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.     

Identity, abundance and physiology of Aquaspirillum-related filamentous bacteria in activated sludge.

Microcolony-forming bacteria closely related to the genus Aquaspirillum in the Betaproteobacteria were recently observed to be abundant in many nutrient removal wastewater treatment plants. The developed oligonucleotide probe, Aqs997, however, occasionally also targeted some filamentous bacteria in activated sludge samples when fluorescence in situ hybridization was performed. In this study, the identity, abundance, and ecophysiology of these Aqs997-positive filamentous bacteria were studied in detail. Most of the Aqs997-positive filamentous bacteria could morphologically be identified as either Eikelboom Type 1701, Type 0041/0675 or possibly Type 1851, all characterized by epiphytic growth. They were found in almost all 21 wastewater treatment plants investigated. Two morphotypes were found. Type A filaments, which seemed to be the same genotype as the microcolony-forming bacteria targeted by probe Aqs997.Type B filaments also hybridized with probe GNS941, specific for the Chloroflexi phylum, so the true identity remains unclear. Aqs997-positive filaments usually stained Gram-negative, but Gram-positive filaments were also found, stressing the difficulties in identifying bacteria from morphology and simple staining results. Studies on the ecophysiology by microautoradiography showed that Aqs997-positive filamentous bacteria did not consume acetate and glucose, while some took up butyrate, mannose, and certain amino acids. Most likely, some Aqs997-positive filamentous bacteria were able to perform full denitrification such as the Aqs997-positive microcolony-forming bacteria, and some were able to store polyhydroxyalkanoates under anaerobic conditions, potentially being glycogen accumulating organisms.     

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.     

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.     

Description of filamentous bacteria present in industrial activated sludge WWTPs by conventional and molecular methods.

Conventional cultivation methods and molecular approaches were utilised to describe the filamentous bacterial population of industrial activated sludge WWTPs. In total 43 strains were isolated by micromanipulation and were affiliated with 12 different species, comprising two new species and a new genus. In particular, a new species of Microthrix, a new genus of a filamentous Alphaproteobacteria morphologically similar to Nostocoida limicola, and a new filamentous species closely related to the opportunistic pathogen Propionibacterium propionicum were obtained. Despite the high number of isolates, the cultivation approach was unable to describe the filamentous bacteria most common in industrial WWTP. A culture-independent approach, termed the cell sorting/RT-PCR method, was therefore applied to identify fastidious or non-culturable filamentous microrganisms from different industrial plants. By this method the relevant filaments were micromanipulated and their 16S rDNA genes were amplified by RT-PCR. This approach was highly efficient. In total 31 16S rRNA sequences were obtained and 16 of them were used for the design of new specific oligonucleotide probes that highlighted dominant filaments in industrial activated sludge plants.     

Interactions between filamentous sulfur bacteria,sulfate reducing bacteria and poly-P accumulating bacteria in anaerobic-oxic activated sludge from a municipal plant

The interactions between filamentous sulfur bacteria (FSB), sulfate reducing bacteria (SRB) and poly-P accumulating bacteria (PAB) in the activated sludge of a municipal plant operated under anaerobic-oxic conditions were examined in batch experiments using return sludge (RAS) and settled sewage. Phosphate release and sulfate reduction occurred simultaneously under anaerobic conditions. SRB were more sensitive to temperature changes than PAB. SRB played an important role in the decomposition of propionate to acetate. When the sulfate reduction rates were high, there was a tendency for the maximum release of phosphate also to be high. This was explained by the fact that PAB utilized the acetate produced by SRB. Sulfur oxidizing bacteria were sensitive to temperature change. When the sulfate reduction rate was high, the sulfide oxidizing rate was also high and filamentous bulking occurred. The results showed that sulfate reduction was a cause of filamentous bulking due to Type 021N that could utilize reduced sulfur.     

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.     

Effect of chemical and biological factors on the densification of filamentous sludge

Densification may be an alternative method for controlling sludge bulking; however, little information is available on the method. Various methods including different oxygen concentrations, surface velocities of aeration, calcium concentrations and extended starvation period were employed for triggering densification of filamentous sludge in this study. It was found that high surface velocity of aeration could effectively improve the settleability of filamentous sludge in the short term, which resulted in filamentous granulation, but could not avoid filamentous bulking even after granulation. Increase of calcium concentration could also improve the settleability by forming pigtail filaments colony in the short term, but could not trigger granulation. Extension of starvation period seems effective neither for further densification nor for granulation. Additionally, increased ovality of meshes formed by filaments was frequently associated with a dense structure of filamentous sludge. It was suggested that pigtail type of filamentous colony that thus increased the resistance to elevated shear force may encourage the densification of sludge, and two kind of densification growth patterns, i.e., granular or cluster growth pattern of filamentous sludge, were identified.     

A national survey of activated sludge separation problems in The Czech republic: Filaments,floc characteristics and activated sludge metabolic properties

The extent of activated sludge separation problems associated with excessive growth of filamentous microorganisms has increased recently in the Czech Republic. Most of the activated sludge plants in the country were contacted by mail with a questionnaire, the aim of which was to obtain the basic technological data on the plants and on the nature and intensity of activated sludge separation problems. Selected activated sludge plants were visited at different periods of the year and samples of activated sludge mixed liquor and foam were collected for microscopic examination and kinetic batch tests. It was found that good settling properties were associated more with good quality activated sludge flocs than with the absence of filaments. On the other hand, a very high abundance of filaments always resulted in bulking or foaming. The most common tilamentous microorganisms in activated sludge mixed liquors were M. parvicella, N. limicola and Types 0092,0041 and 0803 while the foams were mostly dominated by M. parvicella, NALOs (GALOs) and N. limicola. Seasonal shifts in filamentous population diversity were also observed. M. parvicella, Type 0092, N. limicola, Type 0803 and Type 0041 were dominant in both conventional and biological nutrient removal activated sludge plants whereas the other Eikelboom's types of filaments dominated mostly in conventional activated sludge plants. The ability of foams to denitrify depended on the dominating tilamentous microorganism and type of substrate.     

Performances of three R-AN-D-N wastewater treatment plants in the Czech Republic.

The evolution of Czech standards requires higher efficiency of nutrient removal from municipal wastewaters. At the beginning of the last decade of 20th century, a new activated sludge configuration called R-AN-D-N process has been described, successfully tested and now largely used at several wastewater treatment plants (WWTP) in the Czech republic. The main feature of the R-AN-D-N process is the introduction of a regeneration zone in sludge recycle, which enables to increase sludge age in the system without any substantial increase in WWTP volume. Performances of three Czech large WWTP with R-AN-D-N configuration have been monitored and compared within a period of one and a half years. The results confirmed excellent nutrient removal efficiency for wastewaters with different proportion between sewage and industrial effluents. Two of three monitored WWTP received wastewaters from breweries (Budweiser and Pilsner Urquell). The settleability of activated sludge from all three WWTP was correct, with SVI values usually ranging from 50 to 150 ml/g. Monitoring of sludge composition indicated proliferation of several filamentous bacteria, particularly types 0581, 0092 and M. parvicella. No severe bulking events were observed. Finally, the operational costs expressed in CZK (Czech crown: 1 CZK = [see symbol in text]0.0322) per cubic metre of treated sewage or per capita amounts respectively from 2.24 to 6.52, and from 285 to 342.     

Modelling seasonal dynamics of "Microthrix parvicella".

The filamentous bacteria "Microthrix parvicella" can cause serious bulking and scumming in wastewater treatment plants (WWTPs) all over the world. Decades of research have identified Microthrix as a specialized lipid consumer but could not clarify the processes that allow this organism to successfully compete in activated sludge systems. In this study we developed a model, based on ASM3, that describes the pronounced seasonal variations of Microthrix abundance observed in a full-scale WWTP. We hypothesize that low temperatures reduce the solubility of lipids and inhibit their uptake by non-specialized bacteria. The presented model structure and parameters successfully fit the measured data; however they do not necessarily reflect the only and true selection mechanism for Microthrix. This model is not yet to be used for prediction; it is rather a valuable research tool to coordinate the discussion and plan future research activities in order to identify the relevant selection mechanisms favoring Microthrix in activated sludge systems.     

Monitoring activated sludge settling properties using image analysis.

The goal of this study is to develop a monitoring system for activated sludge properties, as this is an essential tool in the battle against filamentous bulking. A fully automatic image analysis procedure for recognising and characterising flocs and filaments in activated sludge images has been optimised and subsequently used to monitor activated sludge properties in a lab-scale installation. The results of two experiments indicate that the image information correlates well with the Sludge Volume Index. It is shown that, at the onset of filamentous bulking, there is an increase in total filament length on the one hand, and a change in floc shape on the other hand.     

Microbial community change of sulfate reduction and sulfur oxidation bacteria in the activated sludge cultivated with acetate and peptone.

The growth of sulfate reducing bacteria (SRB) and filamentous sulfur bacteria was monitored on a laboratory scale in activated sludge reactors using acetate and peptone as the artificial wastewater. When the artificial wastewater contained acetate and peptone, filamentous bacteria increased in the sludge and the SVI values increased. There was a good correlation between sulfate reducing activity and sulfur oxidation activity in the produced sludge. The microbial community change of filamentous sulfur bacteria and sulfate reducing bacteria was analyzed using the fluorescent in situ hybridization (FISH) method. The tendency for the growth of filamentous sulfur bacteria Thiothrix eikelboomii following the growth of SRB was observed. The percentage of SRB385- hybridized cells and DNMA657-hybridized cells found in the total cell area increased from 2-3% to 7-10% when the filamentous bulking occurred.     

Bulking control by low-dose ozonation of returned activated sludge in a full-scale wastewater treatment plant

Sludge bulking is still a problem in the operation of state-of-the-art wastewater treatment plants (WWTPs). The ozonation of returned activated sludge (RAS) is an innovative option as a non-specific measure for the control of filament growth. The applicability of sludge ozonation for bulking control of a large wastewater treatment plant was investigated. At a full-scale WWTP one lane was equipped with a sludge ozonation plant for RAS. The implemented sludge ozonation of RAS was tested against the two identical references lanes of the same WWTP. The positive effect on settleability could be clearly proven. Low-dose sludge ozonation could be a technical alternative in comparison with the established chemical measures for bulking control.     

氧化沟工艺污泥膨胀及出水水质影响因素的研究

针对丝状菌污泥膨胀造成改良式氧化沟工艺处理城市生活污水超标的问题,通过分析进水水质、溶解氧、温度、污泥膨胀指数、出水水质变化的关系,探讨了导致丝状菌膨胀的主要限制因子以及出水水质的变化.研究结果表明,此工艺中进水BOD5、CODcr、TP浓度和pH值变化不是导致污泥膨胀的原因,进水TN和环境温度对污泥膨胀略有影响,DO、NH3-N变化与SVI有较强的相关性,DO和NH3-N越高,SVI越低.     

污泥膨胀的原因分析及预警监测研究

通过分析活性污泥法污水处理系统的实际运行工况,筛选出丝状细菌数量级、污泥沉降比、负荷比、溶解氧、泥龄、温度和营养物类型作为污泥膨胀指标,并根据研究得出污泥膨胀监控指示范围.     

污泥膨胀的原因分析及预警监测研究

通过分析活性污泥法污水处理系统的实际运行工况,筛选出丝状细菌数量级、污泥沉降比、负荷比、溶解氧、泥龄、温度和营养物类型作为污泥膨胀指标,并根据研究得出污泥膨胀监控指示范围。     

Effect of chlorination bulking control on water quality and phosphate release/uptake in an anaerobic-oxic activated sludge system.

This study evaluates the effect of chlorination bulking control on water quality and phosphate release/uptake in an anaerobic-oxic activated sludge system. A series of batch experiments with different specific NaOCl mass dose were conducted to determine the sludge settling properties, supernatant water quality and phosphate metabolism behavior of filamentous bulking sludge. The harvested sludge was from a continuous-flow anaerobic-oxic (A/O) activated sludge pilot-plant, i.e., enhanced biological phosphorus removal (EBPR) system, operated with 15 days of sludge retention time. The filamentous bacteria in the A/O pilot plant were identified to be Thiothrix according to Eikelboom's classification techniques, which was in accordance with the high influent sulfate concentration of this study (50 mg/L sulfate). Increasing NaOCI concentration, as revealed by experimental results, obviously decreased the sludge settling properties (SVI values and zone settling velocities) and meanwhile significantly reduced supernatant water quality (COD, SS, TP) mainly due to higher suspended solids caused by floc disruption. Moreover, the nine-hour batch experiments indicated that high NaOCI dosage (40 mg/gMLSS) completely deteriorated phosphate metabolism of EBPR sludge. Such a high dosage of chlorination further confirmed overdosing through disappearance of intracellular PHB and death of protozoa by microscopic investigation. Still, phosphate release/uptake behavior of EBPR sludge properly functions at low NaOCl dosage (5 mg/g MLSS). Besides, phosphate metabolism worsens rapidly before the SVI value reaches its lowest level. These findings imply that determining NaOCI requirement with merely SVI values can readily result in chlorination overdosing. Proper NaOCI dosage requires a delicately balanced consideration between sludge settling improvement, water quality demand and phosphate metabolism. Batch test of phosphate release/uptake is apparently a prerequisite to conclude an appropriate NaOCl dosage for bulking control.     

Occurrence and significance of filamentous bacteria in pulp and paper activated sludge systems.

A microbial survey of 27 activated sludge (AS) systems included 16 conventional activated sludge (CAS) systems, five sequential batch reactors (SBR) and six oxygen-activated sludge (OAS) systems, all treating pulp and paper effluents. The most prevalent filaments observed were Thiothrix (26%) and Type 021N (22%). The designs of the activated sludge systems seemed to have an effect on the filament types. We found Thiothrix to be the most common filament associated with bulking. For CAS systems, a completely mixed mode of operation promoted Thiothrix and Type 021N growth. Type 021N was favoured in CAS systems with food to microorganism (F/M) ratios higher than 0.2, and with dissolved oxygen (DO) residuals higher than 2 ppm, while Thiothrix generally proliferated at lower F/M ratio and DO residuals. Nutrient deficiencies as well as nutrient dosage variations were suspected in most of the systems having Thiothrix and Type 021N as the most prevalent filaments. Thiothrix appeared to prefer polyphosphate and/or ammonia rather than urea/phosphoric acid as N and P sources. Systems with aerobic selectors showed the lowest filament counts, while systems with no selectors showed the highest filament counts.     

Substrate utilization characteristics of the predominant filamentous and floc-forming bacteria isolated from a chemical fiber factory wastewater treatment plant

To supply theoretical verification of the function of a selector to control aerobic activated sludge bulking in the wastewater treatment plant for a chemical fiber factory in Taiwan, the filamentous and floc-forming bacteria in the aeration tank in the full-scale plant were examined microscopically and isolated. The kinetic characteristics of filamentous and floc-forming bacteria were also investigated.The predominant filamentous organism was Sphaerotilus natans. In addition to this organism, 21 strains of non-filamentous bacteria were isolated using the plate count method. These included Aeromonas jandaei DNA group 9, Acinetobacter johnsonii/genospecies 7, Bacillus pasteurii and Bacillus sp. (using the Biolog identification system). Nine strains showed the ability to form flocs when cultivated in glucose mineral salts medium.With glucose and acetate as sole substrates, the 4 floc-forming bacteria tested showed different substrate utilization characteristics. The 4 strains could be divided into 3 groups. The first group was the substrate degrading bacteria, the second group was the acid degrading bacteria and the other strains were those that had the highest substrate degradation rates at low substrate concentrations (below 800 mg/l). None of the floc-formers could utilize ethylene glycol, which is the major wastewater component. The kinetic characteristics of filamentous bacterium S. natans (Km = 4.0 mg glucose/l, Vm = 0.43 μl O₂/l) and the flocforming bacterium Aeromonas jandaei DNA group 9 (Km = 34.8 mg glucose/l, Vm = 0.59 μl O₂/l) provided information for selector design.