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The DNA fingerprinting techniques, 16S-restriction fragment length polymorphism (16S-RFLP), ribosomal intergenic spacer analysis (RISA) and repetitive extragenic palindrome PCR (Rep-PCR), were used for analyzing the bacterial communities of seven pulp and paper wastewater treatment systems. All three methods generate DNA fingerprints that can be compared using the computer-assisted program, Gelcompar©. Community similarity coefficients were based on quantitative determinations of both the positions of the DNA bands and the band intensities in order to compare the relative differences in the populations. Unique 16S-RFLP DNA fingerprints were observed for each mill suggesting that individual mills contained phylogenetically different communities. However this method was not sensitive enough to detect differences within a mill treatment system from different locations or from different sampling times. The RISA method, which generated more complex fingerprints than 16S-RFLP, could, for some mills, discern differences between samples. The Rep-PCR technique, however, showed the highest degree of resolution and produced not only distinct patterns for each mill but also distinct fingerprints for the temporal and spatial samples from some of the treatment systems. The sensitivity of this method might potentially be used to monitor the stability of the bacterial community within a secondary treatment system.

The abundance of ammonia-oxidizing bacteria and archaea and their amoA genes from the aerobic activated sludge tanks, recycled sludge and anaerobic digesters of a full-scale wastewater treatment plant (WWTP) was determined. Polymerase chain reaction and denaturing gradient gel electrophoresis were used to generate diversity profiles, which showed that each population had a consistent profile although the abundance of individual members varied. In the aerobic tanks, the ammonia-oxidizing bacterial (AOB) population was more than 350 times more abundant than the ammonia-oxidizing archaeal (AOA) population, however in the digesters, the AOA population was more than 10 times more abundant. Measuring the activity of the amoA gene expression of the two populations using RT-PCR also showed that the AOA amoA gene was more active in the digesters than in the activated sludge tanks. Using batch reactors and ddPCR, amoA activity could be measured and it was found that when the AOB amoA activity was inhibited in the anoxic reactors, the expression of the AOA amoA gene increased fourfold. This suggests that these two populations may have a cooperative relationship for the oxidation of ammonia.

To elucidate how widespread antibiotic resistance is in the surface water environment, we studied the prevalence of antibiotic resistance bacteria at four locations in southern Ontario. We found that the percentage of bacteria resistant to the antibiotic tetracycline was higher at the river site, which flows through agricultural land, and lower at the lake sites. A total of 225 colonies were selected for further testing of antibiotic disc susceptibility to eight different antibiotics to calculate the multiple antibiotic resistance (MAR) score and the antibiotic resistance index for each site. Although the isolates from the lake site outside the city displayed resistance to fewer antibiotics, their MAR scores were not significantly different from that of the lake sites adjacent to urban beaches, showing that MAR was widespread in the natural water environments tested. Isolation of colonies under selection pressure to tetracycline was found to have a significant effect on the likelihood that the isolates would contain multiple resistance traits for other antibiotics. Identification of isolates selected on tetracycline was compared with that of isolates that were sensitive to tetracycline, and the community composition was found to be distinctly different, although isolates from the genera Chryseobacterium, Pseudomonas, and Stenotrophomonas were found in both communities.

The activated sludge process in a wastewater treatment plant (WWTP) relies on the activity of microbes to reduce the organic and inorganic matter and produce effluent that is safe to discharge into receiving waters. This research examined the effects of the non-steroidal anti-inflammatory drug ibuprofen and the antibiotic tetracycline on the relative abundance and composition of eukaryotes and bacteria in the microbial population present in activated sludge from a WWTP. The current investigation was designed to observe the impact of these contaminants, at low (environmentally relevant concentrations) as well as high concentrations of the drugs. Using 16S and 18S rRNA gene primer sets and quantitative polymerase chain reaction, the abundance of each population was monitored as well as the relative ratio of the two populations under the various conditions. It was found that current environmentally relevant concentrations of ibuprofen (100 ng/mL) stimulated eukaryotic growth but higher concentrations (2,000 ng/mL, 100,000 ng/mL) reduced their numbers significantly especially in the presence of tetracycline. Finally using denaturing gradient gel electrophoresis, some of the more abundant eukaryotes were identified and it was noted that high ibuprofen and tetracycline concentrations favoured the abundance of some genera.

The reduction and degradation of total organic carbon (TOC) and bacteria from a secondary effluent of synthetic slaughterhouse wastewater using vacuum-ultraviolet (VUV) and ultraviolet-C (UV-C) processes and their combination (UV-C/VUV and VUV/UV-C) were investigated. The TOC removal rates under continuous mode operation ranged from 5.5 to 6.2%. In addition, the treatment with the UV-C/H2O2 and VUV/H2O2 processes under continuous mode operation doubled the TOC removal rates 10.8 and 12.2%, respectively. The optimum molar ratio of H2O2/TOC was found to be 2.5 and 1.5 for the UV-C and VUV processes, respectively. It was observed that all photochemical processes were able to totally inactivate different strains of bacteria with concentrations up to 105 CFU/mL within 27.6 s. Finally, a kinetic model was developed to simulate the TOC degradation from a secondary effluent of synthetic slaughterhouse wastewater.

Recent developments in water quality research have highlighted difficulties in accurately predicting the incidence of pathogens within freshwater based on the viability, culturability and metabolic activity of indicator organisms. QPCR-driven assays are candidates to replace standard culture-based methods, however, protocols suitable for routine use have yet to be sufficiently validated. The objective of this study was to evaluate five oligonucleotide primers sets (ETIR, SINV, exoT, VS1 and ipaH2) for their potential applicability in qPCR assays to detect contamination from five waterborne bacterial pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Campylobacter jejuni, Pseudomonas aeruginosa, and Shigella flexneri). An enrichment-free qPCR protocol was also tested using S. Typhimurium-seeded source water, combining membrane filtration and mechanical, chemical and enzymatic lysis techniques to recover the bacterial cells. All five primer sets were found to have high specificity and sensitivity for the tested organisms. Four of the primers were able to detect pathogen loads as low as 10 cells/mL while 200 cells/mL of C. jejuni were detectable in pure culture. Although sensitivity decreased in an artificially contaminated environmental matrix, it was still possible to detect as few as 10 S. Typhimurium cells without enrichment. The primers and protocols evaluated in this study have demonstrated potential for further validation for possible application alongside traditional indicator techniques.

The conventional biological treatment process can provide a favorable environment for the maintenance and dissemination of antibiotic‐resistant bacteria and the antibiotic resistance genes (ARG) they carry. This study investigated the occurrence of antibiotic resistance in three wastewater treatment plants (WWTP) to determine the role they play in the dissemination of ARGs. Bacterial isolates resistant to tetracycline were collected, and tested against eight antibiotics to determine their resistance profiles and the prevalence of multiple antibiotic resistance. It was found that bacteria resistant to tetracycline were more likely to display resistance to multiple antibiotics compared to those isolates that were not tetracycline resistant. Polymerase chain reaction (PCR) was used to identify the tetracycline resistance determinants present within the bacterial communities of the WWTPs and receiving waters, and it was found that ARGs may not be released from the treatment process. Identification of isolates showed that there was a large diversity of species in both the tetracycline‐resistant and tetracycline‐sensitive populations and that the two groups were significantly different in composition. Antibiotic resistance profiles of each population showed that a large diversity of resistance patterns existed within genera suggesting that transmission of ARG may progress by both horizontal gene and vertical proliferation.

The ultimate specificity in molecular-based assays for pathogen detection relies on the design of the primers and probes. Their ability to hybridize to DNA sequences found only in pathogens can be realized by designing primers and probes that are complementary to pathogen-specific virulence genes. This study evaluates the detection and enumeration strengths of real-time PCR (qPCR) and fluorescent in situ hybridization (FISH) for selected waterborne pathogens and their ultimate applicability within a monitoring framework. Detection limits calculated in the qPCR assay were 150 tir (intimin protein receptor) gene copies for Escherichia coli O157:H7 and 2 × 103invA (inner membrane invasive protein) gene copies for Salmonella enterica serovar Typhimurium. Detection limits were, however, at least 100-fold less sensitive in wastewater extracts, partly because of the inhibitory effect of the wastewater itself. Fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay. While this research demonstrates the potential detection strength of qPCR, it highlights the need for strong dependable primer and probe sets among PCR and FISH methodologies as well as the need for further signal amplification with DNA-targeted FISH for single-copy gene targets within environmental samples.

The bacterial community composition in an activated sludge plant treatment from a bleached kraft pulp mill was monitored over a period of 209 days. Using DGGE and terminal-Restriction Fragment Length Polymorphism (t-RFLP) analysis we generated community DNA fingerprints over the time period. Both methods produce fingerprints that can be used to monitor stability in the system and generate fragments that can be associated with bacterial taxa. Chemical and physical parameters were also collected during that same time frame. We found a number of significant correlations with influent variables such as temperature, chemical oxygen demand (COD), Biochemical oxygen demand (BOD) and chloroform concentrations suggesting that these were the most likely parameters to influence the bacterial community structure. In addition several taxa correlated to important performance indicators such as COD/BOD removals and SVI. Multivariate analysis also confirmed the strong links between taxa variation and temperature, nutrient loads, chloroform and also one class of filaments. Establishing the identity of these taxa and their ecological preferences will greatly enhance our understanding and management of biological treatment systems.

The safety of potable water relies on sensitive and specific testing of samples for pathogenic waterborne agents. Traditional methods often fail to recover or detect the causative pathogen and rely on the detection of indicator organisms to judge the safety of the samples. Molecular technology has grown enormously in the past several decades and lends itself to the development of rapid, sensitive and specific tests for pathogens in all types of samples. This chapter reviews the molecular methods that have been designed and used to detect viruses, bacteria, protozoa, and cyanobacteria from water and environmental samples. It gives a brief description of each method followed by examples of where the method has been used. Detection limits of each method are noted where available along with advantages and disadvantages of each technique.

Community-level characterization of microbial biomass from a municipal activated sludge plant, and two bleached kraft mill effluent (BKME) treatment systems, an oxygenated activated sludge system and an aerated lagoon system, was carried out using the BIOLOG redox-based carbon-substrate utilization assay. Several extraction procedures for separating microbial cells from the dark colloidal samples, including culture enrichment, homogenization, gravel agitation and sonication, were evaluated in order to overcome interference with colour development in the assay. Optimal microbial recovery was achieved by homogenization in the presence of deflocculating agents, sodium pyrophosphate and Tween 80. Principal component analysis was used to differentiate among the microbial communities in the wastewater treatment systems. Biomass obtained from each of the reactors displayed different composite metabolic profiles suggesting a unique indigenous microbial population in each system. Characterization of separate bacterial and protozoal components of one community also showed that each fraction displayed different substrate utilization patterns. Substitution of the already prepared BIOLOG panel substrates with individual chemicals typically associated with BKME allowed for the determination of biodegradation potential in wastewater treatment systems.

The short-term impacts of atrazine (herbicide), tributyltin (organometal) and copper on the behaviour of Euglena gracilis Klebs (Euglenophyta) were assessed. First, the ECOTOX automated image analysis system was used, which measured swimming velocity, cell shape, percentage of cells swimming upwards, and randomness of swimming. Next, visual observation by microscopy was used to measure percentage of cell motility and cell shape. Behavioural changes can be used as an indicator of stress in less than 24 h, potentially making them suitable for inclusion in early-warning systems for water quality. Findings indicate that E. gracilis is a very sensitive organism to copper, showing inhibition of motility with visual observation at 0.8 μmol/L within 1 h. The image analysis system was in general less sensitive than visual observation for detecting behavioural changes after incubation in copper. In contrast, after exposure to organic contaminants atrazine and tributyltin, the ECOTOX system detected small changes in the number of cells swimming upwards (antigravitactic behaviour) at higher concentrations.

During past decades, biomonitors were deployed in lakes and rivers to rapidly detect hazardous chemicals by measuring the endpoints of a single aquatic species at defined short intervals. Most biomonitors, however, are only capable of indicating a departure from baseline water conditions without identifying the cause. In order to provide a more comprehensive assessment, a biomonitoring system which features a library of stereotyped responses of multiple aquatic species in various water conditions is proposed. A preliminary library was constructed by characterizing the behavioural and physiological responses of Daphnia magna, Hyalella azteca, Lumbriculus variegatus, and Pseudokirchneriella subcapitata to various concentrations of atrazine and tributyltin. By employing multivariate statistical tools such as principal component analysis (PCA) and discriminant analysis, this library (which contained responses after 6 h of exposure to contaminants) was used as a template to classify and to model other sets of earlier measurements at 2 and 4 h, resulting in an accuracy of 73 and 97%, respectively. These findings demonstrated the potential capability of the proposed early-warning biomonitoring system to provide real-time water quality assessment and early-warning contaminant detection.

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

Protozoa feed upon free-swimming bacteria and suspended particles inducing flocculation and increasing the turnover rate of nutrients in complex mixed communities. In this study, the effect of protozoan grazing on nitrification was examined in activated sludge in batch cultures maintained over a 14-day period. A reduction in the protozoan grazing pressure was accomplished by using either a dilution series or the protozoan inhibitor cycloheximide. As the dilutions increased, the nitrification rate showed a decline, suggesting that a reduction in protozoan or bacterial concentration may cause a decrease in nitrification potential. In the presence of cycloheximide, where the bacterial concentration was not altered, the rates of production of ammonia, nitrite, and nitrate all were significantly lower in the absence of active protozoans. These results suggest that a reduction in the number or activity of the protozoans reduces nitrification, possibly by limiting the availability of nutrients for slow-growing ammonia and nitrite oxidizers through excretion products. Furthermore, the ability of protozoans to groom the heterotrophic bacterial population in such systems may also play a role in reducing interspecies competition for nitrification substrates and thereby augment nitrification rates.Key words: nitrification, activated sludge, protozoan grazing, ammonia-oxidizing bacteria, cycloheximide.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown α-, β-, and γ-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.Key words: biodiversity, 16S-RFLP, secondary treatment, pulp mills.