Type 021N Research Articles

Variability of type 021N in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes

Bulking sludges were investigated in seven industrial or municipal activated sludge treatment plants from Denmark, Germany and Australia. The dominating filaments were all identified as type 021N according to the Eikelboom key. The extent of variability in the filament taxonomy was assessed using fluorescence in situ hybridization (FISH) with rRNA-targeted nucleic acid probes specific for type 021N, Thiothrix and Leucothrix. Not all of the filaments morphologically identified as type 021N hybridized with the 021N probe. In one treatment plant the predominant filament hybridized with the probe for Thiothrix and in one treatment plant the predominant filament did not hybridize with any of these probes. In none of the plants did filaments hybridize with the probe for Leucothrix. A study of the in situ uptake of different organic substrates by the various filaments was also conducted using microautogradiography. The uptake of 6 different organic substrates under aerobic conditions was studied by providing C-14 or H-3 labeled substrates (acetate, glucose, ethanol, glycine, leucine and oleic acid) in incubations of a period of 3 hours. No filaments took up all the tested substrates, and type 021N from the various treatment plants varied in their uptake abilities. The study demonstrated that strain differences with regard to substrate utilization are likely to occur among bacteria within the same genera and designated types which are indistinguishable on the basis of morphological observations alone and by the molecular probes used in this study for identification. Whether there is a clear correlation between type of wastewater and the capability of taking up the various organic substrates for the filaments remains to be elucidated.

Control of bulking in a full scale plant by addition of talc (PE 8418)

A very severe bulking problem (Type 021N) in the water treatment plant of Bodegraven in The Netherlands was solved by the addition of a specially shaped talqueous powder, PE 8418. After addition of this mineral product, the SVI dropped almost instantaneously from 850 ml/g to 250 ml/g and over the next 2 weeks decreased further to 100 to 125 ml/g. A ratio PE 8418/biomass of 0.6:1 was sufficient to keep the SVI at this level. The settling velocity of the sludge increased from <1.0m/h to 1.5 m/h. These settling properties allowed normal plant operation. During the first 2 weeks of the experiment, scum on the final clarifier and the aeration tank caused some problems. It is assumed that the complete incorporation of PE 8418 particles into the flocs takes some time, if a large quantity of this product is added at once. Meanwhile, free particles may contribute to stabilization of gas bubbles, viz. flotation of activated sludge.

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).

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.

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.

Phylogenetic assessment of five filamentous bacteria isolated from bulking activated sludges

Nearly complete 16S ribosomal RNA (rRNA) 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 organism although it was not possible to designate type 021N 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.

Variability of type 021n in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes

Bulking sludges were investigated in seven industrial or municipal activated sludge treatment plants from Denmark, Germany and Australia. The dominating filaments were all identified as type 021N according to the Eikelboom key. The extent of variability in the filament taxonomy was assessed using fluorescence in situ hybridization (FISH) with rRNA-targeted nucleic acid probes specific for type 021N, Thiothrix and Leucothrix. Not all of the filaments morphologically identified as type 021N hybridized with the 021N probe. In one treatment plant the predominant filament hybridized with the probe for Thiothrix and in one treatment plant the predominant filament did not hybridize with any of these probes. In none of the plants did filaments hybridize with the probe for Leucothrix. A study of the in situ uptake of different organic substrates by the various filaments was also conducted using microautogradiography. The uptake of 6 different organic substrates under aerobic conditions was studied by providing C-14 or H-3 labeled substrates (acetate, glucose, ethanol, glycine, leucme and oleic acid) in incubations of a period of 3 hours. No filaments took up all the tested substrates, and type 021N from the various treatment plants varied in their uptake abilities. The study demonstrated that strain differences with regard to substrate utilization are likely to occur among bacteria within the same genera and designated types which are indistinguishable on the basis of morphological observations alone and by the molecular probes used in this study for identification. Whether there is a clear correlation between type of wastewater and the capability of taking up the various organic substrates for the filaments remains to be elucidated.

合成界面活性剤による糸状性細菌の溶菌

A microbial substance that lyses filamentous bacteria was purified and characterized. It could not be rendered inactive by various enzyme denaturing treatment. The substance had properties similar to those of a biosurfactant that is often produced by B. subtilis. Lysing of filamentous bacteria with synthetic surfactants was also examined. Several synthetic surfactants were found to be capable of lysing filamentous bacteria. Nonionic synthetic surfactants with an HLB of 14 were found to lyse Spaerotilus natans, Type 1701, Thiothrix, Haliscomenobacter, Type 0961 and Type 021N in activated sludges sampled from wastewater treatment plants. Use of the synthetic surfactant to lyse filamentous bacteria was also demonstrated in a continuous-feed aeration tank, and settleability of sludge was improved. The surfactent did not adversely effect floc-forming microorganisms as evidenced by the high rate of TOC removal. The results indicate that synthetic surfactants can be used to prevent filamentous bulking in the activated sludge process.

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).

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.

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.

Application of microautoradiography to the study of substrate uptake by filamentous microorganisms in activated sludge

Excessive growth of filamentous microorganisms in activated-sludge treatment plants is a major operational problem which causes poor settlement of activated sludge. An enhanced understanding of the factors controlling growth of different filamentous microorganisms is necessary in order to establish more successful control strategies. In the present study, the in situ substrate uptake was investigated by means of microautoradiography. It was demonstrated that the uptake of labeled organic substrates by the filamentous microorganisms, during short-term incubation, could be detected by microautoradiography. Viability and respiratory activity of the filaments were also detected by reduction of CTC (5-cyano-2,3-ditolyl tetrazolium chloride) and by incorporation of [(sup3)H]thymidine. Gram, Neisser, and fluorescence staining techniques were used for the localization and identification of the filaments. Activated-sludge samples from five wastewater treatment plants with bulking problems due to filamentous microorganisms were investigated. Microthrix parvicella, Nostocoida limicola, and Eikelboom's type 0041 and type 021N were investigated for their ability to take up organic substrates. A panel of six substrates, i.e., [(sup14)C]acetate, [(sup3)H]glucose, [(sup14)C]ethanol, [(sup3)H]glycine, [(sup3)H]leucine, and [(sup3)H]oleic acid, was tested. The uptake response was found to be very specific not only between the different filamentous types but also among filaments of the same type from different treatment plants. Interestingly, M. parvicella consistently took up only oleic acid among the tested substrates. It is concluded that microautoradiography is a useful method for investigation of in situ substrate uptake by filamentous microorganisms in activated sludge.

Lysis of filamentous bacteria by surfactants

A microbial substance that lyses filamentous bacteria was obtained. The substance also exhibited some properties of a bio-surfactant. Lysing of filamentous bacteria with synthetic surfactants was also examined. Several synthetic surfactants were found to be capable of lysing filamentous bacteria. Nonionic synthetic surfactants with an HLB of 11-15 were found to lyse type 1701 and type 021N in an activated sludge sampled from a sewage treatment plant. Use of the synthetic surfactant to lyse filamentous bacteria was also demonstrated in a continuous-feed aeration tank, and settleability of sludge was improved. The surfactants did not adversely effect floc-forming microorganisms as evidenced by the high rate of TOC removal. The results indicate that synthetic surfactants can be used to prevent filamentous bulking in the activated sludge process.

Stability and variation in sludge properties in two parallel systems for enhanced biological phosphorus removal operated with and without nitrogen removal

In order to introduce nitrogen removal, many conventional activated sludge systems in Sweden have been transformed into low-loaded systems during the 1990's. Occasionally these systems have been combined with enhanced biological phosphorus removal (EBPR). Increased problems of sludge loss from secondary clarifiers have aroused new interest in the properties of activated sludge. The aim of this study was to investigate the long-term variation and stability of sludge properties in two parallel EBPR systems, operating on the same wastewater. One of the systems was designed as a conventional system, and the other as a low-loaded system with nitrogen removal. The study was performed at the Sjölunda plant in Malmö during a two-year period, in a pilot plant which has been in operation since 1986. The diluted sludge volume index (DSVI) was found to be about 150 ml/g in both systems. The highest values were recorded during the winter season. The variation in the DSVI was much greater in the conventional system than in the low-loaded system. The dominating filamentous bacteria in the conventional system were Type 021N and Thiothrix, and in the low loaded system Type 0041/0675 and Type 0092. The EBPR operation induced large amounts of poly-P-containing flocs in both systems. Although it was not possible to determine to what extent the poly-P-containing flocs affected the sludge properties in this study, it was clearly demonstrated that EBPR operation may not always provide conditions which lead to superior sludge properties.

Lysis of filamentous bacteria by surfactants

A microbial substance that lyses filamentous bacteria was obtained. The substance also exhibited some properties of a bio-surfactant. Lysing of filamentous bacteria with synthetic surfactants was also examined. Several synthetic surfactants were found to be capable of lysing filamentous bacteria. Nonionic synthetic surfactants with an HLB of 11–15 were found to lyse type 1701 and type 021N in an activated sludge sampled from a sewage treatment plant. Use of the synthetic surfactant to lyse filamentous bacteria was also demonstrated in a continuous-feed aeration tank, and settleability of sludge was improved. The surfactants did not adversely effect floc-forming microorganisms as evidenced by the high rate of TOC removal. The results indicate that synthetic surfactants can be used to prevent filamentous bulking in the activated sludge process.

Stability and variation in sludge properties in two parallel systems for enhanced biological phosphorus removal operated with and without nitrogen removal

In order to introduce nitrogen removal, many conventional activated sludge systems in Sweden have been transformed into low-loaded systems during the 1990's. Occasionally these systems have been combined with enhanced biological phosphorus removal (EBPR). Increased problems of sludge loss from secondary clarifiers have aroused new interest in the properties of activated sludge. The aim of this study was to investigate the longterm variation and stability of sludge properties in two parallel EBPR systems, operating on the same wastewater. One of the systems was designed as a conventional system, and the other as a low-loaded system with nitrogen removal. The study was performed at the Sjolunda plant in Malmö during a two-year period, in a pilot plant which has been in operation since 1986.The diluted sludge volume index (DSVI) was found to be about 150 ml/g in both systems. The highest values were recorded during the winter season. The variation in the DSVI was much greater in the conventional system than in the low-loaded system. The dominating filamentous bacteria in the conventional system were Type 021N and Thiothriz, and in the low loaded system Type 0041/0675 and Type 0092. The EBPR operation induced large amounts of poly-P-containing flocs in both systems. Although it was not possible to determine to what extent the poly-P-containing flocs affected the sludge properties in this study, it was clearly demonstrated that EBPR operation may not always provide conditions which lead to superior sludge properties.

Growth requirements of filamentous bacteria isolated from bulking and scumming sludge

A total of 68 strains of filamentous bacteria belonging to the genera Sphaerotilus and Haliscomenobacter, Eikelbooms types 021N, 1701, some nocardioform organisms in addition to some unidentified, Gram-negative pink-pigmented isolates were studied qualitatively and quantitatively for their growth requirements and growth response to different nutrients. With the exception of type 021N, all isolates grew better with d-fructose and d-glucose, than with acetate as sole carbon source. Acetate concentrations > 1.0 g 1 −1 inhibited all isolates and Haliscomenobacter and the pink-pigmented strains did not grow at all with acetate. With the exception of Haliscomenobacter isolates, growth of the majority of strains was stimulated by low peptone and yeast extract concentrations (0.05–0.5 g 1 −1). Haliscomenobacter strains did not grow in the presence of yeast extract and peptone. For all isolates a strict requirement for calcium, and (to some extent) magnesium was observed. Below a concentration of 20 mg 1 −1 Ca and 0.1 mg 1 −1 Mg only few strains were able to show visible growth. The Haliscomenobacter isolates were not able to grow in media containing < 25 mg 1 −1 Ca and 5 mg 1 −1 Mg. All isolates did not grow in media containing < 1 mg 1 −1 NH 4N, and 2.5 mg 1 −1 P. Often group or genus specific growth requirements were recorded, indicating that the occurrence of one type of filamentous organisms in bulking sludge can be associated with a specific nutrient mixture in the activated sludge.

Survey on filamentous micro-organisms in activated sludge processes in Bangkok, Thailand

A survey to investigate the proliferation of filamentous micro-organisms, which cause filamentous bulking in activated sludge wastewater treatment processes, was conducted for the first time in the tropical zone. Activated sludge samples were collected from six wastewater treatment processes in Bangkok City, Thailand. The activated sludge plants investigated include those for 1) an industrial wastewater from an industrial estate, 2) a domestic wastewater from apartment houses, 3) a brewery wastewater, 4) a hotel wastewater, 5) a textile industry wastewater and 6) a milk processing wastewater. The identification of proliferating filamentous micro-organisms in the samples was attempted. Type 021N was exclusively dominant in the milk processing wastewater treatment plant. The characteristics of the 021N present were different from “typical” 021N. The most proliferating filament in the textile wastewater treatment plant was suspected to be Nostocoida limicola. There was a predominant filament in the brewery wastewater treatment plant, which is suspected to be a new type. Other major filaments proliferating include Eikelboom Type 021N, 1701, 0041 and 0092. Nocardia was also found in a few plants.

Survey on filamentous micro-organisms in activated sludge processes in Bangkok, Thailand

A survey to investigate the proliferation of filamentous micro-organisms, which cause filamentous bulking in activated sludge wastewater treatment processes, was conducted for the first time in the tropical zone. Activated sludge samples were collected from six wastewater treatment processes in Bangkok City, Thailand. The activated sludge plants investigated include those for 1) an industrial wastewater from an industrial estate, 2) a domestic wastewater from apartment houses, 3) a brewery wastewater, 4) a hotel wastewater, 5) a textile industry wastewater and 6) a milk processing wastewater. The identification of proliferating filamentous micro-organisms in the samples was attempted. Type 021N was exclusively dominant in the milk processing wastewater treatment plant. The characteristics of the 021N present were different from “typical” 021N. The most proliferating filament in the textile wastcwatcr treatment plant was suspected to be Nostocoida limicola. There was a predominant filament in the brewery wastewater treatment plant, which is suspected to be a new type. Other major filaments proliferating include Eikelboom Type 021N, 1701, 0041 and 0092. Nocardia was also found in a few plants.

Ecological interactions among denitrification, poly-P accumulation, sulfate reduction, and filamentous sulfur bacteria in activated sludge

Effects of anoxic-oxic conditions on the growth of sulfate reduction, poly-P accumulation and filamentous sulfur bacteria were examined in the laboratory scale sequential batch reactors. In the anoxic-oxic conditions, denitrification bacteria are dominant. The growth of sulfate reducing bacteria and poly-P accumulating bacteria was suppressed. The number of sulfate reducing bacteria in the activated sludge was below 104 MPN/g MLSS, and the sulfate reduction rate was very low. Filamentous bulking was also suppressed. On the other hand, when nitrate was removed from the artificial wastewater, sulfate reducing bacteria could grow predominantly in the anaerobic conditions. The number of sulfate reducing bacteria was about 106∼107 MPN/g MLSS and the sulfate reduction rate increased (0.17 ∼ 0.21 g SO4/g MLSS·hr). Filamentous bacteria Type 021N increased over 103 cm/mg MLSS. Sodium molybdate was added to the artificial wastewater in order to prevent sulfate reduction. When the concentration of sodium molybdate increased to 980 mg/L, the number of sulfate reducing bacteria decreased to 103 ∼ 104 MPN/g MLSS and the sulfate reduction rate decreased. Filamentous bulking was completely suppressed in these conditions. These results show that sulfate reduction is a main trigger of the filamentous bulking due to Type 021N that can utilize reduced sulfur for an energy source.