Microbiology of the 'G-bacteria' in activated sludge

This review discusses a group of bacteria, the 'G-bacteria', which have a distinctive morphology of cocci in tetrads, sheets or clusters, that are seen in large numbers in many activated sludge biomass samples. Isolates of 'G-bacteria' that have been grown axenically are phylogenetically diverse. The Gram-negative members include several α- and β-proteobacteria, among which is the genus Amaricoccus , while the Gram-positive 'G-bacteria' contain several members of the actinobacteria. It is probable that other, as yet uncharacterized, 'G-bacteria' exist in activated sludge. The hypothesis that these 'G-bacteria' are detrimental to the process of enhanced biological phosphate removal by competing for substrates anaerobically with the phosphate-accumulating bacteria in such systems, based as it is largely on mixed-culture studies, receives little support from studies using those available in pure culture. The evidence on which these conclusions are founded is discussed, as are the arguments used to explain why these 'G-bacteria' all appear to thrive under conditions found in certain activated sludge systems.     

Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

Analysis of the polyphosphate-accumulating microflora in phosphorus-eliminating, anaerobic-aerobic activated sludge systems by using diaminopropane as a biomarker for rapid estimation of Acinetobacter spp.

Polyphosphate-accumulating gram-negative bacteria were isolated from different anaerobic-aerobic activated sludge systems with diverse processes for enhanced biological phosphorus (P) elimination. Of 22 isolates, 10 were allocated to the genus Acinetobacter by using multiple-test systems and soluble protein and polyamine patterns. As diaminopropane (DAP) appears to be the characteristic main polyamine compound produced by Acinetobacter spp., it was used as a biomarker for the genus. The high DAP contents of representative samples from municipal wastes with enhanced biological P elimination indicated that Acinetobacter spp. can be dominant organisms in sewage treatment plants with low organic loading and nitrification and denitrification steps. Contrary to accepted opinion, sludge from treatment plants with efficient P removal and high organic loading had a low DAP content, indicating that bacteria other than Acinetobacter spp. are responsible for enhanced biological P elimination in these plants.     

In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp

Aims: The purpose of this work was to investigate microbial ecology of nitrifiers at the genus level in a typical full-scale activated sludge plant. Methods and Results: Grab samples of mixed liquor were collected from a plug-flow reactor receiving domestic wastewater. Fluorescent in situ hybridization technique (FISH) was used to characterize both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in combination with Confocal Scanning Laser Microscope (CSLM). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes were used in this study. Both Nitrosomonas and Nitrosospira genera as AOB and Nitrobacter and Nitrospira genera as NOB were sought with genus specific probes Nsm156, Nsv443 and NIT3 and NSR1156, respectively. Conclusions: It was shown that Nitrosospira genus was dominant in the activated sludge system studied, although Nitrosomonas is usually assumed to be the dominant genus. At the same time, Nitrobacter genus was detected in activated sludge samples. Significance and Impact of the Study: Previous studies based on laboratory scale pilot plants employing synthetic wastewater suggested that only Nitrospira are found in wastewater treatment plants. We have shown that Nitrobacter genus might also be present. We think that these kinds of studies may not give a valid indication of the microbial diversity of the real full-scale plants fed with domestic wastewater.     

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus

Micromanipulation was used to obtain an isolate (BEN 52) of Eikelboom Type 1851 from a bulking activated sludge plant. Its 16S rDNA sequence reveals its closest relative is 'Roseiflexus castenholzii', a member of the phylum 'Chloroflexi', class 'Chloroflexi', previously called the green non-sulfur bacteria. The 16S rRNA targeted oligonucleotide probe designed for fluorescence in situ hybridisation against this sequence successfully identified filamentous bacteria with the morphological features of Type 1851 in activated sludge samples from plants in several countries and different operational configurations.     

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.     

A quantitative method for measuring the mass concentration of the filamentous bacterium Type 021N in activated sludge using fluorescence in situ hybridization

AIMS: This study aimed to develop a quantitative method for measuring mass concentrations of Type 021N, a bacterium causing bulking in activated sludge. METHODS AND RESULTS: Fluorescence in situ hybridization was used to determine the relationship between the concentration ratio of the mass of the bacterium Type 021N to mass of activated sludge, and the proportion of fluorescence area imparted by probe G123T specific for Type 021N to that obtained with probe EUB338 for bacteria. A linear relationship existed between the cube root of the mass concentration ratio and square root of this area proportion. CONCLUSIONS: A standard curve was obtained for quantifying Type 021N in activated sludge. SIGNIFICANCE AND IMPACT OF THE STUDY: This method may allow the determination of growth rate constant of filamentous bacteria in activated sludge, information that will help in understanding their ecology.     

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.     

Studies on the effect of inoculation of activated sludge with bacteria actively degrading hydrocarbons on the biodegradation of petroleum products

Eighteen strains of bacteria were isolated from activated sludge purifying petroleum-refining wastewaters. These strains were plated on solidified mineral medium supplemented with oil fraction in concentration 1000 mg/l. Four of the strains that grew best in the presence of oil were selected for further studies. The strains were identified based on Bonde's scheme and microscopic observations. Three of them belonged to the genus Arthrobacter and one to the genus Micrococcus. Stationary cultures of single strains and their mixtures were set up in mineral medium containing oil (sterile and non-sterile) as sole carbon source in concentration 1000 mg/l. The oils were found to be removed the most efficiently by a mixture of the strains. After 14 days of culture the amount of oil was utilized by from 63 to 95%. In the next stage of the studies the bacteria were used to inoculate activated sludge. Stationary cultures of the activated sludge were set up in mineral medium with oil. The utilisation of petroleum products by non-inoculated activated sludge (control), activated sludge inoculated with a single strain or a mixture of all four strains was examined. In both inoculated activated sludge cultures approximately 80% of the oils were removed, compared to 60% in the control activated sludge. Therefore, inoculated activated sludge showed 20% higher effectiveness of removal of petroleum derivatives.     

Predominant Bacteria in an Activated Sludge Reactor for the Degradation of Cutting Fluids

For the first time, an activated sludge reactor, established for the degradation of cutting fluids, was examined for predominant bacteria. In addition, both total and viable numbers of bacteria in the reactor were determined so that the percentage of each predominant type in the total reactor population could be determined. Three samples were studied, and a total of 15 genera were detected. In each sample, the genus Pseudomonas and the genus Microcyclus were present in high numbers. Three other genera, Acinetobacter, Alcaligenes, and Corynebacterium, were also found in every sample but in lower numbers. In one sample, numerous appendaged bacteria were present, and one of these, the genus Seliberia, was the most predominant organism in that sample. However, in the other two samples no appendaged bacteria were detected. Six genera were found in this reactor which have not been previously reported in either cutting fluids in use or in other activated sludge systems. These genera were Aeromonas, Hyphomonas, Listeria, Microcyclus, Moraxella, and Spirosoma. None of the predominant bacteria belonged to groups of strict pathogens.     

The in situ physiology of Skermania piniformis in foams in Australian activated sludge plants

The in situ physiology of the filamentous bacterium Skermania piniformis frequently seen in activated sludge foams in Australia was investigated. An oligonucleotide probe, Spin1449, targeting the 16S rRNA of S. piniformis was designed for its identification by fluorescence in situ hybridization (FISH), validated with pure cultures and applied successfully to foam samples from two geographically distant Australian plants. While filaments of this bacterium appeared to be comparatively hydrophobic, the organism had no clear preference for hydrophobic or hydrophilic substrates. In both foams examined using microautoradiography (MAR), filaments selectively took up substrates under aerobic and anoxic (NO(3) (-)) but not anaerobic or anoxic (NO(2) (-)) conditions. Skermania piniformis assimilated oleic acid, palmitic acid, glycerol and glycine. Ectoenzyme activities detected suggest that S. piniformis has an ability to assimilate a greater range of substrates than might be concluded from the MAR data obtained here. Based on the substrate uptake data presented here, an anaerobic selector may work for controlling S. piniformis in activated sludge systems.     

Using respirometric techniques and fluorescent in situ hybridization to evaluate the heterotrophic active biomass in activated sludge

The separation and accurate quantification of active biomass components in activated sludge is of paramount importance in models, used for the management and design of waste water (WW) treatment plants. Accurate estimates of microbial population concentrations and the direct, in situ determination of kinetic parameters could improve the calibration and validation of existing models of biological nutrient removal activated sludge systems. The aim of this study was to obtain correlations between heterotrophic active biomass (Z(BH)) concentrations predicted by mathematical models and quantitative information obtained by Fluorescent in situ hybridizations (FISH). Respirometric batch test were applied to mixed liquors drawn from a well-defined parent anoxic/aerobic activated sludge system to quantify the Z(BH) concentrations. Similarly fluorescent labeled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidizers were used in combination with DAPI staining to validate the Z(BH) active biomass component in activate sludge respirometric batch tests. For the direct enumeration and simultaneous in situ analysis of the distribution of nitrifying bacteria, in situ hybridization with oligonucleotide probes were used. Probes (NSO 1225, NSR 1156, and NIT3) were used to target the nitrifiers and the universal probe (EUB MIX) was used to target all Eubacteria. Deducting the lithoautotrophic population from the total bacteria population revealed the Z(BH) population. A conversion factor of 8.49 x 10(-11) mg VSS/cell was applied to express the Z(BH) in terms of COD concentration. Z(BH) values obtained by molecular probing correlated closely with values obtained from the modified batch test. However, the trend of consistently poor correspondence of measured and theoretical concentrations were evident. Therefore, the focus of this study was to investigate alternative technology, such as FISH to validate or replace kinetic parameters which are invariably incorporated into models.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: I. Enumeration1

Agar plating media containing solely activated sludge extracts yielded, in general, higher viable counts of activated sludge bacteria than any other culture medium tested. Activated sludge extracts made from different treatment plants varied in efficacy in evoking maximal viable counts. Frequently, homologous plating, i.e., plating inocula of activated sludges on extracts made from the same activated sludges, tended to yield lower counts than the heterologous platings tried in this investigation. The counts obtained by homologous plating of activated sludge were not significantly lower and sometimes were even significantly higher than the counts obtained on standard Nutrient Agar, which had been found by previous workers to be a good medium for counting activated sludge bacteria. The higher counts obtained with activated sludge extracts set objectives for formulating reproducible or defined culture media for the enumeration of activated sludge bacteria.     

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。     

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     

Isolation and Characterization of Cellulolytic Bacteria from Activated Sludge

Cellulolytic aerobic bacteria were isolated from activated sludge systems. Of the media tested for enumeration, only filter paper media gave reliable counts. Five isolates were studied further for characterization. It was found that one strain (DK) belonged to the genus Cellulomonas. The other four strains expressed similarity to the genus Pseudomonas. The different characteristics that were studied, however, do not permit them to be identified with any recognized species. Based on certain characters we believe that they are alcaligenes-like pseudomonads.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

Numerical taxonomy of Skermania piniformis and related isolates from activated sludge

The microscopic morphology of nocardioforms causing foaming problems in activated sludge usually consists of filaments with branches at either right angles ( Nocardia amarae -Like Organisms, NALO) or acute angles (Pine Tree-Like Organisms, PTLO). Fifty-nine nocardioforms, mainly with PTLO morphology, isolated from mixed liquor and foam samples from Australian activated sludge plants, and 39 reference strains of nocardioforms, including type strains, were characterized using 109 morphological and physiological characters. Cluster Analysis and Principal Component Analysis showed that the activated sludge isolates clustered in six groups. All isolates that had typical PTLO morphology clustered unambiguously with the Skermania piniformis type strain (formerly called Nocardia pinensis ) showing that, unlike NALO, reliable unequivocal identification of S. piniformis , based on microscopic morphology in activated sludge, was possible. Other foam isolates whose morphology consisted of branches with both acute angles and right angles clustered as two separate groups, probably representing new species. These could not be confused microscopically with S. piniformis , despite some branches showing acute angles. The remaining three groups had typical NALO morphology. One of these groups did not cluster with any reference cultures and may be a new species or genus.