Detection of filamentous and nitrifying bacteria in activated sludge with 16S rRNA probes

Activated sludge samples from wastewater treatment plants from potato starch and starch derivatives factory and from a municipal sewage treatment plant were analyzed with DNA probes specific for several filamentous bacteria. It was found that Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix sp. and Eikelboom Type 021N were common in the activated sludges. Fluorescent in situ Hybridization (FISH) analysis could detect more types of sheathed bacteria and yielded a more accurate quantification of bacteria than conventional microscopy.In a pilot and a full scale wastewater treatment plant (WWTP) clear correlations were found between the SVI and growth of a Sphaerotilus natans and a Thiothrix sp. Addition of chlorine to the bulking sludge resulted in an improved SVI of the sludge but only damaged filamentous cells outside the floc. Nitrification was measured with substrate depletion and FISH analysis. Signal interpretation of FISH analysis was demonstrated both manually and with automated image analysis.     

Evaluation of filamentous microorganism growth factors in an industrial wastewater activated sludge system

An investigation comprising four studies was undertaken to determine possible factors affecting the growth of several different types of filamentous microorganisms present in a bulking industrial wastewater activated sludge. Results from laboratory-scale continuous-flow and full-scale studies suggested that DO concentration and F:M ratio were the likely key factors affecting filamentous growth in the activated sludge. From the results of two laboratory studies isolating the effects of DO concentration and F:M ratio on filamentous growth, favorable growth ranges of DO concentration or F:M ratio were estimated for the following filaments: Microthrix parvicella, Nocardia spp., Nostocoida limicola II, and Types 0041, 1851, and 1863. Most of the bacteria causing filamentous bulking of the activated sludge were found to be filaments typically associated with low F:M, and increasing the F:M ratio appeared to cause N. limicola II to lose its competitive advantage in the activated sludge system. Type 1863, on the other hand, was found to be a low DO filament, as DO concentrations of 0.1 mg O₂/l or less appeared to be a necessary condition for its filamentous growth. Though Nocardia was found to be a low F:M filament, its growth also seemed to be affected by DO concentration, as its growth was stimulated by concentrations of 1.0 mg O₂/l or greater, with a near linear relationship up to at least 5 mg O₂/l.     

Phylogenetic assessment of five filamentous bacteria isolated from bulking activated sludges

Nearly complete 16S ribosomal RNA (TRNA) sequences were determined for fully characterised axenic strains of Thiothrix, Eikelboom type O21N, and Eikelboom type 1701 originally isolated from bulking activated sludges. Thiothrix strains formed a monophyletic group (100% bootstrap support) with previously described Thiothrix nivea strain JP2 and Thiothrix ramosa. Eikelboom type O21N strain AP3 revealed a sufficiently strong relationship to the Thiothrix group to suggest a common ancestry for the two organisms although it was not possible to designate type O21N as a species of Thiothrix. Eikelboom type 1701 contained within its sequence the target sequence of an oligonucleotide probe for the detection of Sphaerotilus natans.     

Effectiveness of oligonucleotide probes targeted against Thiothrix nivea and type 021n 16s rRNA for in situ identification and population monitoring in activated sludges

In situ hybridization (FISH) of two fluorescent oligonucleotide probes, TNI and 21N, directed against 16S rRNA fragments of Thiothrix nivea and type 021N filamentous bacteria, was used to study activated sludge samples of various origins. Comparison of the results obtained by this technique with morphological data enabled us to evaluate the sensitivity and specificity of the probes, as well as to propose practical classification criteria, in an effort to correlate the identification and ecology of these two microorganisms. We applied the two oligonucleotide probes to the study of Thiothrix sp. in pilot and laboratory batch reactors fed a substrate that was moderately deficient in rapidly available phosphorus. We monitored the growth dynamics of Thiothrix sp. in the pilot reactor, using various hydraulic configurations and after certain perturbations, such as transient substrate overloads. The results provided data which led us to question the importance of the length of time oxygen-deficient sludges spend in clarifier tanks and we discuss the effects of both moderate reductions in oxygen and of very intermittent feedings on the occurrence of Thiothrix sp. growth peaks. The identification and quantification of these filamentous bacteria by size class, made possible by in situ hybridization, are shown to be powerful tools for the early detection and evaluation of episodes of proliferation of filamentous bacteria, revealing them to be much more sensitive indicators than the sludge volume index (SVI).     

自然河道中沉水植物苦草对水流的生理响应

通过野外现场研究苦草生长和生理对水流的响应规律。结果表明:30~40 cm/s的水流速度对苦草的生长形态有影响;与静水区相比,动水区中苦草植株矮小、叶片较窄。在植物生长旺季和生殖生长期间,动水对植物细胞内的蛋白质和可溶性糖含量有促进作用,同时也诱导过氧化氢(H2O2)等活性氧上升,使得超氧化物歧化酶(SOD)、过氧化氢酶(CAT)等抗氧化酶活性相应提高以降低活性氧伤害;30~40 cm/s的流速对苦草生长有一定的影响,但不影响苦草在河道生态修复中的应用。     

RNA/DNA ratio as an indicator of metabolic activity in resin acid-degrading bacteria

We investigated the relationship between the growth rate and the ratio of RNA to DNA in of four resin acid degrading bacteria isolated from a sequencing batch reactor (SBR). Chemical assays as well as slot blot hybndizations with species-specific oligonucleotide probes were used to quantify the nucleic acids. These slow-growing bacteria have a positive linear correlation between growth rate and RNA/DNA ratio, similar to faster-growing bacteria like E. coli. We propose to use this correlation to measure metabolic activities of selected resin acid degrading bacteria in the complex community of the SBR in situ using species-specific hybridization probes. Preliminary experiments suggest that hybridization probes can be used to detect growth rate-dependent changes in the RNA/DNA ratio.     

Biodegradation of phenolic compounds by an acclimated activated sludge and isolated bacteria

The degradation of a mixture of phenol, 4-chlorophenol (4CP), 2,4-dichlorophenol (24DCP) and 2,4,6-trichlorophenol (246TCP) by acclimated activated sludge and by isolated bacteria was studied. Activated sludge was acclimated for 70 days to 40 mg phenols/1 then the microorganisms responsible for the CP degradation were isolated and identified. Four types of Gram-negative bacteria (Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, and Chryseomonas luteola) were identified. Also, two acid-fast bacilli with distinct glycolipid patterns were isolated. From their chemical composition and their growth characteristics, both isolates appeared to be mycobacteria closely related to Mycobacterium peregrinum. The degradation kinetics of each phenol by Aeromonas sp., Pseudomonas sp. Flavomonas oryzihabitans, Chrvseomonas luteola and activated sludge were determined. The acclimated activated sludge degradation rates were from one to two orders of magnitude higher than those of pure strains when uptake rates were calculated in terms of the viable biomass (CFU). The specific substrate uptake rate for acclimated activated sludge varied between 8.2 and 15.8 ß 10⁻⁷ mg/CFU·d (407-784 mg/BVSS·d). Aeromonas sp. had the highest specific substrate uptake rate of the pure strains, based on a VSS basis (33–57 mg/gVSS·d) but, in terms of viable biomass (5.0–15.6 × 10⁻⁸ mg/CFU·d), the Pseudomonas sp. rate was the highest. Specific substrate uptake rates were 1.8 mg chlorinated phenoWg VSS-d for unacclimated activated sludge.     

Autecology of scum producing bacteria

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.     

Survey of filamentous populations in nutrient removal plants in four European countries

A joint EU research project aimed at solving activated sludge bulking in nutrient removal plants was initiated in 1993. The project started with a survey of the size and composition of the filamentous population in nutrient removal plants in Denmark, Germany, Greece and the Netherlands.The results show that biological nutrient removal process conditions indeed favour filamentous microorganisms in their competition with floc forming organisms. An increase in the size of the filamentous population resulted in a deterioration of the settling properties of the biomass, except for plants with Bio-P removal conditions. It is assumed that in the latter case the dense clusters of Bio-P bacteria increase the weight of the flocs, and compensate for the effect of the larger number of filaments.Although exceptions frequently occur, the following sequence in decreasing filamentous organism population size was observed for the process conditions indicated:

• -completely mixed + simultaneous denitrification;
• -completely mixed + intermittent aeration/denitrification;
• -alternating anoxic/oxic process conditions, with an anaerobic tank for biological phosphate removal (Bio-Denipho);
• -alternating anoxic/oxic process conditions (Bio-Denitro);
• -predenitrification

The surveys provided little information about the effect of nutrient removal in plants with plug flow aeration basins. Simultaneous precipitation with aluminium salts nearly always resulted in a low number of filaments and a good settling sludge.The size of the filamentous organism population showed a seasonal pattern with a maximum in winter/early spring and a minimum during summer (in Greece: during autumn). This seasonal variation is primarily caused by the effect of the season on the population sizes of M. parvicella, N. limicola and Type 0092.M. parvicella is by far the most important filamentous species in nutrient removal plants. In Denmark only, Type 0041 also frequently dominates the filamentous population, but seldom causes severe bulking. Considering their frequency of occurrence, approx. 10 other filamentous micro-organisms are of minor importance. Growth of some of these species, viz. those which use soluble substrate, can be prevented by the introduction of Bio-P process conditions.M. parvicella and Type 0041 (and probably also Actinomycetes and the Types 1851 and 0092) seem to compete for the same substrates i.e. the influent particulate fraction. Most of the differences in composition of the filamentous microorganism population can be explained by whether or not premixing of influent and recycled sludge is used. In general, premixing for a short period of time followed by anoxic conditions favours Type 0041. M. parvicella seems to proliferate if the particulate fraction is first hydrolysed or if it enters the plant via an oxic zone. It is concluded that bulking in nutrient removal plants is mainly caused by filamentous species requiring the particulate fraction for their growth.     

Isolation and characterization of Lake Erie bacteria that degrade the cyanobacterial microcystin toxin MC-LR

Microcystin-LR (MC-LR) is a cyclic hepatotoxin produced by cyanobacteria, including Microcystis sp. and Planktothrix sp. Harmful algal blooms (HABs) in Lake Erie have become a major human health concern in recent years, highlighted by the August 2014 City of Toledo, Ohio, municipal water “do not drink” order that affected nearly 500,000 residents for 3?days. Given that microcystin degrading bacteria have been reported from HAB-affected waters around the world, we hypothesized that MC-LR degrading bacteria could be isolated from Lake Erie. To test this hypothesis, 13 water samples were collected from various Lake Erie locations during the summers of 2014 and 2015, MC-LR was continuously added to each water sample for 3 to 5?weeks to enrich for MC-LR-degrading bacteria, and MC-LR was quantitated over time. Whereas MC-LR was relatively stable in sterile-filtered lake water, robust MC-LR degradation (up to 19?ppb/day) was observed in some water samples. Following the MC-LR selection process, 67 individual bacterial isolates were isolated from MC-LR degrading water samples and genotyped to exclude potential human pathogens, and MC-LR degradation by smaller groups of bacterial isolates (e.g., groups of 22 isolates, groups of 11 isolates, etc.) was examined. Of those smaller groups, selected groups of four to five bacterial isolates were found to degrade MC-LR into non-toxic forms and form robust biofilms on siliconized glass tubes. Taken together, these studies support the potential use of isolated bacterial isolates to remove MC-LR from drinking water.     

Horizontal Distribution and Density of Yeasts and Filamentous Fungi in Lake St. Clair Water

Yeasts and filamentous fungi were collected from 67 stations throughout Lake St. Clair. Organisms from water samples were isolated by filtration through membrane filters and cultured on a selective agar medium. Representative colonies of the predominant types of yeasts and filamentous fungi and certain bacteria were identified. Yeasts and yeast-like fungi, including Rhodotorula, Candida, Cryptococcus, and Aureobasidium species, were widely distributed in densities of 1 to 1,000 colony forming units per 100 mL. The presence of these quantities and types of yeasts in the water may be related to nutrients of municipal and industrial inputs into these waters.     

Nearshore benthic blooms of filamentous green algae in Lake Baikal

For the first time, species of the genus Spirogyra, non-typical of the open nearshore waters of Lake Baikal, formed algal mats with Ulothrix zonata, Ulothrix tenerrima, and Ulothrix tenuissima near the village of Listvyanka, Russia. Normally widely distributed in the 0- to 1.5-m depth range, the growth of U. zonata was now evident and dominant (63% of the biomass) in the 2- to 5-m depth range. The overgrowth of the lake bottom by filamentous green algae, changes in distributional boundaries, the emergence and mass development of species of the genus Spirogyra, the presence of the eutrophic diatom indicator Fragilaria capucina var. vaucheriae, elevated abundances of coliform bacteria, and elevated levels of nutrients suggest an early stage of cultural eutrophication in the nearshore of Lake Baikal near Listvyanka, a popular tourist destination. The unusual abundance of Fragilaria associated with the filamentous green algae consisted of long-ribbon colonies of F. capucina var. vaucheriae, a eutrophic species, wound around the filamentous green algae, enhancing the dense algae mats. Historically dominant species, such as Didymosphenia geminata, Tetraspora cylindrica var. bullosa, and Draparnaldioides baicalensis typically observed at deeper depths of Lake Baikal, are now subdominants or minor species in the nearshore along the shoreline near Listvyanka.     

Impact of septic compounds and operational conditions on the microbiology of an activated sludge system.

In activated sludge (AS) biotreatment, septic compounds such as volatile organic acids and reduced sulphur compounds have been frequently cited as a major cause of Thiothrix and Type 021N filamentous bulking. These filaments are common in Canadian pulp and paper biotreatment systems, where they cause settling problems in secondary clarifiers. We conducted a 14-week study of a TMP/newsprint mill effluent to characterize the septic compounds entering the biotreatment, and to determine correlations with AS biomass characteristics and biotreatment operating parameters. A significant correlation was found between the sludge volume index, the abundance of Type 021N, and the propionic acid (PA) concentration in the primary clarified effluent. PA also induced a significant change in the flocculating bacteria size distribution determined by digital imaging. Consequently, the correlation observed between PA and Type 021N bulking is an indirect effect of inhibition of floc-forming microorganisms, giving a competitive advantage to filaments.     

Causes of,and control strategies for,Microthrix parvicella bulking and foaming in nutrient removal activated sludge systems

Bench and pilot scale nutrient removal activated sludge units were used to examine the effect of factors such as temperature, substrate type (easily biodegradable in the form of acetate and slowly biodegradable in the form of oleic acid) on Microthrix parvicella growth. The configurations examined include complete mix with and without selectors (anoxic and anaerobic) and plug flow reactors. The results indicate that low temperatures and substrates in the form of long chain fatty acids favour the growth of M. parvicella. With respect to reactor configuration, a plug flow configuration was shown to be quite effective in controlling the growth of M. parvicella and producing a sludge with good settling characteristics, while the presence of a selector, either anoxic or anaerobic, had no significant effect on the growth of M. parvicella.     

Modification of ASM3 for the determination of biomass adsorption/storage capacity in bulking sludge control.

The selector activated sludge (SAS) systems are known to prevent excessive growth of filamentous microorganisms responsible for bulking sludge, but these systems were hardly ever modelled. This study aimed to develop a model capable of predicting rapid substrate removal in the SAS systems. For this purpose, the Activated Sludge Model No. 3 (ASM3) was extended with three processes (adsorption, direct growth on the adsorbed substrate under aerobic or anoxic conditions). The modified ASM3 was tested against the results of batch experiments with the biomass originating from two full-scale SAS systems in Germany. The endogenous biomass was mixed with various readily biodegradable substrates (acetate, peptone, glucose and wastewater) and the utilisation of substrate (expresses as COD) and oxygen uptake rates (OURs) were measured during the experiments. In general, model predictions fitted to the experimental data, but a considerable number of kinetic (5) and stoichiometric (2) parameters needed to be adjusted during model calibration. The simulation results revealed that storage was generally a dominating process compared to direct growth in terms of the adsorbed substrate utilisation. The contribution of storage ranged from 65-71% (Plant A) and 69-92% (Plant B).     

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O₂ within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters arc not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 165 rDNA sequence analysts confirmed the affiliation suggested by FISH.     

新开河4种水生植物表面附着微生物群落特征

以水生植物金鱼藻、伊乐藻、萍蓬草和菱角为研究对象,利用扫描电镜、荧光显微镜和16S rRNA高通量测序技术,探究不同水生植物表面微生物群落特征。结果表明:沉水植物表面附生藻类密度比浮叶植物的高,且植物表面附生藻类组成与周围水体明显不同;浮叶植物萍蓬草和菱角根部的微生物群落结构与其叶表面的差异较大。4种水生植物表面附着细菌群落优势门类依次为变形菌门、厚壁菌门、绿弯菌门、拟杆菌门、酸杆菌门和疣微菌门,具有水体污染物质净化功能;两种类型水生植物表面附着有大量微生物,且不同植物之间和同种植物不同器官之间的优势种存在一定的差异。     

Laboratory evaluation of spawning substrate type on potential egg predation by round goby (Neogobius melanostomus)

Spawning habitat structure may protect demersal eggs of broadcast spawning species from depredation. Egg predation by round goby (Neogobius melanostomus) is commonly referenced as a concern associated with their invasion of the Laurentian Great Lakes. Whereas nest-building species have received some attention in egg predation studies, broadcast spawning species may be particularly vulnerable as they do not guard their eggs. This study used a 2?×?7 factorial experiment to investigate how substrate characteristics influenced the rate that fertilized eggs are lost when exposed to round goby. Eggs for two recreationally important broadcast spawning species, northern pike (Esox lucius) and muskellunge (Esox masquinongy), were placed within spawning habitat treatments (bare, silt, sand, rubble, gravel, filamentous algae, and submerged aquatic vegetation) representing a range (least to most complex) of habitat complexity. Regardless of substrate type, egg loss was similar between the two esocid species. Across all substrates and species, the number of eggs lost varied two-fold over the 24-hour experiment, with the lowest rate of egg loss observed in the most complex substrate (submerged aquatic vegetation) and the highest over the least complex substrates (bare and silt). Both northern pike and muskellunge are known to spawn over structurally complex submerged aquatic vegetation and filamentous algae. Although eggs spawned over these substrates likely offer some protection from predation, eggs that settle over less complex ancillary habitats may face higher predation risk.     

The establishment of the nuisance cyanobacteria Lyngbya wollei in Lake St. Clair and its potential to harbor fecal indicator bacteria

Lyngbya wollei is a filamentous cyanobacterium which forms large nuisance mats and has infested eastern and southeastern U.S. Lakes and reservoirs for over 100 years. Lyngbya was recently identified in the Great Lakes system in the St. Lawrence River, and Western Lake Erie. Here we report on large deposits of L. wollei washing onshore at a popular recreational beach in Lake Saint Clair, part of the Great Lakes system. The amount of L. wollei deposited on shore was quantified and evaluated for the presence of fecal indicator bacteria (FIB). High concentrations of Escherichia coli, enterococci and Clostridium perfringens were found in the L. wollei in nearshore waters. The densities of E. coli (MPN), enterococci (MPN) and C. perfringens (CFU) attached to L. wollei averaged 3.5, 3.2 and 3.2 log/g, respectively. In contrast, nearshore waters contained nearly 10 times less FIB, averaging 2.6, 2.4 and 2.6 log/100 ml of E. coli (MPN), enterococci (MPN) and C. perfringens (CFU), respectively. DNA fingerprint analysis was used to examine the population structure of E. coli isolates obtained from L. wollei mats. The L. wollei-borne E. coli strains were genetically diverse, suggesting a causal relationship between E. coli and L. wollei. Results from this study indicate that in addition to the macroalga such as Cladophora, cyanobacteria like L. wollei also harbor FIB, potentially impacting water quality and human health in the Great Lakes.     

Sources and Sinks of Escherichia coli in Benthic and Pelagic Fish

Escherichia coli and fecal coliform bacteria were isolated from five benthic and four pelagic fish species to determine their role in the fecal contamination of recreational waters. All fish were collected during fall 2006 from Southworth Marsh in the Duluth-Superior Harbor, a public beach that is commonly posted to minimize water contact due to high E. coli levels. Although fecal coliform bacteria were isolated from each fish species, they were only isolated from 66% and 72% of the individual benthic and pelagic fish, respectively. While 42% of the fecal coliforms from benthic fish were E. coli, only 4% of these bacteria from pelagic fish were E. coli. Cluster analysis showed different fish species harbored identical strains of E. coli and some fish contained multiple E. coli strains. The potential source for 65% of the E. coli isolates obtained from fish were identified by using the HFERP DNA fingerprinting method and libraries of E. coli DNA fingerprints from warm-blooded animals and environmental isolates collected in the area. The E. coli strains whose source could be identified were most similar to strains isolated from sediments, Canada geese, mallard ducks, and wastewater. None of the fish E. coli had DNA fingerprints matching those from any water or beach sand isolates. Although our results demonstrate that benthic fish contain E. coli, it may be more appropriate to consider these fish as a vector of E. coli from other sources, rather than a new source of E. coli contamination in aquatic environments.