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.     

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.     

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).     

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.     

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.     

Occurrence and control of filamentous bulking in aerated wastewater treatment plants of the French paper industry.

The occurrence of filamentous bacteria was investigated in 15 French pulp and paper activated sludge wastewater treatment plant (WWTP). Large filamentous populations were present in most of the plants. Identification carried out with conventional methods based on morphological features and staining techniques showed that the four main filamentous bacteria encountered in these industrial WWTP and responsible for bulking belong to the genera Thiothrix sp., Type 021 N, Haliscomenobacter hydrossis and Type 0092. During two years a specific survey was performed for three of these WWTP showing recurrent bulking phenomena. Data from WWTP performance, chemical data and filaments characterization were compared to correlate the presence of specific filaments with process operating conditions.     

Comparison of some characteristics of aerobic granules and sludge flocs from sequencing batch reactors.

Physical, chemical and biological characteristics were investigated for aerobic granules and sludge flocs from three laboratory-scale sequencing batch reactors (SBRs). One reactor was operated as normal SBR (N-SBR) and two reactors were operated as granular SBRs (G-SBR1 and G-SBR2). G-SBR1 was inoculated with activated sludge and G-SBR2 with granules from the municipal wastewater plant in Garching (Germany). The following major parameters and functions were measured and compared between the three reactors: morphology, settling velocity, specific gravity (SG), sludge volume index (SVI), specific oxygen uptake rate (SOUR), distribution of the volume fraction of extracellular polymeric substances (EPS) and bacteria, organic carbon and nitrogen removal. Compared with sludge flocs, granular sludge had excellent settling properties, good solid-liquid separation, high biomass concentration, simultaneous nitrification and denitrification. Aerobic granular sludge does not have a higher microbial activity and there are some problems including higher effluent suspended solids, lower ratio of VSS/SS and no nitrification at the beginning of cultivation. Measurement with CLSM and additional image analysis showed that EPS glycoconjugates build one main fraction inside the granules. The aerobic granules from G-SBR1 prove to be heavier, smaller and have a higher microbial activity compared with G-SBR2. Furthermore, the granules were more compact, with lower SVI and less filamentous bacteria.     

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。     

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.     

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.     

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.     

Fluorescence in situ hybridization analysis of nitrifiers in piggery wastewater treatment reactors.

Fluorescence in situ hybridization (FISH) was performed to analyze the nitrifying microbial communities in an activated sludge reactor (ASR) and a fixed biofilm reactor (FBR) for piggery wastewater treatment. Heterotrophic oxidation and nitrification were occurring simultaneously in the ASR and the COD and nitrification efficiencies depend on the loads. In the FBR nitrification efficiency also depends on ammonium load to the reactor and nitrite was accumulated when free ammonia concentration was higher than 0.2 mg NH3-N/L. FISH analysis showed that ammonia-oxidizing bacteria (NSO1225) and denitrifying bacteria (RRP1088) were less abundant than other bacteria (EUB338) in ASR. Further analysis on nitrifying bacteria in the FBR showed that Nitrosomonas species (NSM156) and Nitrospira species (NSR1156) were the dominant ammonia-oxidizing and nitrite-oxidizing bacteria, respectively, in the piggery wastewater nitrification system.     

Aerobic granular sludge: a promising technology for decentralised wastewater treatment.

In order to evaluate the characteristics of aerobic granular sludge, a sequencing batch reactor, feeding with synthetic wastewater at the organic loading rate of 8 kg COD/m3 d, was employed on the laboratory scale. Granules occurred in the reactor within 1 week after the inoculation from conventional flocculent sludge. Aerobic granular sludge was characterised by the outstanding settling properties and considerable contaminates removal efficiencies. The SVI30 values were in the range of 20 to 40 ml g(-1). However, the sludge volume index of short settling time (e.g. SVI10--10 min) is suggested to describe the fast settling properties of aerobic granular sludge. The potential application in the decentralised system is evaluated from the point view of footprint and high bioactivity. The occurrence of sloughing, resulting from the outgrowth of filamentous organisms, would be responsible for the instability of aerobic granules. The starvation phase should therefore be carefully controlled for the maintenance and stability of aerobic granular sludge system.     

Influence of temperature and sludge loading on activated sludge settling,especially on Microthrix parvicella

During recent years modern full scale wastewater treatment plants with biological nitrification, denitrification and phosphorus removal have had increasing problems with foam formation on the surfaces of aerobic tanks and with bulking activated sludge. The results of a survey in 1995 (Kunst and Knoop, 1996) showed that most often the filamentous bacterium Microthrix parvicella is responsible for these problems. Up to today there is only little knowledge about its selection criteria in activated sludge. Therefore several expenments were done in full scale activated sludge plants and in laboratory systems under defined conditions to investigate the influence of low (< 0.1 kg/(kg·d)) and high (≤ 0.2 kg/(kg·d)) BOD₅-sludge loading rates on the growth and morphology of M. parvicella and the settlement of activated sludge. Furthermore the influence of temperatures of 5°C, 12°C and 20°C on the growth of M. parvicella was investigated. It was shown that M. parvicella grows at low BOD₅-sludge loading rate and low temperature and is the main causative organism of bulking and foaming sludge in nutrient removal plants. On the basis of this investigation it was concluded that the growth of M. parvicella and the settling problems of the activated sludge resulting from excessive growth of this filament will always appear in modern municipal wastewater treatment plants with BOD₅-sludge loading rate ≤ 0.1 kg/(kg·d) especially under low temperature conditions.     

Factors affecting the activity of anammox bacteria during start up in the continuous culture reactor.

Factors affecting cultivation of extremely slow-growing bacteria (anaerobic ammonium oxidiser, doubling time 11 days) were investigated by using upflow anaerobic sludge blanket (UASB) reactors which can maintain high solid retention time. The effects of concentrations of DO, free ammonia (FA), and nitrite on activation of anammox activity were tested during the start-up period. The reactor was inoculated with granular sludge collected from a full-scale UASB reactor used for treating brewery wastewater, and sludge from a piggery wastewater treatment plant and rotating biological contactor treating sewage. Results of continuous operation showed that concentrations of DO, free ammonia (FA) and nitrite in the reactors played a key role in stimulating the anammox activity during start-up period. It is crucial to keep DO below 0.2 ppm, FA below 2 mg/L and nitrite nitrogen below 35 mg/L to cultivate anammox cells in the continuous bioreactor. When the levels of DO, FA and nitrite in the influent were controlled at less than the inhibition levels, the anammox activity increased gradually in the anaerobic condition. Addition of hydrogen sulphide into the reactor enhanced anammox activity in the continuous culture. Through the SEM, TEM and FISH analysis, anammox bacteria were detected in the granular sludge after 3 months of continuous operation.     

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.     

Foaming in anaerobic digesters caused by Microthrix parvicella

Three large wastewater treatment plants in the greater Stockholm area have experienced serious anaerobic digester foaming. Microscopic studies of the sludge from the foam phase showed a network of the filamentous organism Microthrix parvicella. The morphology of the long, coiled filament appeared to be affected by the anaerobic conditions where it became broken up into to shorter and thicker filaments. The operating strategy to prevent foam in the anaerobic digesters at these plants is to control the growth of M. parvicella in the activated sludge tanks by increasing the sludge load. Top installed stirrers and the addition of poly-aluminium salt have also been used to prevent foam formation.     

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.