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Efficient biodegradation of 5-tolyltriazole (5-TTri) in wastewater treatment would minimize its potential detrimental effects on aquatic systems. Therefore, in order to profile 5-TTri biodegrading activated sludge communities (ASC) by DGGE and NGS, acclimation experiments with (i) easily degradable substrates, and (ii) various complex substrates mimicking wastewater conditions were performed. DGGE revealed four genera: Aminobacter (family Phyllobacteriaceae), Flavobacterium (family Flavobacteriaceae), Pseudomonas (family Pseudomonaceae), and Hydrogenophaga (family Comamonadaceae). Metagenomics (DNA) revealed the dominant families Alcaligenaceae, Pseudomonadaceae and Comamonadaceae that also represented the most active families at the RNA level (metatranscriptomics), which might indicate their importance for 5-TTri biodegradation. ASC acclimation and the composition of the substrate significantly affected 5-TTri biodegradation and the development of biodegrading communities. Using acetate only, a moderate 5-TTri degrading community was detected with a very low biodiversity and Pseudomonas spp. as dominant organisms. In contrast, setups fed 'sludge supernatant' (a complex substrate) efficiently biodegraded 5-TTri and formed a more diverse microbial community but with Hydrogenophaga spp. as the dominant group. Finally, a hypothetical 5-TTri biodegradation pathway was constructed based exclusively on the detected, biodegradation-related, Hydrogenophaga spp. genes.

The corrosion inhibitor 5-tolyltriazole (5-TTri) can have a detrimental impact on aquatic systems thus implying an acute need to reduce the effluent concentrations of 5-TTri. In this study, 5-TTri biodegradation was enhanced through acclimation and nutrient supply. Activated sludge communities (ASC) were setup in nine subsequent ASC generations. While generation two showed a lag phase of five days without biodegradation, generations four to nine utilized 5-TTri right after inoculation, with biodegradation rates from 3.3 to 5.2 mg L(-1)d(-1). Additionally, centrifuged AS supernatant was used to simulate the nutrient conditions in wastewater. This sludge supernatant (SS) significantly enhanced biodegradation, resulting in removal rates ranging from 3.2 to 5.0 mg L(-1)d(-1) without acclimation while the control groups without SS observed lower rates of ⩽ 2.2 mg L(-1)d(-1).

Benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) were monitored over 1 year in three wastewater treatment plants (WWTPs) with a membrane bioreactor (MBR-MH) and two conventional activated sludge systems (CAS-E, CAS-M). The influent/effluent concentrations and treatment stages removal efficiencies were monitored. 5-TTri was removed best (mean removal 80%) in the WWTP mainly by biodegradation followed by BTri (mean removal 45%) and 4-TTri (mean removal 15%) that showed a significant lower elimination. High removal fluctuations for all three benzotriazoles occurred over the four seasons with lowest removal during winter. All three WWTPs constituted a point source for BTs in the aquatic environment as concentration measurements in the receiving rivers upstream and downstream of the WWTP proved. While MBR-MH and CAS-M significantly increased the downstream concentrations, CAS-E only slightly increased the downstream concentrations as the receiving river was already contaminated with benzotriazoles from hydropower. 5-TTri was detected in lowest concentrations due to its good removal compared to BTri and 4-TTri that contribute to high effluent concentrations with the potential to accumulate due to insufficient self-purification.

Potential aerobic biodegradation mechanisms of the widely used polar, low-adsorptive sulfonamide antibiotic sulfamethoxazole (SMX) were investigated in activated sludge at bench scale. The study focused on (i) SMX co-metabolism with acetate and ammonium nitrate and (ii) SMX utilization when present as the sole carbon and nitrogen source. With SMX adsorption being negligible, elimination was primarily based on biodegradation. Activated sludge was able to utilize SMX both as a carbon and/or nitrogen source. SMX biodegradation was enhanced when a readily degradable energy supply (acetate) was provided which fostered metabolic activity. Moreover, it was raised under nitrogen deficiency conditions. The mass balance for dissolved organic carbon showed an incomplete SMX mineralization with two scenarios: (i) with SMX as a co-substrate, 3-amino-5-methyl-isoxazole represented the main stable metabolite and (ii) SMX as sole carbon and nitrogen source possibly yielded hydroxyl-N-(5-methyl-1,2-oxazole-3-yl)benzene-1-sulfonamide as a further metabolite.

In situ phosphatase, esterase, lipase, and β-glucuronidase activities were investigated in filamentous scum bacteria by ELF (enzyme labeled fluorescence)-technology. Microthrix parvicella exhibited significant activities for all four enzymes with lipase activity being the highest. In situ activities were considerably higher in activated sludge as compared to scum indicating M. parvicella growth to occur mainly in the sludge fraction. "Nocardioform actinomycetes" showed significant activities for phosphatase, esterase, and β-glucuronidase, lipase activity was only moderate. Activities revealed to be similar for activated sludge and scum. As population densities of enzyme-active actinomycetes were noticeably higher in scum they presumably find good growth conditions in the scum layer. Enzyme activities in Nostocoida limicola morphotypes were only low with no lipase activity being detected. Due to their phylogenetic diversity activity assignment should be combined with fluorescence in situ hybridization (FISH). Numerous type 0041/0675 and type 1851 filaments showed all but lipase activity. However, some of their attached bacteria revealed to be lipase-active. Only a few morphotype 0092 filaments revealed phosphatase, esterase, and β-glucuronidase activity. ELF investigations proved suitable for monitoring in situ filamentous activity. Present imponderabilities of Eikelboom morphotype phylogenetic affiliation are discussed.

Scum biocenoses in activated sludge nutrient removal plants besides nocardioform actinomycetes and Microthrix parvicella frequently accommodate other conspicuous organisms such as Eikelboom types 0041/0675, 0581, 0092, and 1851, as well as Nostocoida limicola-like organisms. In the scum fraction of high load plants often bacteria of type 1863 and non-filamentous actinomycetes are found. Many of these morphologically distinguishable scum bacteria are so-called morphotypes belonging to various groups or genera of bacteria as was proved by fluorescence in situ hybridization (FISH). Aim of this paper is to summarize our respective actual findings and the difficulties to provide practitioners with effective troubleshooting measures for these organisms.

To evaluate the effects of aluminum salts on floating sludge caused by Microthrix parvicella, the addition of AlCl3 was tested in a pilot scale activated sludge treatment plant fed with municipal wastewater. After enriching M. parvicella by dosing an emulsion of vegetable oil and Tween80, an average aluminum application rate per mass of mixed liquor suspended solids of 3.5 g kg-1 d-1 was applied. Specific sludge parameters such as the settling ability and the filament index were measured during the experiment. At the same time the effect of aluminum on the tendency to float and the hydrophobicity of activated sludge as well as on the morphology of M. parvicella was investigated. Microscopic investigations revealed a morphological modification of the filamentous bacterium during adding of AlCl3. M. parvicella was found mostly inside of the activated sludge flocs and appeared as a short filament. Concurrently, an improvement of the settling characteristics of the activated sludge was observed. The tendency to float and the hydrophobicity of activated sludge decreased during the dosing. This is of significant relevance in the attempt to prevent floating sludge accumulation in activated sludge systems. The decrease of hydrophobicity of activated sludge during AlCl3 dosage is probably related to the interaction of the aluminum species with the lipophilic substances and hydrophobic cell surface of M. parvicella (co-precipitation process).

The objective of this study was to quantify scum bacteria by FISH-technique and to correlate their numbers to the hydrophobicity and EPS compounds extracted from sludge and scum. Screening experiments from various wastewater treatment plants (WWTPs) demonstrated that in most plants examined Microthrix parvicella was found in high numbers both in activated sludge and scum. Further nocardioform actinomycetes and type 1863 identified as Acinetobacter spp. could be characterized as important scum bacteria growing in low numbers in activated sludge and being enriched in scum layers. On the basis of these screening results, one nocardioform actinomycetes/Acinetobacter spp.-dominated WWTP1 and one M. parvicella-dominated WWTP2 were selected for detailed investigations. In actinomycetes/Acinetobacter-dominated WWTP1 both lipophilic substances in the EPS and hydrophobic cell surfaces stabilized the scum layer. In M. parvicella- dominated WWTP2 the hydrophobic cell surfaces of these organisms were responsible for the hydrophobic sludge properties and sludge floating. In both of the selected WWTPs the detected lipophilic substances in the EPS improved growth rate and development of hydrophobic cell surfaces and therefore promoted scum processes.

Scum formation is a widespread problem in activated sludge nutrient removal plants. It often comes along with an excessive development of the filamentous bacterium “Microthrix parvicella” stabilizing the flotation process. As “M. parvicella” was found to depend on long-chain fatty acids (LCFA) as sole carbon source not only in vitro but also in situ, some options of in-situ substrate supply are discussed. Wastewater concentrations of fatty acids in the range of 2 to 15 mg L-1 and homologue concentrations from synthetic surfactant degradation below 10 mg L-1 rule out these substrates as source for excessive biomass production. They might, however, well be suitable for start-up of a “M. parvicella” population. Build-up of excessive biomass might rely on fatty acid supply originating in cell walls of lysed stationary phase bacteria of long residence time sludge fractions such as scum layers. Moreover, biogenic surfactants such as rhamnolipids have been proved to be an excellent carbon source for excessive biomass production in vitro. Schaum in nahrstoffeliminierenden Klaranlagen: Der Einfluss von Kohlenstoffquellen auf das Wachstum von „Microthrix parvicella” Starke Schaumbildung ist gegenwartig ein weit verbreitetes Phanomen in Belebungsanlagen mit Nahrstoffelimination. Es geht oft mit einem ubermasigen Wachstum des Fadenbakteriums „Microthrix parvicella” einher, das den Flotationsprozess stabilisiert. Bisherige Befunde zeigen, dass „M. parvicella” auf langkettige Fettsauren als einzige Kohlenstoffquelle angewiesen ist, nicht nur in vitro, d.h. im Labor, sondern auch in situ, d.h. im Belebtschlamm selbst. Daher werden im Folgenden einige Moglichkeiten der Substratversorgung des Organismus in situ diskutiert. Fettsaurekonzentrationen im Abwasser im Bereich von 2 bis 15 mg L-1 und Homologenkonzentrationen aus dem Abbau synthetischer Tenside unter 10 mg L-1 machen diese Quellen als Grund fur exzessives Wachstum unwahrscheinlich. Sie konnten aber sehr wohl eine Rolle beim Aufbau einer „M. parvicella”-Population spielen. Die Bildung exzessiver Biomasse ist dann wahrscheinlich, wenn eine hohe Fettsaurekonzentration aus der Zellwand lysierter Zellen in der stationaren Wachstumsphase zur Verfugung steht, wie dies in Schaumschichten mit extrem hohem Schlammalter gegeben ist. Weiter haben sich biogene, d.h. organismenburtige Tenside, wie z.B. Rhamnolipide, als exzellente Kohlenstoffquelle fur starken Biomassenzuwachs in vitro herausgestellt.

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.

Vitamin supplementation is advertised in wastewater treatment to compensate for a deficiency of growth factors and thereby increase sludge activity and purification efficiency. Addition of vitamins of the B-complex was tested with activated sludge from 5 plants and compared to municipal sludge. Auxotrophy of bacteria isolates turned out to be compensated in most cases by vitamin producers in the activated sludge biocenoses.

Non-filamentous hydrophobic scum bacteria were isolated from scumming wastewater treatment plants (WWTP) by means of adhesion to hydrocarbons. They were characterized with respect to taxonomy, substrate preferences, cell surface hydrophobicity, and emulsification capability. Their role during flotation events is discussed. Rhodococci are selected by hydrolysable substrates and contribute to flotation both by cell surface hydrophobicity and emulsifying activity at long mean cell residence times (MCRT). Saprophytic Acinetobacter strains are able to promote flotation by hydrophobicity and producing emulsifying agents under conditions when hydrophobic substrates are predominant. Hydrogenophaga and Acidovorax species as well as members of the Cytophaga/Flavobacterium group are prone to proliferate under low loading conditions and contribute to flotation mainly by emulsification.

In a municipal two-stage biological wastewater treatment plant with full nitrification, methanol-fed post-denitrification was investigated in a succeeding sand filter system originally designed for the retention of suspended solids in the secondary effluent. Temperature, pH, redox potential, concentration of oxygen and nitrogen-compounds and gas production were determined in situ in the filter bed. In addition, biological parameters such as volatile solids, biofilm thickness, and nitrate elimination efficiency of denitrifying bacteria isolates were determined. The data characterize the microbial processes underlying denitrification in the filter cells and support operation optimization by avoiding detrimental effects on denitrification efficiency such as accumulation of biomass, pH drop and accumulation of undesired intermediates such as nitrite and nitrous oxide.

Background Evaluation of xenobiotics biodegradation potential, shown here for benzotriazoles (corrosion inhibitors) and sulfamethoxazole (sulfonamide antibiotic) by microbial communities and/or pure cultures normally requires time intensive and money consuming LC/GC methods that are, in case of laboratory setups, not always needed. Results The usage of high concentrations to apply a high selective pressure on the microbial communities/pure cultures in laboratory setups, a simple UV-absorbance measurement (UV-AM) was developed and validated for screening a large number of setups, requiring almost no preparation and significantly less time and money compared to LC/GC methods. This rapid and easy to use method was evaluated by comparing its measured values to LC-UV and GC-MS/MS results. Furthermore, its application for monitoring and screening unknown activated sludge communities (ASC) and mixed pure cultures has been tested and approved to detect biodegradation of benzotriazole (BTri), 4- and 5-tolyltriazole (4-TTri, 5-TTri) as well as SMX. Conclusions In laboratory setups, xenobiotics concentrations above 1.0 mg L-1 without any enrichment or preparation could be detected after optimization of the method. As UV-AM does not require much preparatory work and can be conducted in 96 or even 384 well plate formats, the number of possible parallel setups and screening efficiency was significantly increased while analytic and laboratory costs were reduced to a minimum.

Background Sulfamethoxazole (SMX, sulfonamide antibiotic) biodegradation by activated sludge communities (ASC) is still only partly understood. The present work is focusing on nine different bacteria species capable of SMX biodegradation that were isolated from SMX-acclimated ASC. Results Initially 110 pure cultures, isolated from activated sludge, were screened by UV-absorbance measurements (UV-AM) for their SMX biodegradation potential. Identification via almost complete 16S rRNA gene sequencing revealed five Pseudomonas spp., one Brevundimonas sp., one Variovorax sp. and two Microbacterium spp.. Thus seven species belonged to the phylum Proteobacteria and two to Actinobacteria. These cultures were subsequently incubated in media containing 10 mg L-1 SMX and different concentrations of carbon (sodium-acetate) and nitrogen (ammonium-nitrate). Different biodegradation patterns were revealed with respect to media composition and bacterial species. Biodegradation, validated by LC-UV measurements to verify UV-AM, occurred very fast with 2.5 mg L-1 d-1 SMX being biodegraded in all pure cultures in, for UV-AM modified, R2A-UV medium under aerobic conditions and room temperature. However, reduced and different biodegradation rates were observed for setups with SMX provided as co-substrate together with a carbon/nitrogen source at a ratio of DOC:N – 33:1 with rates ranging from 1.25 to 2.5 mg L-1 d-1. Conclusions Media containing only SMX as carbon and nitrogen source proved the organisms’ ability to use SMX as sole nutrient source where biodegradation rates decreased to 1.0 – 1.7 mg L-1 d-1. The different taxonomically identified species showed specific biodegradation rates and behaviours at various nutrient conditions. Readily degradable energy sources seem to be crucial for efficient SMX biodegradation.

The aerobic biodegradation of 5-methyl-benzotriazole (5-TTri) was optimized using lab-scale setups and activated sludge communities (ASC) collected from three wastewater treatment plants (WWTP) MBR-MH, CAS-E and CAS-M being different in their treatment technologies. ASC inocula were diluted to rule out non-biodegrading species and incubated under two nutrient conditions: A) mineral salt media (MSM) and B) carbon and nitrogen supplied MSM giving MSM-CN. 5-TTri removal with the ASC ranged from 60% to 100% in only 10 days. 100 μL suspended biomass from the biodegrading setups was subsequently plated on solid media to eliminate possible activated sludge remnants. After growth occurred, mixed colonies were harvested and inoculated in fresh liquid MSM containing 20 mg L(-1) 5-TTri. These bacterial consortia showed good 5-TTri removal in MSM-CN rather than in MSM, indicating nutrient supply being required for efficient biodegradation. In addition, experiments with high 5-TTri concentrations ranging from 20 to 1,000 mg L(-1) were conducted in both, MSM and MSM-CN and the maximal 5-TTri removal capacity of the ASC evaluated. 50 mg L(-1) 5-TTri was still removed in both media whereas 100 mg L(-1) was solely removed in MSM-CN. 5-TTri biodegradation patterns also indicated that 5-TTri might be co-metabolized by microbial consortia. Furthermore, experiments with gradient-solid-media-plates showed 5-TTri to be inhibitory for the ASC in concentrations above 50 mg L(-1) and revealed the optimal conditions regarding carbon and nitrogen concentration and pH value for effective 5-TTri biodegradation by ASC. Nitrogen proved a crucial factor for enhancing organisms' biodegradation capacity with an optimal pH around 7 while carbon showed no such effect.

The intensive use of benzotriazoles as corrosion inhibitors for various applications and their application in dishwasher detergents result in an almost omnipresence of benzotriazole (BTri), 4-methyl- and 5-methyl-benzotriazole (4-TTri and 5-TTri, respectively) in aquatic systems. This study aims on the evaluation of the biodegradation potential of activated sludge communities (ASCs) toward the three benzotriazoles regarding aerobic, anoxic, and anaerobic conditions and different nutrients. ASCs were taken from three wastewater treatment plants with different technologies, namely, a membrane bioreactor (MBR-MH), a conventional activated sludge plant CAS-E (intermittent nitrification/denitrification), and CAS-M (two-stage activated sludge treatment) and used for inoculation of biodegradation setups. All ASCs eliminated up to 30 mg L(-1) 5-TTri and BTri under aerobic conditions within 2-7 and 21-49 days, respectively, but not under anoxic or anaerobic conditions. 4-TTri was refractory at all conditions tested. Significant differences were observed for BTri biodegradation with non-acclimated ASCs from MBR-MH with 21 days, CAS-E with 41 days, and CAS-M with 49 days. Acclimated ASCs removed BTri within 7 days. Furthermore, different carbon and nitrogen concentrations revealed that nitrogen was implicitly required for biodegradation while carbon showed no such effect. The fastest biodegradation occurred for 5-TTri with no need for acclimatization, followed by BTri. BTri showed sludge-specific biodegradation patterns, but, after sludge acclimation, was removed with the same pattern, regardless of the sludge used. Under anaerobic conditions in the presence of different electron acceptors, none of the three compounds showed biological removal. Thus, presumably, aerobic biodegradation is the major removal mechanism in aquatic systems.

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.

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.

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.

Detection of filamentous bacteria morphotypes involved in scum formation in activated sludge wastewater treatment plants by conventional sludge microscopy is often doomed to fail because of morphological and taxonomical variations. The aim of this study is to compare detection, identification, and quantification quality of filamentous "scum bacteria" found by conventional activated sludge microscopy and fluorescence in situ hybridization (FISH). In the case of filamentous Microthrix parvicella and Eikelboom morphotypes 1863 and 1851, conventional activated sludge microscopy and FISH results correspond well. In contrast, conventional activated sludge microscopy overlooks nocardioform actinomycete and type 1863 single cells. On the other hand, FISH underestimates filamentous nocardioform actinomycetes and morphotypes 0041/0675 or 0092 because of insufficient cell wall permeability or because of their taxonomic variability, with a resulting inadequacy of previously published probes. Nostocoida limicola morphotype results are still inconclusive because of low bacteria numbers being available in situ and the enormous taxonomic variability within this group.

The content of the water-soluble vitamins thiamine, riboflavin and folic acid was determined in the dry solids and the water phase of activated sludge and digested sludge respectively, in municipal and industrial wastewater treatment plants. Thiamine and riboflavin were determined by fluorimetric detection. Folic acid was determined by means of microbiological assays. Dry solids of activated sludge from municipal wastewater treatment plants contained 1.1–6.6 mg kg−1 thiamine, 17.6–43.2 mg kg−1 riboflavin and 10–13 mg kg−1 folic acid with 1–6 μg l−1 thiamine and 9–25 μg l−1 riboflavin in the water phase. Dry digested sludge matter contained 0.9–6.0 mg kg−1 thiamine with a content of the water phase after filtration of 18–386 μg l−1, riboflavin was 3.2–10.8 mg kg−1 and 6–116 μg l−1, respectively, folic acid was 0.25–0.62 mg kg−1 in the dry solid matter, none was found in the water phase. Dry solids of activated sludge from industrial sewage treatment plants contained 1.9–29.1 mg kg−1 thiamine, the water phase after filtration contained 3–14 μg l−1, riboflavin was 21.3–41 mg kg−1 with 1–26 μg l−1 in the water phase, folic acid was 2 mg kg−1 in the dry solids of activated sludge. The vitamin content varies in different sludge fractions. The requirements of heterotrophic saprophytes for thiamine and riboflavin should usually be met by the vitamin content in the sludge. Before dosing the vitamin content should be monitored.

Summary From 14 sewage treatment plants in southern Germany, Berlin and Switzerland sufferingfrom scum production in the aeration tank, 14 strains of nocardioform actinomycetes have been isolated. 11 strains belonged to the Rhodococcus rhodochrous group and fell into three different species. 2 strains belonged to the Gordona aurantiaca group, 1 strain was identified as Nocardia amarae . Physiological tests revealed marked differences between the strains indicating that specific scum control methods are needed depending on the actinomycete strain responsible for scumming. Some Rhodococcus strains were susceptible to heavy metals such as FeCl 3 . Growth of Nocardia amarae could be limited by supernatant of the anaerobic digester. No connection of scum production with the nitrogen and phosphorus content of the influent was found as is often assumed in the literature. The dependence of scum production on the grease and oil content of the influent is discussed. Since the isolated actinomycetes were found to have the ability to synthesize lipidic cell material from proteins and carbohydrates, there is no indication of a strong relation.