) and hTPO expression was measured immunologically in an enzyme-linked immunosorbent assay (ELISA). Progressive overexpression of secreted hTPO occurred up to a final MTX concentration of 10,000 nM. Slot-blot analysis of genomic DNA from CHO cells expressing truncated hTPO revealed amplification profiles of the DHFR and hTPO genes to be similar, in parallel with hTPO protein production. High-level expression of secreted hTPO offers the potential for obtaining large amounts of biologically and immunologically active protein for future study.