Novel filamentous spore-forming iron bacteria causes bulking in activated sludge

Several Gram-positive iron bacteria were isolated from bulking sludge. They were filamentous and had false branching. They had sheaths with iron deposits and formed spores on modified sucrose casitone yeast extract agar. They did not grow on influsion agar for longer than 1 month but could withstand 80°C for 1 h on the same medium. Adding them to sewage before aeration increased the biochemical oxygen demand of waste water and caused poor settling properties of activated sludges. They were the predominant filamentous micro-organisms in bulking activated sludges. At present, these strains cannot be assigned to recognized taxa of Bacillus spp. or sheathed iron bacteria.     

Relationship of species-specific filament levels to filamentous bulking in activated sludge

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.     

Clarifying the roles of kinetics and diffusion in activated sludge filamentous bulking

We hypothesized that the growth rates of filaments and floc formers in activated sludge are affected by the combination of kinetic selection (Lou and de los Reyes, Biotechnol Bioeng 92(6): 729-739, 2005b) and substrate diffusion limitation (Martins et al., Water Res 37:2555-2570, 2003). To clarify the influence of these factors in explaining filamentous bulking, a conceptual framework was developed in this study. The framework suggests the existence of three different regions corresponding to bulking, non-bulking, and intermediate regions, based on substrate concentration. In the bulking and non-bulking regions, kinetic growth differences control the competition process, and filaments or floc formers dominate, respectively. In the intermediate region, substrate diffusion limitation, determined by the floc size, plays the major role in causing bulking. To test this framework, sequencing batch reactors (SBRs) were operated with influent COD of 100, 300, 600, and 1,000 mg/L, and the sludge settleability was measured at various floc size distributions that were developed using different mixing strengths. The experimental data in the bulking and intermediate regions supported the proposed framework. A model integrating the two factors was developed to simulate the substrate concentrations at different depths and floc sizes under intermittently feeding conditions. The modeling results confirmed that substrate diffusion limitation occurs inside the flocs at a certain range of activated sludge floc sizes over the operation cycle, and provided additional support for the proposed framework.     

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.     

活性污泥中群体感应淬灭菌的分离纯化及功能验证

【背景】近年来,群体感应淬灭(Quorum Quenching,QQ)技术在膜生物污堵防控中的应用研究受到了广泛关注。然而,目前已成功分离纯化的高效QQ菌有限,更多高效QQ菌资源亟待挖掘。【目的】从实际运行的膜生物反应器(MembraneBioreactor,MBR)活性污泥中采样,分离并富集高效QQ菌。【方法】以根瘤农杆菌(Agrobacterium tumefaciens) A136为报告菌株,使用指示琼脂平板法测定各菌株的N-辛酰基高丝氨酸内酯(N-Octanoyl-DL-Homoserine Lactone,C8-HSL)降解能力。以紫色色杆菌(Chromobacterium violaceum) VIR24为报告菌株,定量测定所得QQ菌降解N-己酰高丝氨酸内酯(N-Hexanoyl-DL-Homoserine Lactone,C6-HSL)信号分子的能力。通过微生物形态、生理生化及16SrRNA基因序列测定、构建系统发育树、扫描电子显微镜形态观测等方法对菌株进行分类学鉴定。用共培养法分析QQ菌对生物膜形成的抑制能力,通过聚乙烯醇和海藻酸钠包埋固定化QQ菌。【结果】筛选出了6株高效QQ菌,其中对C8-HSL分解能力最强的为杆状、革兰氏阴性戴尔福特菌属(Delftia sp.) JL5。定量分析结果表明菌株JL5能在10 h内完全降解C6-HSL。菌株JL5显著抑制铜绿假单胞菌(Pseudomonas aeruginosa) PAO1和菠萝泛菌(Pantoea ananatis) SK-1生物膜的形成。固定化后的JL5微球仍具有高效的C6-HSL和C8-HSL信号分子分解能力,而且分解速度较被广泛报道的红球菌(Rhodococcussp.)BH4更快。【结论】研究分离得到了高效的QQ菌,能够有效抑制N-酰基高丝氨酸内酯(N-Acyl-HomoserineLactones,AHL)型群体感应菌生物膜的形成,固定化后仍然具有强QQ活性,具备广泛的应用前景,为后续QQ膜生物污堵防控技术的实践应用奠定了基础。     

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.     

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.     

Bacteriology of activated sludge,in particular the filamentous bacteria

Microscopic examination of bulking activated sludge samples showed the presence of a variety of filamentous microorganisms, some of which have not yet been described in the literature. A method was developed to obtain pure cultures of these threaded bacteria. To date, five clearly different groups of filamentous bacteria may be distinguished by the determination of a few morphological and physiological characteristics of the isolates. A variety of sheathed bacteria are included in Group I. Group II includes non-motile, gram-negative, orange- or yellow-pigmented filamentous bacteria. These microorganisms are thought to be related to some species of the genusFlavobacterium. The gram-negative, threaded bacteria of Group III show a more or less distinct gliding movement and form red colonies on rich agar media. These bacteria may apparently be related to species described in the generaMicroscilla andFlexibacter. The filamentous bacteria of Group IV structurally resemble someCyanophyceae, but do not contain photosynthetic pigments. They are gram-positive and non-motile. A number of unknown, non-motile bacteria which stain gram-positive with a variable number of gram-negative autolyzed cells in the filaments, are assigned to Group V. The properties of the isolated bacteria are described briefly and their occurrence in bulking activated sludge is discussed.     

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.     

Isolation of rhodoquinone-containing chemoorganotrophic bacteria from activated sludge

Abstract Several strains of Gram-negative aerobic chemoheterotrophic bacteria which produced rhodoquinone were isolated from activated sludge. All isolates contained a rhodoquinone with eight isoprene units as a major component in addition to the corresponding homologue of ubiquinone. The isolates seemed to make up a single taxon because of their homogeneity in phenotypic and chemotaxonomic properties, but could not be allocated to any known taxa of Gram-negative bacteria.     

Gram-staining characterisation of activated sludge filamentous bacteria by automated colour analysis

An automated image analysis method has been developed for the monitoring of the Gram-staining characteristics of filamentous bacteria in activated sludge. The binary method of pixel classification agreed with manual estimation (level of correlation of 0.9 for Gram-positive bacteria). Its robustness has been assessed by repeatability tests. Population shifts in terms of Gram-staining characteristics have been monitored in laboratory-scale experiments with two feeding schedules using this technique.Revisions requested 22 September 2004; Revisions received 11 October 2004     

Isolation of some new sulphur bacteria from activated sludge

E mtiazi , G., H abibi , M.H. & S etareh , M. 1990. Isolation of some new sulphur bacteria from activated sludge. Journal of Applied Bacteriology 69 , 864–870.
During studies on bulking of activated sludge some new sulphur micro-organisms, which were able to grow aerobically and anaerobically on reduced sulphur compounds, were isolated on thiosulphate agar. These were capable of autotrophic and heterotrophic growth on a wide range of substrates. In view of their ability to oxidize reduced sulphur compounds, and because one of them was an oxidase- and catalase-positive, Gram-negative, motile coccus (0.36–0.48 μm) it was named Thiosphaera persica . The second one was an oxidase- and catalase-positive, Gram-negative, motile rod (1.32–1.80 μm) and was named Thiobacillus persica . The third one was oxidase-negative, catalase-positive, motile, Gram-negative and polymorphic and was named Sulphobacter polymorpha .     

Relations between substrate feeding pattern and development of filamentous bacteria in activated sludge processes

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of substrate. It was found that continuously fed systems repeatedly resulted in the development of filamentous bacteria and bulking of the sludge. Intermittently fed systems did form good settling sludges, without filamentous bacteria. The same results were found using different sludge loadings and different concentrations of mixed liquor suspended solids. High dissolved oxygen concentration did not prevent bulking in continuous systems while low dissolved oxygen concentration resulted in bulking with intermittently fed systems. It was found that the substrate removal rate of intermittently operated systems was always higher than for continuously fed systems. The hypothesis is formulated that intermittent feeding leads to higher substrate removal rates by floc forming bacteria and their predominance in intermittently fed systems, which can be compared to plug flow systems.     

Phage-host associations in a full-scale activated sludge plant during sludge bulking

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of “Candidatus Microthrix parvicella” led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with “Candidatus Accumulibacter phosphatis,” suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.     

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.     

Relations between substrate feeding pattern and development of filamentous bacteria in activated sludge processes:

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of substrate. In a previous paper it was shown that continuously operated systems resulted in the development of filamentous bacteria and bulking sludges. Intermittently fed sludges resulted in good settling. These results are now confirmed when substrates other than glucose are present in the influent, such as nutrient broth, acetate and starch. With casein deflocculation occurred. For intermittent systems the substrate removal rates were higher than for continuous systems. Based on the results a theory is presented to account for the growth of filamentous bacteria (and bulking) in continuous systems (completely mixed systems). This theory assumes that in intermittently fed systems (plug flow systems) floc forming bacteria become dominant as a result of higher substrate uptake rates and the possibility to survive a starvation phase by thriving on accumulated intracellular metabolites.     

Characterization of a new gram-negative filamentous bacterium isolated from bulking sludge

Summary A new gram-negative filamentous and sheath-forming bacterium with hardly visible rod-shaped single cells was isolated from bulking sludge. Three strains of this type grow aerobically, produce carotenoid pigments especially, on rich media and exhibit gliding motility on solid surfaces. Even numbered straight-chain acid were predominating in total fatty acid patterns, whereas a branched-chain odd numbered fatty acids, although occurring in lower amounts, is discussed as an appropriate chemotaxonomical marker for this bacterium. As respiratory quinones only menaquinones could be detected. The whole cell proteins of the isolates showed identical electropherograms on polyacrylamide slabs. The G+C-content was found to be 59 mol%. Morphological, physiological, and chemotaxonomical features imply that the strains belong to a new genus of the gliding bacteria. Taxonomically they have to be placed near Herpetosiphon and Chloroflexus These results could be confirmed by the sequence analysis of the 16 S r-RNA performed by Ludwig and Stackebrandt (1984, personal communication).