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.     

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.     

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.     

In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography

Microthrix parvicella is a filamentous microorganism responsible for bulking and foaming problems in many activated sludge treatment plants. The problems have increased with the introduction of nutrient removal in many countries, and presently, there is no reliable control strategy for M. parvicella. Little is known about the physiology of M. parvicella, and conflicting data exist about its preferred organic substrates, and whether it is able to be physiologically active under anaerobic and anoxic conditions. In this study, the ability of M. parvicella to take up various radioactively labeled organic compounds was investigated in situ at three nutrient removal plants using a microautoradiographic technique. Of 12 compounds tested under aerobic conditions only the long chain fatty acids (LCFA), oleic acid and palmitic acid, and to some extent a lipid, trioleic acid, were assimilated. None of the simple substrates such as acetate, propionate, butyrate, glucose, ethanol, glycine and leucine were taken up. Furthermore, the uptake of oleic acid was compared under anaerobic, anoxic and aerobic conditions, and it was demonstrated that in addition to aerobic conditions M. parvicella was also able to take up oleic acid under anaerobic and anoxic conditions. No difference in substrate uptake pattern for M. parvicella was found among the tested activated sludge plants. The results strongly indicate that a better control strategy against M. parvicella must rely on a better understanding of presence and availability of triglycerides and LCFA, and an improved knowledge of the physiology of M. parvicella under anaerobic and anoxic conditions.     

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.     

Molecular biological methods to detect “Microthrix parvicella” and to determine its abundance in activated sludge

Molecular biological methods were evaluated in attempts to detect and quantitate levels of “Microthrix parvicella” in activated sludges. Approximately 66% of the 23S rRNA gene sequence of a strain (Ben43) of the Gram positive bulking and foaming organism “Microthrix parvicella” was determined, while a lesser amount was determined for “M. parvicella” strain RNI. The high mo1%G+C Gram positive bacteria (HGCGPBs) possess two powerfully diagnostic regions in the 23S rDNA and these were investigated in both strains. Firstly, the 18 nucleotide HGCGPB probe sequence (HGC69a) varied in at least two nucleotides with the sequence from both strains of “M. parvicella”. Secondly, an approximately 100 nucleotide stable insert between helices 54 and 55 in the 23S rRNA of HGCGPBs was discovered to be present in “M. parvicella”, but in both strains it was unique in length (79 nucleotides) and sequence. The region of the 23S rDNA with the stable insert was exploited to develop a polymerase chain reaction assay in which amplicons from “M. parvicella” were larger than those from nonHGCGPBs (i.e. all Bacteria except the HGCGPBs), and smaller than those from HGCGPBs. This assay was evaluated with DNAs extracted from activated sludges but although “M. parvicella” was morphologically identified, and was a dominant filament in at least one of the samples, no “M. parvicella” specific sized amplicons could be recovered from it. Amplicons of sizes generated by nonHGCGPBs and HGCGPBs were routinely produced in the stable insert PCR with DNAs from activated sludges where the highest yield was of amplicons from nonHGCGPBS.A second series of experiments were undertaken with the objective of evaluating the use of a non-radioactive hybridization method, based on extraction of bacterial RNA, for quantifying “M. parvicella” in activated sludge samples. Total nucleic acids were extracted from activated sludge samples and immobilized on nylon membranes. Probing with 16S rRNA-directed DIG-labelled oligonucleotide probes, detection of chemiluminescent signals on membranes and densitometry allowed hybridization signals to be quantified. The relationship between the amount of nucleic acid hybridized and the hybridization signal intensity observed was found to be linear over a specified range of signal intensities. A range of activated sludge samples were analysed for “M. parvicella” and variations in levels could be distinguished.     

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.     

Anaerobic processes in activated sludge

We found anoxic zones in aerated activated sludge flocs, and demonstrated denitrification under normal operating conditions. Sulfate reduction was not found. Micro-environments and microbial conversions in flocs from bulking and non-bulking activated sludge were determined with microsensors for H₂S, O₂, NO₂₋ and NO₃₋. Denitriftcation and sulfate reduction rates were measured with ¹⁵N- and ³⁵S-tracer techniques. We showed that under normal reactor conditions (ca. 20% air saturation) anoxic zones develop within flocs allowing denitrification. The denitrtftcation rates amounted to 40% of the rates under anoxic conditions. At 100% air saturation no anoxic zones were found and no denitrification occurred. However, in flocs from bulking sludge (at 20% air saturation) anoxic zones were absent and denitrification did not occur. In bulking sludge only at total anoxia was denitrification found. Confocal microscopy showed that flocs from bulking sludge were much looser than those from non-bulking sludge. The absence of anoxic zones and of denitrification was attributed to the open floc structure, allowing advective oxygen transport.Sulfate reduction was not detected in any of the sludges tested by microsensors or by tracer techniques even under anoxic conditions. This indicates that the sulfur cycle (sulfate reduction and sulfide oxidation) does not play a role in mineralization processes and bulking in activated sludge. Preliminary molecular work (in situ hybridization with the 16S-rRNA probe SRB385) indicated the presence of small amounts of sulfate reducing bacteria in all sludges. Either the probe is not specific or the sulfate reducers present are not active under reactor conditions.     

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.     

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.     

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.     

Some physiological properties of an Italian isolate of “Microthrix parvicellai”

“Microthrir parvicella” strain RN1, isolated by micromanipulation from an activated sludge treatment plant in Italy, is phylogenetically identical to two other isolates from Australia. Our Italian isolate of “M. parvicella” is a slow growing (μmax ranging from 0.3 to 0.5 d^-1), aerobic organism that exhibits nutritional versatility, being able to utilize simple and complex compounds as carbon and nitrogen sources. In pure culture, the organism cannot grow anaerobically nor is it able to denitrify. Strain RN1 is able to grow both in aerobic and in microaerophilic conditions and this metabolic versatility could give it a metabolic advantage in the activated sludge milieu.     

活性炭促进UASB处理高浓度啤酒废水效果研究

高浓度啤酒废水在厌氧消化过程中容易酸化,抑制甲烷转化。为提高UASB反应器的稳定性和处理效果,本研究通过序批式试验的方法,以模拟啤酒废水为底物,考察了两种不同粒径的活性炭对加速UASB污泥颗粒化进程和提高甲烷产率的影响。试验运行95 d,进水有机负荷从2.9 kg/(m3·d)增加到12.0 kg/(m3·d),监测反应器内产气量、出水VFA、出水TOC及污泥特性等参数的变化趋势。结果表明:投加活性炭能有效缩短厌氧污泥颗粒化的时间,增强产甲烷菌活性,大幅提升甲烷产量,出水TOC和VFA都维持在较低水平;并且较小粒径的粉末活性炭对UASB反应器的促进作用优于较大粒径的颗粒活性炭,能有效促进底物向甲烷气体转化。     

Horizontal transfer of bacterial heavy metal resistance genes and its applications in activated sludge systems

The bacterial nickel (Ni) resistance determinant ncc-nre of Alcaligenes 31A strain cloned to an IncQ broadhost-range plasmid pKT240 gave rise to pMOL222. The plasmid was subsequently mobilized into various Eubacteria and found to confer an increased Ni resistance on these recipients. An increase of Ni resistance was also observed after the transfer of pMOL222 into activated sludge bacteria by plate mating. The dissemination of pMOL222 into an activated sludge pilot stabilized the system during a heavy metal shock loading with 0.25 mM Ni.     

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.     

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.     

Hydrolysis of return sludge for production of easily biodegradable carbon: effect of pre-treatment, sludge age and temperature.

Return sludge from two Swedish and two Danish wastewater treatment plants were hydrolysed in laboratory reactors. Treatment plants with/without pre-sedimentation and with/without nitrification were represented. Soluble organic matter was produced from all types of sludge, but the yield was to a large extent dependent on what type of sludge was hydrolysed. Activated sludge from wastewater treatment plants without pre-treatment returned more soluble carbon after hydrolysis than sludge from treatment plants with pre-sedimentation. In addition, more soluble carbon was formed from non-nitrifying activated sludge than from nitrifying sludge. Moreover, the maximum yield of soluble COD at 10 degrees C was less than the yield at 20 degrees C. The initial hydrolysis rate was found to be between 0.35 and 1.8 mg soluble COD/(g VS x h). With the exception of one case, between 15 and 50% of the produced soluble COD was shown to be volatile fatty acids, a suitable carbon source for biological phosphorus removal. Nitrification rate measurements indicated that the viability of the activated sludge was not affected by the hydrolysis.     

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.     

Phosphorus and polyhydroxyalkanoates variation in a combined process with activated sludge and biofilm

This study investigates variations of phosphorus and polyhydroxyalkanoates (PHAs) in a combined activated sludge - biofilm process, operating under various sludge retention times (5, 10 and 15 days) and different dissolved oxygen conditions (0.1, 0.5, 1.0 and 2.0 mg/l in aerobic stage). Experimental results indicate that phosphorus uptake closely corresponds to utilization of PHAs during anoxic and aerobic stages. Moreover, the sludge in the anoxic stage exhibits a higher PHAs utilization efficiency with respect to phosphorus uptake than sludge in the aerobic stage, when it is under low COD-SS loading conditions. The values of r_P/PHA, representing sludge capacity on phosphorus uptake, range from 0.1–1.0 mg P/mg PHAs. In addition, analyzing the distribution of 3-hydroxybutyrate (3HB), 3-hydroxy-2-methylbutyrate (3H2MB), 3hydroxyvalerate (3HV) and 3-hydroxy-2-methylvalerate (3H2MV) reveals that 3HB and 3HV are the major components of PHAs. The values of 3HB/PHAs and 3HV/PHAs vary with COD-SS loading of the process. When F/M ratio increases, 3HV/PHAs value increases and 3HB/PHAs value decreases simultaneously. This phenomenon implies that more bacteria accumulated 3HV as storage matter under high COD-SS loading conditions. The kind of bacteria population shift would intensify the competition of “G bacteria” with polyphosphate accumulating organisms, possibly causing process deterioration during phosphorus removal.     

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.