Application of Image Analysis Techniques in Activated Sludge Wastewater Treatment Processes

Image analytical techniques have been extensively developed to evaluate complex microbial aggregates such as sludge flocs and biofilms. This review covers the latest contributions concerning the application of image analysis to the activated sludge systems with respect to the most frequently used morphological parameters and relations between them and traditional wastewater treatment parameters. Recent developments have indicated that image analysis can be successfully used for the quantification of flocs and filamentous bacteria in the operating wastewater treatment plants, which enables prediction of bulking events and pinpoint flocs formation.     

Effects of synthetic polymer on the filamentous bacteria in activated sludge

Filamentous bulking is one of the solid-liquid separation problems always seen in activated sludge process. The addition of synthetic polymer is always one of the popular ways for the treatment plant operator to immediately solve the poor sludge settling problem. Therefore, it may be interesting to understand the effects of synthetic polymer on the filamentous bacteria in activated sludge. In this study, synthetic polymer was applied to a lab-scale wastewater treatment system with the filamentous bulking problem. The population structure of filamentous bacteria and sludge characteristics were investigated under different conditions. When synthetic polymer was added into the system, it was found that poor sludge settleability caused by filamentous bulking was temporarily solved and filamentous branches growing outside the flocs were damaged or inhibited. However, filamentous growth was still observed inside the flocs. After the addition of polymer was halted, filamentous branches extended out of the flocs immediately. Very serious filamentous bulking occurred and sludge settleability became much worse than that occurring before the addition of polymer. And, it took several weeks for the system to return to normal operation.     

分子检测技术对活性污泥中氨氧化细菌的比较研究

采用PCR扩增、随机克隆测序等技术,分析处理含高浓度氨氮的废水处理系统不同驯化时期的4个活性污泥样品,对样品中氨氧化细菌（AOB）的种类和氨单加氧酶（AMO）的活性进行分析比较,并在国内首次采用PCR变性梯度凝胶电泳（DGGE）相结合的技术对样品中总的细菌类群的差异进行研究。结果表明所检测到的氨氧化细菌优势菌群均属于变形细菌的β亚类,与Nitrosomonas sp.具有较高的相似性。活性污泥驯化成熟后,废水处理系统中AMO的活性有明显提高,活性污泥中的细菌类群更加集中,优势菌群相对稳定,系统对废水的处理效率也相应提高。结果表明采用分子检测技术有利于更全面地了解AOB的类群和功能,进而改善废水处理系统的处理效果。     

Multiple approaches to assess filamentous bacterial growth in activated sludge under different carbon source conditions

Aims:  To compare molecular and microscopic approaches in determining which filamentous bacteria grow in activated sludge reactors when different carbon sources and different activated sludge mixed liquor inocula are used.
Methods and Results:  Microscopic and molecular (Denaturing Gradient Gel Electrophoresis and Fluorescent In Situ Hybridization) techniques were used to determine which filamentous bacteria became dominant in lab scale reactors treating wastewater composed of different carbon sources. Molecular analysis indicated the presence of Sphaerotilus natans and Thiothrix -related organisms . Microscopy indicated the presence of Nostocoida limicola in some reactors. Sludge volume index increased as filament abundance increased. The detection level of DGGE analysis increased when the abundance levels of the filaments were high.
Conclusions:  Simultaneous application of traditional and molecular methods was effective, and highlighted the advantages and limitations of each method. Readily biodegradable substances favoured the growth of specific filaments in a mixed liquor environment. The origin of inoculum influenced which specific filamentous bacteria grew.
Significance and Impact of the Study:  The study shows the potential problems when using particular techniques, and highlights the need for multiple approaches when studying filaments. The study also provides more information on which filaments will grow under different carbon source conditions for a given inoculum.     

Sludge minimisation technologies

The treatment and disposal of excess sludge represents a bottleneck of wastewater treatment plants all over the world, due to environmental, economic, social and legal factors. There is therefore a growing interest in developing technologies to reduce the wastewater sludge generation. The goal of this paper is to present the state-of-the-art of current minimisation techniques for reducing sludge production in biological wastewater treatment processes. An overview of the main technologies is given considering three different strategies: The first option is to reduce the production of sludge by introducing in the wastewater treatment stage additional stages with a lower cellular yield coefficient compared to the one corresponding to the activated sludge process (lysis-cryptic growth, uncoupling and maintenance metabolism, predation on bacteria, anaerobic treatment). The second choice is to act on the sludge stage. As anaerobic digestion is the main process in sewage sludge treatment for reducing and stabilising the organic solids, two possibilities can be considered: introducing a pre-treatment process before the anaerobic reaction (physical, chemical or biological pre-treatments), or modifying the digestion configuration (two-stage and temperature-phased anaerobic digestion, anoxic gas flotation). And, finally, the last minimisation strategy is the removal of the sludge generated in the activated sludge plant (incineration, gasification, pyrolysis, wet air oxidation, supercritical water oxidation).     

Relationship between protozoan and metazoan communities and operation and performance parameters in a textile sewage activated sludge system

The present study aims at investigating the possibility of assessing performance and depuration conditions of an activated sludge wastewater treatment plant through an exploration of the microfauna. The plant, receiving textile industrial (70%) and domestic (30%) sewage, consists of a two-step biological depurating plant, with activated sludge followed by a percolating system. A total of 35 samples were analyzed during five months, and 30 taxa of protozoa and small metazoa were found. Epistylis rotans, Vorticella microstoma, Aspidisca cicada and Arcella sp. were the most frequent protozoa identified. Several significant correlations between biological, physical–chemical and operational parameters were determined, but no significant correlations could be established between biological parameters and removal efficiencies. The Sludge Biotic Index (SBI) reflected the overall state of the community but only presented statistically significant correlations with the influent total suspended solids (TSS), total suspended solids in mixed-liquor (MLTSS) and dissolved oxygen (DO). The determination of key groups and taxa along with general community parameters showed to have potential value as indicators of the depuration conditions. Despite the impossibility of correlating biological parameters and the removal efficiencies, the present study attests the value of the microfauna to assess the operation of the activated sludge systems even in the case of non-conventional plants and/or plants receiving industrial sewage.     

Monitoring filamentous bulking in activated sludge systems fed by synthetic or municipal wastewater

The stability with respect to filamentous bulking of two activated sludge fully-aerobic systems, one with a completely mixed tank and one with a channel reactor, fed either by a synthetic wastewater or by a primary-settled municipal wastewater, of variable composition and flow rate, has been investigated. The morphological characteristics of the biomass in terms of floc size and roughness and of filamentous bacteria abundance have been monitored by image analysis. Severe bulking was only observed in the well-mixed tank fed at a constant flow rate by synthetic substrate of constant concentration, when the channel reactor fed in a similar manner was fully stable. Variations of biomass characteristics as well as of settling properties were observed on both systems fed with the real wastewater, but these events were related to the characteristics of the wastewater, as similar changes were observed on the full-scale plant fed with the same substrate. In any case, automated image analysis was an efficient way to monitor in detail the fate of the activated sludge at pilot and full scale.     

活性污泥丝状膨胀的模拟试验与控制对策研究

以新鲜的生活污水作为水样,采用同步培菌法,在培养活性污泥的基础上,模拟一定的条件促进丝状菌生长直至形成丝状膨胀,并探索消除丝状膨胀的控制措施,同时通过对丝状菌进行分析鉴定及生活特性显微观察,确定产生丝状膨胀的丝状菌的种类。因为实验能够较好地接近污水厂的实际情况,其结论对污水厂曝气池的正常运行具有实际指导作用。     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

Novel Actinomycete Isolated from Bulking Industrial Sludge

A novel actinomycete was the predominant filamentous microorganism in bulking activated sludge in a bench-scale reactor treating coke plant wastewater. The bacterium was isolated and identified as an actinomycete that is biochemically and morphologically similar to Amycolatopsis orientalis; however, a lack of DNA homology excludes true relatedness. At present, the isolate (NRRL B 16216) cannot be assigned to the recognized taxa of actinomycetes.     

Limitation of sludge biotic index application for control of a wastewater treatment plant working with shock organic and ammonium loadings

This study aimed to determine the relationship between activated sludge microfauna, the sludge biotic index (SBI) and the effluent quality of a full-scale municipal wastewater treatment plant (WWTP) working with shock organic and ammonium loadings caused by periodic wastewater delivery from septic tanks. Irrespective of high/low effluent quality in terms of COD, BOD₅, ammonium and suspended solids, high SBI values (8–10), which correspond to the first quality class of sludge, were observed. High SBI values were connected with abundant taxonomic composition and the domination of crawling ciliates with shelled amoebae and attached ciliates. High SBI values, even at a low effluent quality, limit the usefulness of the index for monitoring the status of an activated sludge system and the effluent quality in municipal WWTP-treated wastewater from septic tanks. It was shown that a more sensitive indicator of effluent quality was a change in the abundance of attached ciliates with a narrow peristome (Vorticella infusionum and Opercularia coarctata), small flagellates and crawling ciliates (Acineria uncinata) feeding on flagellates.     

Impact of operational parameters on bacterial community in a full-scale municipal wastewater treatment plant

A bacterial community in activated sludge from a full-scale municipal wastewater treatment plant was monitored throughout the year with the use of FISH, RISA and DGGE techniques. In the investigated range of temperatures (11.9-21.6 degrees C), a rise in temperature resulted in a lower total bacteria richness, while organic load rate changes from 0.09 to 0.21 g COD x g TSS(-1) x d(-1) were positively correlated with the number of bands in RISA patterns. The most diverse pattern (29 different bands) was characteristic for the activated sludge sample collected at the end of January at wastewater temperature of 11.9 degrees C. The ammonia-oxidising bacteria community did not change during the study, and comprised of 4 different bacterial populations with one dominant species closely related to Nitrosospira sp. REGAU (GenBank accession number AY635572.1). The percentage of ammonia-oxidising bacteria in the activated sludge varied from 6.2 to 19.5% and depended on temperature (R = 0.61, p = 0:02) and organic load rate (R = -0.55, p = 0.04).     

Application of the Fluorescent-Antibody Technique for the Detection of Sphaerotilus natans in Activated Sludge

Sphaerotilus natans, one of the most widely reported causes of bulking in activated sludge, can exist both within and outside of a sheath. It can easily be confused with similar activated sludge bacteria and thus can be overlooked when present in low numbers. Fluorescent antiserum was successfully prepared against the nonfilamentous form and was shown to be highly specific, showing no reaction with either pure cultures of similar filamentous bacteria or entirely unrelated organisms. It did, however, show a lack of strain specificity since it reacted with S. natans isolates from the Federal Republic of Germany and the United States and with filamentous bacteria in South African activated sludges. Fluorescent antibody is capable of penetrating the filaments of S. natans to stain the cells individually. The use of fluorescent antiserum in the identification of S. natans filaments obscured by activated sludge flocs and other suspended matter was simple since the cells stained brightly and could be observed through the less dense matter, while the use of other microscope techniques would be hampered by these obstructions. The use of fluorescent antibody will facilitate ecological studies of S. natans in activated sludge and other aqueous environments.     

活性污泥中菌群多样性及其功能调控研究进展

活性污泥是污水处理厂生物处理工艺的功能主体,活性污泥中菌群的种类、数量及活性是提高污水处理能力与效果的重要基础。本文综述了活性污泥处理工艺中的主要功能细菌(絮凝菌、脱氮菌、除磷菌等)生物群落的多样性与生态特征,并对目前主流的菌群鉴定方式进行总结,最后从运行条件、定向驯化及生物强化3个方面对菌群调控进行论述,以期为活性污泥法污水处理工艺提供一些理论指导。     

Characterization of PHB storage in activated sludge extended filamentous bacteria by automated colour image analysis

The storage of poly-β-hydroxybutyrate (PHB) in extended filamentous bacteria from activated sludge was monitored by Sudan Black staining: PHB granules were blue in the reddish filaments counterstained by safranin. By quantitative image analysis of colour images grabbed on an optical microscope, the distribution of the PHB loading of the extended filaments was estimated by determination of the proportion of blue pixels of their skeleton. The method was applied for different feed compositions to demonstrate its ability to monitor the PHB synthesis and storage capacity of filamentous bacteria in mixed cultures. Fast PHB storage, within a few hours, could be observed with acetate-based feeding solutions but the storage rate decreased with more complex feeds (meat extract based feed, wastewater).     

Sludge bulking impact on relevant bacterial populations in a full-scale municipal wastewater treatment plant

We have investigated the changes of microbial community structures and the concomitant performance in two biological wastewater treatment systems (conventional and inverted A²/O processes) over a whole cycle of sludge bulking. A low level of filament abundance was observed during non-bulking period, with types 0092 and 0041 as the dominant filamentous bacteria. With the increase of the sludge volume index values from 76 (73) to 275 (300) mg/L, the filament abundance estimated by microscopic examination increased from 1 (few) to 5 (abundant), with Microthrix parvicella becoming the dominant filament bacteria. Sludge bulking resulted in a significant shift in bacterial compositions from Proteobacteria to Actinobacteria dominance, characterized by the significant presence of filamentous M. parvicella (from not detected to higher than 60% of clones) and decrease of the dominant Betaproteobacterial population (from higher than 40% to less than 1%). Important relevant bacterial populations including polyphosphate-accumulating organism (PAO, Candidatus Accumulibacter phosphatis), ammonia-oxidizing bacteria (AOB, Nitrosomonas), nitrite-oxidizing bacteria (NOB, Nitrospira) and denitrifying bacteria (Thauera) were absent under the serious bulking condition. Accumulation of nitrite and ammonia was observed during serious bulking, while the phosphorus removal performance was not decreased because M. parvicella could behave as a PAO.     

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.     

Sludge wash-out as strategy for Anammox process start-up

In this study sludge wash-out was evaluated as a strategy to start-up the Anammox process in order to establish it in a shorter period of time. Sludge from a domestic wastewater treatment plant (WTP) was used to seed two (RI and RII) anaerobic sequencing batch reactors (ASBR). During the start-up period RI was operated as a continuous stirred tank reactor (CSTR) using a dilution rate of 0.2 d⁻¹, which promoted the sludge wash-out. After this period, the remaining sludge was retained in the reactor. The reactor RII was operated as an ASBR throughout the study period with a high cell retention. The performance of the two reactors in terms of nitrogen removal was compared over a period of 380 days. During the last RI operation phase the specific nitrogen removal rate increased exponentially, attaining values of 85 mg N/g TSS d. However, a rate of 190 mg N/g TSS d in the batch test under optimal conditions was achieved. The specific nitrogen removal rate remained almost constant for RII with a mean value of 6 mg N/g TSS d being observed during the operation period. The rate for the RII batch test was 20 mg N/g TSS d. These results confirm that the higher total suspended solids (TSS) in RII (reactor with high cell retention) was not effective in terms of N removal improvement. Anammox-like bacteria were found using fluorescence in situ hybridization (FISH) in reactor RI after 225 days and a new Anammox species was identified.     

Upgrading activated sludge systems and reduction in excess sludge

Most of 200 Activated Sludge Plant in Iran are overloaded and as a result, their efficiency is low. In this work, a pilot plant is manufactured and put into operation in one of the wastewater treatment plants in the west of Tehran. Instead of conventional activated sludge, a membrane bioreactor and an upflow anaerobic sludge blanket reactor used as a pretreatment unit in this pilot. For the sake of data accuracy and precision, an enriched municipal wastewater was opted as an influent to the pilot. Based on the attained result, the optimum retention time in this system was 4h, and the overall COD removal efficiency was 98%. As a whole, the application of this retrofit would increase the plant's capacity by a factor of 5 and reducing the excess sludge by a factor of 10. The sludge volume index in the anaerobic reactor was about 12 after granulation occurred.     

Ecophysiology of the filamentous Alphaproteobacterium Meganema perideroedes in activated sludge

A comprehensive study of the ecophysiology of the filamentous Meganema perideroedes affiliated to the Alphaproteobacteria, possessing a "Nostocoida limicola Type II" filamentous morphology was conducted. This morphotype often causes serious bulking problems in activated sludge wastewater treatment plants, and hardly anything is known about its physiology. The study was carried out by applying a suite of in situ methods in an industrial activated sludge treatment plant with excessive growth of this species. The experiments revealed a very versatile organism able to take up a large variety of organic substrates under aerobic conditions. It had a remarkably high storage capacity forming polyhydroxyalkanoates from most substrates tested. When nitrate was present as e-acceptor, the number of substrates to be consumed by M. perideroedes was more restricted compared to aerobic conditions. With nitrite as e-acceptor, only acetate and glucose among the substrates tested could be assimilated and used for storage and possibly growth. This indicated that M. perideroedes might be able to denitrify under certain conditions, which is unusual for filamentous bacteria in activated sludge. No substrate uptake or storage was seen under anaerobic conditions. M. perideroedes was relatively hydrophobic, compared to other filamentous bacteria and microcolonies present in the sludge, indicating the presence of a hydrophobic sheath. Several excreted surface-associated exoenzymes were detected in the sludge, but M. perideroedes never showed any activity, except once after a breakdown in the production facility. This confirmed that M. perideroedes mainly grows on soluble substrates. Based on the studies of the ecophysiology of M. perideroedes, potential control strategies are suggested.