Identity and ecophysiology of filamentous bacteria in activated sludge

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.     

Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.     

Phylogeny and in situ identification of a novel gammaproteobacterium in activated sludge

Failure of a continuously aerated sequencing batch reactor (SBR) pilot plant-enhanced biological phosphorus removal (EBPR) process, designed to remove phosphorus from the clarified effluent from a conventional non-EBPR wastewater treatment plant, was associated with the dominance ( c . 50% of the biovolume) of gammaproteobacterial coccobacilli. Flow cytometry and subsequent clone library generation from an enriched population of these Gammaproteobacteria showed that their 16S rRNA genes were most similar to partial clone sequences obtained from an actively denitrifying SBR community, and from anaerobic : aerobic EBPR communities. Under the SBR operating conditions used here, these cells stained for poly-β-hydroxyalkanoates, but never polyphosphate. Applying FISH probes designed against them in combination with microautoradiography showed that they could also assimilate acetate 'aerobically'. FISH analyses of biomass samples from the full-scale treatment plant providing the pilot plant feed showed that they were present there in high numbers. However, they were not detected by FISH in laboratory-scale communities of the same aerated laboratory-scale EBPR process even when EBPR had failed, or from several full-scale EBPR plants or other activated sludge processes.     

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.     

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.     

Microbial ecology of autothermal thermophilic aerobic digester (ATAD) systems for treating waste activated sludge

Despite their widespread use, our understanding of the microbial ecology of the autothermal thermophilic aerobic digesters (ATAD) used to dispose of sludge from wastewater treatment plants is poor. Applying both culture-dependent and molecular methods to two ATAD systems in Victoria, Australia treating different wastewaters revealed that their communities were highly specialized. Denaturing gradient gel electrophoresis (DGGE) profiling suggested differences in their population compositions and both changed over time. However, both showed low level biodiversity, and contained several novel bacterial populations. 16S rRNA clone library data and FISH analyses showed that Thermus thermophilus dominated both communities and that of a third ATAD plant in NSW (more than 90% of the total bacterial biovolume in repeated samples taken from each of the three ATAD plants). Culture-dependent methods also showed Geobacillus spp. were present in both Victorian communities. Nevertheless, the ecophysiology of these populations and their putative roles in sludge digestion remain unclear. FISH/microautoradiographic studies did not provide conclusive data elucidating which substrate/s T. thermophilus might utilize in the ATAD reactors.     

Control of Microthrix parvicella growth in activated sludge

Abstract Effects of aerobic and anaerobic conditions on the growth of Microthrix parvicella in the activated sludge were studied to prevent bulking caused by this filamentous bacterium. The study was conducted on a pilot plant with selector and the data were compared with those observed in a full scale plant subjected to severe bulking due to a massive growth of M. parvicella . Both plants were fed with the same settled waste water. A substantial suppression of the growth of M. parvicella was observed in only the experiments where returned activated sludge was mixed with waste water under aerobic conditions. Both the number of filaments and the sludge volume index (SVI) were lower in the pilot plant than in the full scale plant. Under anerobic conditions, the selector was not able to improve the settleability and avoid the growth of M. parvicella .     

Ecophysiology of Crassulacean Acid Metabolism (CAM)

BACKGROUND AND SCOPE: Crassulacean Acid Metabolism (CAM) as an ecophysiological modification of photosynthetic carbon acquisition has been reviewed extensively before. Cell biology, enzymology and the flow of carbon along various pathways and through various cellular compartments have been well documented and discussed. The present attempt at reviewing CAM once again tries to use a different approach, considering a wide range of inputs, receivers and outputs. INPUT: Input is given by a network of environmental parameters. Six major ones, CO(2), H(2)O, light, temperature, nutrients and salinity, are considered in detail, which allows discussion of the effects of these factors, and combinations thereof, at the individual plant level ('physiological aut-ecology'). RECEIVERS: Receivers of the environmental cues are the plant types genotypes and phenotypes, the latter including morphotypes and physiotypes. CAM genotypes largely remain 'black boxes', and research endeavours of genomics, producing mutants and following molecular phylogeny, are just beginning. There is no special development of CAM morphotypes except for a strong tendency for leaf or stem succulence with large cells with big vacuoles and often, but not always, special water storage tissues. Various CAM physiotypes with differing degrees of CAM expression are well characterized. OUTPUT: Output is the shaping of habitats, ecosystems and communities by CAM. A number of systems are briefly surveyed, namely aquatic systems, deserts, salinas, savannas, restingas, various types of forests, inselbergs and paramós. CONCLUSIONS: While quantitative census data for CAM diversity and biomass are largely missing, intuition suggests that the larger CAM domains are those systems which are governed by a network of interacting stress factors requiring versatile responses and not systems where a single stress factor strongly prevails. CAM is noted to be a strategy for variable, flexible and plastic niche occupation rather than lush productivity. 'Physiological syn-ecology' reveals that phenotypic plasticity constitutes the ecophysiological advantage of CAM.     

Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR

AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.     

Enhancement of the activated sludge process by activated carbon produced from surplus biological sludge

Surplus biological sludge from wastewater treatment operations was converted into activated carbon and then added to the aerated vessel of an activated sludge process treating phenol and glucose. The addition of activated carbon, either sludge-based or commercial, enhanced phenol removal from 58 to 98.7% and from 87 to 93% for COD with feed concentrations of 100 mg phenol l^–1 and 2500 mg COD l^–1. No differences were found between the activated sludge-activated carbon bench scale continuous reactors operating with either commercial or sludge-based activated carbon in spite of the higher adsorption capacity of the former.     

Growth inhibition of activated sludge by humus

Humus at 0.5 to 1 mg l–1 inhibited growth of activated sludge by about 55% in batch and long-term repeated batch cultures without decreasing sugar utilization. The growth inhibition was considerable when concentrations of substrates in the medium supplied per unit weight of activated sludge were low.     

In situ detection of protein-hydrolysing microorganisms in activated sludge

Protein hydrolysis plays an important role in the transformation of organic matter in activated sludge wastewater treatment plants, but no information is currently available regarding the identity and ecophysiology of protein-hydrolysing organisms (PHOs). In this study, fluorescence in situ enzyme staining with casein and bovine serum albumin conjugated with BODIPY dye was applied and optimized to label PHOs in activated sludge plants. A strong fluorescent labeling of the surface of microorganisms expressing protease activity was achieved. Metabolic inhibitors were applied to inhibit the metabolic activity to prevent uptake of the fluorescent hydrolysates by oligopeptide-consuming bacteria. In five full-scale, nutrient-removing activated sludge plants examined, the dominant PHOs were always different morphotypes of filamentous bacteria and the epiflora attached to many of these. The PHOs were identified by FISH using a range of available oligonucleotide probes. The filamentous PHOs belonged to the candidate phylum TM7, the phylum Chloroflexi and the class Betaproteobacteria. In total they comprised 1-5% of the bacterial biovolume. Most of the epiflora-PHOs hybridized with probe SAP-309 targeting Saprospiraceae in the phylum Bacteroidetes and accounted for 8-12% of the total bacterial biovolume in most plants and were thus an important and dominant part of the microbial communities.     

Examination into the gamma irradiation of activated sludge

This study has shown that the treatment of activated sludge by gamma irradiation resulted in a deterioration in the filterability, a decrease in the size of the floc particles and an increase in the organic matter present in the sludge supernatant. A significant difference was found between the results obtained for filamentous and non-filamentous sludges in relation to the amount of soluble polysaccharide produced.     

Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

The operating line for activated sludge

The mass balance for O2 on an activated sludge process has been re-arranged to identify the O2 limited boundary for any given treatment installation and for any combination of feed flow rate and input bCOD. The boundary is described graphically as the operating line.     

Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge process

The effects of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 1,2,4-trichlorobenzene (1,2,4-TCB) on the biodegradation kinetics of the conventional activated sludge system (CASS) and the selector activated sludge system (SASS) were investigated. Experiments were carried out using a respirometric method on unacclimated biomass from two lab-scale systems that were operated with the sludge age of 8 days. Toxicity of the test compounds for both reactors were arranged according to EC₅₀ (effective concentration) values in order as: 1,2,4-TCB > 2,4-DCP > 2-CP > phenol. All selected test compounds induced higher inhibition effect in the CASS. The SASS appeared to reduce inhibition effect in comparison to the CASS, by 21.36%, 66.95%, 64.37% and 33.33% for phenol, 2-CP, 2,4-DCP and 1,2,4-TCB, respectively. Consequently, the SASS may be recommended as a promising configuration alternative for the waste streams containing toxic compounds.     

聚丙烯二氧化钛负载膜固定化活性污泥对污水处理的研究

利用聚丙烯二氧化钛负载膜为载体,吸附固定活性污泥微生物,处理有机废水,考察了聚丙烯膜的组成、吸咐固定时间以及聚丙烯二氧化钛活性污泥负载膜对污水有机物的降解。由全有机碳分析仪对降解过程的有机物进行检测;结果表明;活性污泥负载膜处理废水8小时后,废水中的有机物含量下降达50％-60％,对所处理的废水的降解效果明显。     

Quantitative use of fluorescent in situ hybridization to examine relationships between mycolic acid-containing actinomycetes and foaming in activated sludge plants

The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P < 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m(3) completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 x 10(6) cells ml(-1) or 4 x 10(12) cells m(-2). We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.     

Comparative analysis of biological phosphate removal (BPR) and non-BPR activated sludge bacterial communities with particular reference to Acinetobacter

The bacterial community of a biological phosphate removal (BPR) activated sludge process was studied and compared to that of a non-BPR process treating the same municipal waste water. Bacterial isolates from the BPR process, as characterized by whole cell fatty acids, belonged to more than twenty genera, with Micrococcus, Staphylococcus and Acidovorax scoring highest. Acinetobacter spp represented 4% of cultured bacteria, ≤3% as estimated by fluorescence in situ hybridization, and well under 10% on the basis of the proportion of ubiquinone Q9 in the sludge. The mole proportions of ubiquinones, Q8 : Q10 : Q9 in the sludge were maintained fairly stable at approximately 9:4:1. The spectra of the isolated strains and the proportions of ubiquinones in the processes (BPR vs non-BPR) were otherwise similar, but a significant number of isolates related to actinomycetes were obtained from the BPR sludge only. The BPR process did not enrich Acinetobacter. Pure cultures of Acinetobacter isolated from the sludge stained for polyphosphate, but Acinetobacter cells responding to the ACA probe in native sludge from the BPR process did not. Instead, the bulk of the polyphosphate in the BPR sludge was located in a distinct morphotype of large, coccoid, highly clustered cells. Received 3 August 1998/ Accepted in revised form 29 October 1998     

Extraction of activated sludge bacteria exopolymers by ultrasonication

Ultrasonication for the extraction of activated sludge exopolymers was evaluated by total cell count, exopolymer extraction and transmission electron microscopy (TEM). A high deflocculation was achieved after 30 s of sonication in PBS (phosphate-buffered saline). TEM showed that cell lysis was minimal only when sludges were sonicated for 30 s. For sludges sonicated for 30, 90 and 420 s and stained with Ruthenium Red, exopolymers were not extracted on a large scale without considerable cell lysis. Sludges sonicated for 30 s in EDTA gave a larger fraction of damaged cells and also showed copious amounts of attached exopolymers.