Margit Schade

Performance characteristics of qPCR assays targeting human- and ruminant-associated Bacteroidetes for microbial source tracking across sixteen countries on six continents

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Population density and enzyme activities of heterotrophic bacteria in sewer biofilms and activated sludge

Abstract The heterotrophic activity of different wastewater biocenoses is assessed. The biofilms originate from a sewer discharging domestic wastewater and from a sewer discharging trade wastewater with chromium and nickel contamination. The activated sludge was taken from the aeration tank from the high load stage (F:M ratio 0.8 kg kg −1 d −1 ) and from the low load stage (F:M ratio 0.1 kg kg −1 d −1 ) of a two-stage municipal wastewater treatment plant. We determined the population density of heterotrophic saprophytes, polymer degrading bacteria, ammonifying bacteria as well as of nitrate reducers and denitrifiers. Enzyme activities were determined as relative substrate turnover rates for esterase, α- and β-glucosidase, for phosphatase and for l -alanine-aminopeptidase. The population density of heterotrophic saprophytes was one order of magnitude higher in the high load stage compared to the low load activated sludge. The sewer biofilms showed one to two orders of magnitude higher counts per g dry weight for the heterotrophic bacteria as compared to the activated sludges. The turnover rates of esterase and l -alanine-aminopeptidase were higher than those of α- and β-glucosidase and phosphatase. The sewer biofilms are highly active biocenoses both with respect to population densities as with respect to enzyme activities. Their heterotrophic activity is comparable with the one in high load activated sludge. Even the bacteria in the biocenosis which is exposed to high concentrations of chromium and nickel showed a high activity.

Scum in Activated Sludge Plants: Impact of Non‐filamentous and Filamentous Bacteria

Excessive scum production is a widespread phenomenon in present activated sludge wastewater treatment. The question how foaming is initiated and stabilized is still unanswered. Hydrophobic wastewater ingredients and surface active material such as synthetic surfactants are discussed among others as major causative agents for scum production. Focusing on biological impacts non-filamentous bacteria isolated from scum turned out to contribute to flotation by both cell surface hydrophobicity and emulsification activity, depending on the prevailing substrate and milieu conditions. The biological characterization of scum based on microscopic sludge investigation of conspicuous microorganisms resulted in a significant shift of filamentous and non-filamentous organism populations with Gram-positive bacteria prevailing in present nutrient removal plants as compared to the situation ten years ago. Their hydrophobic cell surface is supposed to support adherence and stabilization of interfaces and thus promote sludge flotation. In scum six types of filamentous bacteria turned out to be numerous: Microthrix parvicella and nocardioform actinomycetes, both of them being enriched in the scum fraction, moreover Nostocoida limicola and Eikelboom types 0041/0675, 1851, and 0092. Possible interactive mechanisms between non-filamentous and filamentous scum bacteria and their selection factors are discussed in order to contribute to a better understanding of scum formation and to provide efficient troubleshooting measures. Schaum in Belebungsanlagen: Einfluss nicht-fadiger und fadiger Bakterien Starke Schaumbildung ist gegenwartig ein weit verbreitetes Phanomen in Klaranlagen nach dem Belebungsverfahren. Die Frage nach Entstehung und Stabilisierung dieser Schaume ist nach wie vor ungeklart. Hydrophobe Abwasserbestandteile und oberflachenaktive Stoffe wie etwa Tenside werden unter anderem als wichtige Ursache der Schaumbildung diskutiert. Die Suche nach biologischen Einflussen ergab, dass aus Schaum isolierte Bakterien ohne Fadenbildung sowohl durch Oberflachenhydrophobierung als auch durch die Bildung stabiler Emulsionen zur Flotation beitragen, abhangig von den vorherrschenden Substrat- und Milieubedingungen. Die biologische Charakterisierung von Schaum, basierend auf mikroskopischen Untersuchungen auffalliger Organismen in belebtem Schlamm, ergab in Anlagen mit Stickstoff- und Phosphorentfernung eine deutliche Verschiebung hin zu Gram-positiven Bakterien – Fadenbakterien und solche, die nicht fadig wachsen – im Vergleich zu den vor etwa 10 Jahren ublichen Anlagen zur reinen Elimination von Kohlenstoffverbindungen. Die hydrophobe Oberflache dieser Organismen scheint der Adhasion an und Stabilisierung von Grenzflachen und damit der Flotation forderlich zu sein. In Schaum wurden sechs wichtige Fadenorganismen festgestellt: Microthrix parvicella und nocardioforme Actinomyceten, die beide in der Schaumfraktion angereichert werden, des Weiteren Nostocoida limicola und die zunachst noch mit Nummern benannten Fadenbakterien Eikelboom Typ 0041/0675, 1851 und 0092. Mogliche Interaktionen zwischen nicht-fadigen und fadigen Schaumbakterien sowie ihre Selektionsfaktoren werden diskutiert. Damit wird angestrebt, dem Praktiker uber ein besseres Verstandnis der Schaumbildung effektive Bekampfungsmasnahmen an die Hand zu geben.

Occurrence of human-associated Bacteroidetes genetic source tracking markers in raw and treated wastewater of municipal and domestic origin and comparison to standard and alternative indicators of faecal pollution.

This was a detailed investigation of the seasonal occurrence, dynamics, removal and resistance of human-associated genetic Bacteroidetes faecal markers (GeBaM) compared with ISO-based standard faecal indicator bacteria (SFIB), human-specific viral faecal markers and one human-associated Bacteroidetes phage in raw and treated wastewater of municipal and domestic origin. Characteristics of the selected activated sludge wastewater treatment plants (WWTPs) from Austria and Germany were studied in detail (WWTPs, n = 13, connected populations from 3 to 49000 individuals), supported by volume-proportional automated 24-h sampling and chemical water quality analysis. GeBaM were consistently detected in high concentrations in raw (median log10 8.6 marker equivalents (ME) 100 ml−1) and biologically treated wastewater samples (median log10 6.2–6.5 ME 100 ml−1), irrespective of plant size, type and time of the season (n = 53–65). GeBaM, Escherichia coli, and enterococci concentrations revealed the same range of statistical variability for raw (multiplicative standard deviations s* = 2.3–3.0) and treated wastewater (s* = 3.7–4.5), with increased variability after treatment. Clostridium perfringens spores revealed the lowest variability for raw wastewater (s* = 1.5). In raw wastewater correlations among microbiological parameters were only detectable between GeBaM, C. perfringens and JC polyomaviruses. Statistical associations amongst microbial parameters increased during wastewater treatment. Two plants with advanced treatment were also investigated, revealing a minimum log10 5.0 (10th percentile) reduction of GeBaM in the activated sludge membrane bioreactor, but no reduction of the genetic markers during UV irradiation (254 nm). This study highlights the potential of human-associated GeBaM to complement wastewater impact monitoring based on the determination of SFIB. In addition, human-specific JC polyomaviruses and adenoviruses seem to be a valuable support if highly specific markers are needed.

Specific binding of aluminium and iron ions to a cation-selective cell wall channel of Microthrix parvicella.

Heavy metal salts containing aluminium or iron are used in wastewater treatment to control excessive growth of the Gram-positive bacterium Microthrix parvicella, frequently observed in wastewater plants suffering from bulking, foaming and scum. Microthrix parvicella belongs to the class Actinobacteria but not to mycolata, although its taxonomic position in this class is not identified. Investigations using the microspheres adhesion to cells method (MAC) suggested that M. parvicella is as strongly hydrophobic as the mycolic acid containing actinomycetes. The cell wall of M. parvicella was investigated for the presence of water-filled channels using the lipid bilayer assay. An ion-permeable channel called MppA with a conductance of 600 pS in 1 M KCl was identified in cell wall extracts and purified to homogeneity. The cation-selective channel showed no voltage-dependent closure at higher voltages. Interestingly, MPPA could be blocked by heavy metal ions. Binding of polyvalent cations such as iron and aluminium was studied in titration experiments and revealed stability constants for their binding to MppA up to 700 M(-1). The cell wall channel of M. parvicella contains a binding site for polyvalent cations which may play a crucial role for the effect of heavy metals salts on M. parvicella-dominated activated sludge.

Global Distribution of Human-Associated Fecal Genetic Markers in Reference Samples from Six Continents

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750–4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log10 7.2–8.0 marker equivalents (ME) 100 mL–1) and biologically treated wastewater samples (median log10 4.6–6.0 ME 100 mL–1) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.