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This review discusses critically how seasonal changes might affect the community composition and dynamics of activated sludge wastewater treatment plants (WWTP), and examines the factors thought more generally to control microbial community assembly, including the role of taxa-time relationships and stochastic and deterministic influences. The review also questions the differences in protocols used in these studies, which make any subsequent attempts at data comparisons problematic. These include bacterial DNA extraction and PCR methodologies, 16S rRNA sequencing and especially its depth, and subsequent statistical analyses of the data, which together often fail to reveal seasonal dynamic community shifts. Suggestions are given as to how experimental protocols need to be improved and standardized, and especially the requirement to examine bacterial populations at the species level. This review looks critically at what is known currently about seasonal influences on key members of this community, including viruses, the bacteria responsible for nitrogen and phosphorus removal and those causing bulking and foaming. The data show many of these species exhibit replicative seasonal abundances over several years, but not under all conditions, illustrating how complex these community dynamics are. Fungal and protozoal/metazoal seasonal community dynamics, less studied, are also discussed. The current data suggest that seasonal temperature fluctuations are responsible for most of seasonal community dynamics by selectively favouring or otherwise individual populations. However, more longer term studies carried out under much stricter controlled conditions are required., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

The taxonomic status of two Gordonia strains, designated BEN371 and CON9 T , isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9 T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia . The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9 T represent a novel species of the genus Gordonia . The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9 T (=DSM 43602 T =JCM 35249 T ) as the type strain.

The production of a stable foam on the surfaces of reactors is a global operating problem in activated sludge plants. In many cases, these foams are stabilized by hydrophobic members of the Mycolata, a group of Actinobacteria whose outer membranes contain long-chain hydroxylated mycolic acids. There is currently no single strategy which works for all foams. One attractive approach is to use lytic bacteriophages specific for the foam stabilizing Mycolata population. Such phages are present in activated sludge mixed liquor and can be recovered readily from it. However, no phage has been recovered which lyses Gordonia amarae and Gordonia pseudoamarae, probably the most common foaming Mycolata members. Whole genome sequencing revealed that both G. amarae and G. pseudoamarae from plants around the world are particularly well endowed with genes encoding antiviral defence mechanisms. However, both these populations were lysed rapidly by a parasitic nanobacterium isolated from a plant in Australia. This organism, a member of the Saccharibacteria, was also effective against many other Mycolata, thus providing a potential agent for control of foams stabilized by them., (© 2022 The Society for Applied Microbiology.)

This comprehensive review looks critically what is known about members of the genus Defluviicoccus, an example of a glycogen accumulating organism (GAO), in wastewater treatment plants, but found also in other habitats. It considers the operating conditions thought to affect its performance in activated sludge plants designed to remove phosphorus microbiologically, including the still controversial view that it competes with the polyphosphate accumulating bacterium Ca. Accumulibacter for readily biodegradable substrates in the anaerobic zone receiving the influent raw sewage. It looks at its present phylogeny and what is known about it's physiology and biochemistry under the highly selective conditions of these plants, where the biomass is recycled continuously through alternative anaerobic (feed); aerobic (famine) conditions encountered there. The impact of whole genome sequence data, which have revealed considerable intra- and interclade genotypic diversity, on our understanding of its in situ behaviour is also addressed. Particular attention is paid to the problems in much of the literature data based on clone library and next generation DNA sequencing data, where Defluviicoccus identification is restricted to genus level only. Equally problematic, in many publications no attempt has been made to distinguish between Defluviicoccus and the other known GAO, especially Ca. Competibacter, which, as shown here, has a very different ecophysiology. The impact this has had and continues to have on our understanding of members of this genus is discussed, as is the present controversy over its taxonomy. It also suggests where research should be directed to answer some of the important research questions raised in this review., (Copyright © 2022. Published by Elsevier Ltd.)

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca . Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published ( Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca . Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanus T , the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bessarab, Maszenan, Haryono, Arumugam, Saw, Seviour and Williams.)

Many wastewater treatment plants around the world suffer from the operational problem of foaming. This is characterized by a persistent stable foam that forms on the aeration basin, which reduces effluent quality. The foam is often stabilized by a highly hydrophobic group of Actinobacteria known as the Mycolata 1 . Gordonia amarae is one of the most frequently reported foaming members 1 . With no currently reliable method for treating foams, phage biocontrol has been suggested as an attractive treatment strategy 2 . Phages isolated from related foaming bacteria can destabilize foams at the laboratory scale 3,4 ; however, no phage has been isolated that lyses G. amarae. Here, we assemble the complete genomes of G. amarae and a previously undescribed species, Gordonia pseudoamarae, to examine mechanisms that encourage stable foam production. We show that both of these species are recalcitrant to phage infection via a number of antiviral mechanisms including restriction, CRISPR-Cas and bacteriophage exclusion. Instead, we isolate and cocultivate an environmental ultrasmall epiparasitic bacterium from the phylum Saccharibacteria that lyses G. amarae and G. pseudoamarae and several other Mycolata commonly associated with wastewater foams. The application of this parasitic bacterium, 'Candidatus Mycosynbacter amalyticus', may represent a promising strategy for the biocontrol of bacteria responsible for stabilizing wastewater foams.

Amplicon sequence fingerprinting of communities in activated sludge systems have provided data revealing the true level of their microbial biodiversity and led to suggestions of which intrinsic and extrinsic parameters might affect the dynamics of community assemblage. Most studies have been performed in China and Denmark, and comparatively little information is available for plants in other countries. This study looked at how the communities of three plants in Victoria, Australia, treating domestic sewage changed with season. All were designed to remove nitrogen microbiologically. They were all located close together to minimise any influence that climate and demographics might have on their operation, and samples were taken at weekly intervals for 12 months. 16S rRNA amplicon sequencing revealed that each plant community was distinctively different to the others and changed over the 12-month sampling period. Many of the factors suggested in other similar studies to be important in determining community composition in activated sludge systems could not explain the changes noted here. The most likely influential factors were considered to be temperature and influent composition reflecting changes in dietary intake by the populations served by each plant, since in all three, the most noticeable changes corresponded to seasonal shifts. KEY POINTS: • Monitoring microbial communities in 3 wastewater treatment plants removing nitrogen • Temperature is the most influential factor in dynamic changes in community composition.

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

We report the complete genome sequence of Moraxella osloensis strain YV1, which was isolated from a wastewater treatment plant in Australia. The YV1 genome comprises a 2,615,801-bp chromosome and four plasmids. Moraxella osloensis strain YV1 displays the distinctive morphology of Eikelboom morphotype 1863., (Copyright © 2020 Batinovic et al.)

It is now clear that several of the filamentous bacteria in activated sludge wastewater treatment plants globally, are members of the phylum Chloroflexi. They appear to be more commonly found in treatment plants designed to remove nitrogen (N) and phosphorus (P), most of which operate at long sludge ages and expose the biomass to anaerobic conditions. The Chloroflexi seem to play an important beneficial role in providing the filamentous scaffolding around which flocs are formed, to feed on the debris from lysed bacterial cells, to ferment carbohydrates and to degrade other complex polymeric organic compounds to low molecular weight substrates to support their growth and that of other bacterial populations. A few commonly extend beyond the floc surface, while others can align in bundles, which may facilitate interfloc bridging and hence generate a bulking sludge. Although several recent papers have examined the phylogeny and in situ physiology of Chloroflexi in activated sludge plants in Denmark, this review takes a wider look at what we now know about these filaments, especially their global distribution in activated sludge plants, and what their functional roles there might be. It also attempts to outline why such information might provide us with clues as to how their population levels may be manipulated, and the main research questions that need addressing to achieve these outcomes., (Copyright © 2019 Speirs, Rice, Petrovski and Seviour.)

Bacteriophages (phages) are biological entities that have attracted a great deal of attention in recent years. They have been reported as the most abundant biological entities on the planet and their ability to impact the composition of bacterial communities is of great interest. In this review, we aim to explore where phages exist in natural and artificial environments and how they impact communities. The natural environment in this review will focus on the human body, soils, and the marine environment. In these naturally occurring environments there is an abundance of phages suggesting a role in the maintenance of bacterial community homeostasis. The artificial environment focuses on wastewater treatment plants, industrial processes, followed by pharmaceutical formulations. As in natural environments, the existence of bacteria in manmade wastewater treatment plants and industrial processes inevitably attracts phages. The presence of phages in these environments can inhibit the bacteria required for efficient water treatment or food production. Alternatively, they can have a positive impact by eliminating recalcitrant organisms. Finally, we conclude by describing how phages can be manipulated or formulated into pharmaceutical products in the laboratory for use in natural or artificial environments.

We describe here the isolation and characterization of the bacteriophage, NTR1 from activated sludge. This phage is lytic for Nocardia transvalensis, Nocardia brasiliensis and Nocardia farcinica. NTR1 phage has a genome sequence of 65,275 bp in length, and its closest match is to the Skermania piniformis phage SPI1 sharing over 36% of its genome. The phage belongs to the Siphoviridae family, possessing a long non-contractile tail and icosahedral head. Annotation of the genome reveals 97 putative open reading frames arranged in the characteristic modular organization of Siphoviridae phages and contains a single tRNA-Met gene.

Previous studies have shown that enhanced biological phosphorus removal (EBPR) performance under continuous aerobic conditions always eventually deteriorates; however, the speed at which this happens depends on the carbon source supplied. The published data suggest that propionate is a better carbon source than acetate is for maintaining operational stability, although it is not clear why. A lab-scale sequencing batch reactor was run initially under conventional anaerobic/aerobic conditions with either acetate or propionate as the carbon source. Chemical and microbiological analyses revealed that both sources performed as expected for such systems. When continuous aerobic conditions were imposed on both these established communities, marked shifts of the "Candidatus Accumulibacter" clades were recorded for both carbon sources. Here, we discuss whether this shift could explain the prolonged EBPR stability observed with propionate.

Although the phylogeny of many of the filamentous bacteria responsible for bulking in activated sludge plants is now known, and fluorescence in situ hybridisation (FISH) probes have been designed for their in situ identification, there are some noticeable exceptions. This study reports the identification of the Eikelboom morphotypes 0041 and 0675. Because these morphotypes differ only in their filament diameters, they are often considered together in surveys based on microscopic identifications. Here we show that they are phylogenetically distinct, and so should be viewed no longer as morphological variants of a single population. Amplicon sequencing data of Australian EBPR plant biomass containing types 0041 and 0675, and phylogenetic analysis have revealed that both, like many other bulking filament morphotypes, are members of the phylum Chloroflexi and probably representatives of two different genera. FISH probes are described here targeting each. Surveys carried out on Australian activated sludge plants suggest that type 0675 occurs more in plants designed to remove phosphorus, while type 0041 shows no such preference, and was seen in biomass samples from a wide range of plant configurations., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g -1 of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.

Overgrowth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) leads to impaired sludge settleability, a condition known as bulking, which is a common operational problem worldwide. Filaments with the Eikelboom 0092 morphotype are commonly associated with such bulking episodes. Members of the uncultured B45 phylotype, which is embraced within the phylum Chloroflexi, were recently shown to exhibit this morphology. Although these organisms are among the most abundant populations recorded in activated sludge processes, nothing is known about their metabolic characteristics. In this study, a genome sequence, representing the B45 phylotype, was retrieved from a metagenome generated from an activated sludge WWTP. The genome consisted of two chromosomes and one plasmid, which were 4.0, 1.0 and 0.04 Mbps in size, respectively. A metabolic model was constructed for this organism, based on annotation of its genome, showing its ability to generate energy by respiration, utilizing oxygen, nitrite or nitrous oxide as electron acceptors, or by fermentation of sugars. The ability of B45 members to ferment sugars under anaerobic conditions was validated in situ with microautoradiography-fluorescence in situ hybridization. The provisional name of 'Candidatus Promineofilum breve' is proposed for this species. This study represents the first detailed information on an uncultured genus of filamentous organisms from activated sludge.

Little is known about the prevalence, functionality and ecological roles of temperate phages for members of the mycolic acid producing bacteria, the Mycolata. While many lytic phages infective for these organisms have been isolated, and assessed for their suitability for use as biological control agents of activated sludge foaming, no studies have investigated how temperate phages might be induced for this purpose. Bioinformatic analysis using the PHAge Search Tool (PHAST) on Mycolata whole genome sequence data in GenBank for members of the genera Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella revealed 83% contained putative prophage DNA sequences. Subsequent prophage inductions using mitomycin C were conducted on 17 Mycolata strains. This led to the isolation and genome characterization of three novel Caudovirales temperate phages, namely GAL1, GMA1, and TPA4, induced from Gordonia alkanivorans, Gordonia malaquae, and Tsukamurella paurometabola, respectively. All possessed highly distinctive dsDNA genome sequences.

We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA., (Copyright © 2016 Dyson et al.)

Foaming in activated sludge plants is a worldwide problem commonly caused by proliferation of bacteria of the order Corynebacteriales. These include Skermania piniformis, a filamentous bacterium that has been documented to be a major cause of foaming globally, and particularly in Australian treatment plants. Phage SPI1 is the first phage that was isolated and shown to infect this organism. It targets seven of the nine strains of S. piniformis held in our culture collection, but none of the other 73 mycolata strains of different genera, mostly isolated from wastewater, against which it was tested. Phage SPI1 is a member of the family Siphoviridae and has a circularly permuted dsDNA genome of 55,748 bp with a G+C content of 67.8 mol %. It appears to be obligatorily lytic, with no evidence of genes related to a lysogenic mode of existence.

Three Tsukamurella phages, TIN2, TIN3, and TIN4, were isolated from activated sludge treatment plants located in Victoria, Australia, using conventional enrichment techniques. Illumina and 454 whole-genome sequencing of these Siphoviridae viruses revealed that they had similar genome sequences, ranging in size between 76,268 bp and 76,964 bp. All three phages shared 74% nucleotide sequence identity to the previously described Gordonia phage GTE7. Genome sequencing suggested that phage TIN3 had suffered a mutation in one of its lysis genes compared to the sequence of phage TIN4, to which it is genetically very similar. Mass spectroscopy data showed the unusual presence of a virion structural gene in the DNA replication module of phage TIN4, disrupting the characteristic modular genome architecture of Siphoviridae phages. All three phages appeared highly virulent on strains of Tsukamurella inchonensis and Tsukamurella paurometabola., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The Eikelboom filamentous morphotype 0803 is commonly found in activated sludge systems globally, where it contributes to sludge bulking events. Earlier reports have suggested that it is a member of both the Proteobacteria and Chloroflexi. This study shows that this filament contributing to a period of poor sludge settleability in an Australian activated sludge plant is a member of the Chloroflexi, but not within the Caldilinea, as reported for this morphotype in Danish plants. Instead, it is a member of the Anaerolineae. The fluorescent signals generated in these filaments using the FISH probes designed here were unevenly distributed, a situation similar to that seen earlier in the Anaerolineae morphotype 0092 to which it is more closely related phylogenetically than it is to the Caldilinea morphotype 0803. FISH-based surveys showed that this 0803 phylotype is uncommon in Australian activated sludge systems, and where seen is present usually at low abundances. The FISH probes described here will facilitate attempts to map the distribution and impact of this Australian filament morphotype 0803 in activated sludge systems of different configurations in plants around the world., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

Aims: Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence variant is present in strains of Acinetobacter junii, sequence data shows differences in ITS copies between and among them. This paper set out to explain why these ITS variants arise, and whether their presence compromises the reliability of the ITS targeted methods currently available for typing Ac. junii strains., Methods and Results: ITS sequences from a number of strains of Ac. junii were either downloaded from public databases or generated here by cloning and sequencing ITS PCR amplicons. ITS copies of Ac. junii strain 97338 were all 666 bp long, with identical sequences. In Ac. junii ATCC 17908(T) /BCRC 14854(T) ), ITS copies were also all identical in their lengths but now were 706/7 bp long. Two sequence variants of these 707 bp ITS were detected. One was identical in its sequence to the nine ITS copies downloaded from the whole genome sequence of Ac. junii CIP 64·5, and those in several other Ac. junii strains. The other 707 bp ITS variant occurred elsewhere only in Ac. junii strain DSM 14968 of those examined. The six ITS copies from the genome sequence of Ac. junii NIPH 182 were all 685 bp, and with identical sequences. Ac. junii strain 178 also possessed this same 685 bp ITS variant, one of six variants detected there. At least five ITS sequence variants were seen in Ac. junii strain 97380, four in strain DSM 14968 and two in the whole genome of strain 107470., Conclusions: As with those of other Acinetobacter species, such ITS variants arise not from intragenomic recombination events but from the presence of different length indels. These arise from horizontal gene transfers involving ITS fragments of other Acinetobacter species., Significance and Impact of the Study: The presence of these indels compromises the reliability of the ITS targeted methods available for typing Acinetobacter junii. It also precludes the value of using ITS sequences as phylogenetic markers in members of the genus Acinetobacter, since the outcomes in both cases depends on which copy variant is chosen., (© 2015 The Society for Applied Microbiology.)

The FISH probe TM7-305 is thought to target the filamentous Eikelboom morphotype 0041 as a member of the Candidatus ‘Saccharibacteria’ (formerly TM7) phylum. However, with activated sludge samples in both Japan and Australia, this probe hybridized consistently with filamentous bacteria fitting the description of the morphotype 1851, which also responded positively to the CHL1851 FISH probe designed to target Chloroflexi members of this morphotype. 16S rRNA clone libraries from samples containing type 1851 TM7-305-positive filaments yielded Chloroflexi clones with high sequence similarity to Kouleothrix aurantiaca. These contained a variant TM7-305 probe target site possessing weakly destabilizing mismatches insufficient to prevent probe hybridization. Furthermore, the TM7-905 FISH probe, designed to target members of the entire Candidatus ‘Saccharibacteria’ phylum, also hybridized with the filament morphotypes 0041/0675, which responded also to the phylum level Chloroflexi probes. Many Chloroflexi sequences have only a single base mismatch to the TM7-905 probe target sequence. When competitor probes for both the TM7-305 and TM7-905 Chloroflexi non-target sites were applied, no fluorescent signal was seen in any of the filamentous organisms also hybridizing with the aforementioned Chloroflexi probes. These data indicate that these competitor probes must be included in hybridizations when both the TM7-905 and TM7-305 FISH probes are applied, to minimize potential false positive FISH results.

To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2-13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species.

We compared the relative values of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) for profiling fungal communities in wastewater treatment plants using both ITS and 18S rRNA gene fragments as phylogenetic markers. A similar number of fungal ribotypes was obtained with both methods for the same treatment plant when the ITS primer set was used, while a greater number of ribotypes was obtained with T-RFLP compared to DGGE with the 18S rRNA primer set. Non-metric multi-dimensional scaling of presence/absence data and analysis of similarity showed that both methods could distinguish between the different plant communities at a statistically significant level (p < 0.05), regardless of which phylogenetic marker was used. The data suggest that both methods can be used preferably together to profile activated sludge fungal communities. A comparison of profiles generated with both these phylogenetic markers based on the number of ribotypes/bands, suggests that the 18S rRNA region is more discriminatory than the ITS region. Detected differences in fungal community compositions between plants probably reflect differences in their influent compositions and operational parameters.

We here characterize a novel bacteriophage (NBR1) that is lytic for Nocardia otitidiscaviarum and N. brasiliensis. NBR1 is a member of the family Siphoviridae and appears to have a structurally more complex tail than previously reported Siphoviridae phages. NBR1 has a linear genome of 46,140 bp and a sequence that appears novel when compared to those of other phage sequences in GenBank. Annotation of the genome reveals 68 putative open reading frames. The phage genome organization appears to be similar to other Siphoviridae phage genomes in that it has a modular arrangement.

'Candidatus Microthrix parvicella' is a lipid-accumulating, filamentous bacterium so far found only in activated sludge wastewater treatment plants, where it is a common causative agent of sludge separation problems. Despite attracting considerable interest, its detailed physiology is still unclear. In this study, the genome of the RN1 strain was sequenced and annotated, which facilitated the construction of a theoretical metabolic model based on available in situ and axenic experimental data. This model proposes that under anaerobic conditions, this organism accumulates preferentially long-chain fatty acids as triacylglycerols. Utilisation of trehalose and/or polyphosphate stores or partial oxidation of long-chain fatty acids may supply the energy required for anaerobic lipid uptake and storage. Comparing the genome sequence of this isolate with metagenomes from two full-scale wastewater treatment plants with enhanced biological phosphorus removal reveals high similarity, with few metabolic differences between the axenic and the dominant community 'Ca. M. parvicella' strains. Hence, the metabolic model presented in this paper could be considered generally applicable to strains in full-scale treatment systems. The genomic information obtained here will provide the basis for future research into in situ gene expression and regulation. Such information will give substantial insight into the ecophysiology of this unusual and biotechnologically important filamentous bacterium.

Rhodococcus equi is a pathogenic member of the Actinobacteria responsible for causing serious infections in equines. A novel Siphoviridae bacteriophage (REQ1) lytic in R. equi was isolated and characterized. The genome size of REQ1 is 51,342 bp, and its sequence shares 7 % similarity to other DNA sequence in GenBank. Putative open reading frames were identified, and their functions were identified based on their predicted amino acid similarities. REQ1 phage has a modular genome, a feature common in double-stranded DNA phages.

We report here the isolation and genome sequences of two novel phages, lytic for Rhodococcus and Nocardia species. Named RER2 and RGL3, both are members of the family Siphoviridae, and each possesses a novel genome of 46,586 bp and 48,072 bp, respectively. RER2 and RGL3 phages share a modular genome organization, as seen in other sequenced Siphoviridae phage genomes, and appear to share a common evolutionary origin. The genomes of these phages share no similarity with other Rhodococcus or Nocardia phages but are related to Mycobacterium phages. The data presented here extend our understanding of Rhodococcus phage genomics.

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

The Nosticoida limicola filamentous morphotype is held responsible for incidents of bulking and foaming in activated sludge. Members of the actinobacterial N. limicola II have been isolated and grown in pure culture and shown to belong to the genus Tetrasphaera, and play an important role in phosphorus removal. This article describes the isolation and genomic characterization of a phage able to lyse Tetrasphaera jenkinsii, TJE1. This lytic phage is a member of the Caudovirales specific for T. jenkinsii. The complete DNA sequence of TJE1 phage revealed it to have a circularly permuted genome (49,219 bp) with 66 putative open reading frames, a single transcriptional terminator, and 6 pairs of inverted repeats within the genome sequence. The TJE1 phage genome is organised into a modular gene structure, but shares only limited sequence identity with other phages so far described.

Fungal diversity of communities in several activated sludge plants treating different influent wastes was determined by comparative sequence analyses of their 18S rRNA genes. Methods for DNA extraction and choice of primers for PCR amplification were both optimised using denaturing gradient gel electrophoresis profile patterns. Phylogenetic analysis revealed that the levels of fungal biodiversity in some communities, like those treating paper pulp wastes, were low, and most of the fungi detected in all communities examined were novel uncultured representatives of the major fungal subdivisions, in particular, the newly described clade Cryptomycota. The fungal populations in activated sludge revealed by these culture-independent methods were markedly different to those based on culture-dependent data. Members of the genera Penicillium, Cladosporium, Aspergillus and Mucor, which have been commonly identified in mixed liquor, were not identified in any of these plant communities. Non-fungal eukaryotic 18S rRNA genes were also amplified with the primer sets used. This is the first report where culture-independent methods have been applied to flocculated activated sludge biomass samples to estimate fungal community composition and, as expected, the data obtained gave a markedly different view of their population biodiversity compared to that based on culture-dependent methods.

Activated sludge plants suffer frequently from the operational problem of stable foam formation on aerobic reactor surfaces, which can be difficult to prevent. Many foams are stabilized by mycolic acid-containing Actinobacteria, the mycolata. The in situ biocontrol of foaming using phages is an attractive strategy. We describe two polyvalent phages, GTE5 and GRU1, targeting Gordonia terrae and Gordonia rubrupertincta, respectively, isolated from activated sludge. Phage GRU1 also propagates on Nocardia nova. Both phages belong to the family Siphoviridae and have similar-size icosahedral heads that encapsulate double-stranded DNA genomes (∼65 kb). Their genome sequences are similar to each other but markedly different from those of other sequenced phages. Both are arranged in a modular fashion. These phages can reduce or eliminate foam formation by their host cells under laboratory conditions.

Bacteriophages are considered to be the most abundant biological entities on the planet. The Siphoviridae are the most commonly encountered tailed phages and contain double-stranded DNA with an average genome size of ∼50 kb. This paper describes the isolation from four different activated sludge plants of the phage RRH1, which is polyvalent, lysing five Rhodococcus species. It has a capsid diameter of only ∼43 nm. Whole-genome sequencing of RRH1 revealed a novel circularly permuted DNA sequence (14,270 bp) carrying 20 putative open reading frames. The genome has a modular arrangement, as reported for those of most Siphoviridae phages, but appears to encode only structural proteins and carry a single lysis gene. All genes are transcribed in the same direction. RRH1 has the smallest genome yet of any described functional Siphoviridae phage. We demonstrate that lytic phage can be recovered from transforming naked DNA into its host bacterium, thus making it a potentially useful model for studying gene function in phages.

Previous research has suggested that enhanced biological phosphorus removal (EBPR) from wastewater can be achieved under continuous aerobic conditions over the short term. However, little is known how environmental conditions might affect aerobic EBPR performance. Consequently we have investigated the impact of temperature, pH and dissolved oxygen (DO) concentrations on EBPR performance under strictly aerobic conditions. A sequencing batch reactor (SBR) was operated for 108 days on a six-hour cycle (four cycles a day). The SBR ran under alternating anaerobic-aerobic conditions as standard and then operated under strictly aerobic conditions for one cycle every three or four days. SBR operational temperature (10, 15, 20, 25 and 30°C), pH (6, 7, 8 and 9) and DO concentration (0.5, 2.0 and 3.5mg/L) were changed consecutively during the aerobic cycle. Recorded increases in mixed liquor phosphorus (P) concentrations during aerobic carbon source uptake (P release) were affected by the biomass P content rather than the imposed changes in the operational conditions. Thus, P release levels increased with biomass P content. By contrast, subsequent aerobic P assimilation (P uptake) levels were both affected by changes in operational temperature and pH, and peaked at 20-25°C and pH 7-8. Highest P uptake detected under these SBR operating conditions was 15.4 mg Pg-MLSS(-1) (at 25°C, pH 7 and DO 2.0mg/L). The ability of the community for linked aerobic P release and P uptake required the presence of acetate in the medium, a finding which differs from previous data, where these are reported to occur in the absence of any exogenous carbon source. Fluorescence in situ hybridization was performed on samples collected from the SBR, and Candidatus 'Accumulibacter phosphatis' cells were detected with PAOmix probes through the operational periods. Thus, Candidatus 'Accumulibacter phosphatis' seemed to perform P removal in the SBR as shown in previous studies on P removal under strictly aerobic conditions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Most activated sludge treatment plants suffer from the presence of foams on the surfaces of their aeration reactors. These are often stabilized by hydrophobic mycolic acid-synthesizing actinobacterial species. A polyvalent Siphoviridae phage, GTE7, which lysed several Gordonia and Nocardia species, is described here. Its genome has a modular structure similar to that described for Rhodococcus phage ReqiDocB7. In laboratory-scale experiments, we showed that GTE7 prevents stabilization of foams by these Gordonia and Nocardia species.

On-site aerobic sequencing batch reactor (SBR) treatment plants are implemented in many Australian wineries to treat the large volumes of associated wastewater they generate. Yet very little is known about their microbiology. This paper represents the first attempt to analyze the communities of three such systems sampled during both vintage and nonvintage operational periods using molecular methods. Alphaproteobacterial tetrad forming organisms (TFO) related to members of the genus Defluviicoccus and Amaricoccus dominated all three systems in both operational periods. Candidatus 'Alysiosphaera europaea' and Zoogloea were codominant in two communities. Production of high levels of exocellular capsular material by Zoogloea and Amaricoccus is thought to explain the poor settleability of solids in one of these plants. The dominance of these organisms is thought to result from the high COD to N/P ratios that characterize winery wastes, and it is suggested that manipulating this ratio with nutrient dosing may help control the problems they cause.

There is considerable interest in exploiting the novel physical and biological properties of microbial exopolysaccharides in industry and medicine. For economic and scientific reasons, large scale production under carefully monitored and controlled conditions is required. Producing exopolysaccharides in industrial fermenters poses several complex bioengineering and microbiological challenges relating primarily to the very high viscosities of such culture media, which are often exacerbated by the producing organism's morphology. What these problems are, and the strategies for dealing with them are discussed critically in this review, using pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides. The role of fermenter configuration in their production is also examined.

Hydrophobic Actinobacteria are commonly associated with the stabilization of foams in activated sludge systems. One possible attractive approach to control these foam-stabilizing organisms is the use of specific bacteriophages. We describe the genome characterization of a novel polyvalent DNA phage, GTE2, isolated from activated sludge. This phage is lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis. Phage GTE2 belongs to the family Siphoviridae, possessing a characteristic icosahedral head encapsulating a double-stranded DNA linear genome (45,530 bp) having 10-bp 3'-protruding cohesive ends. The genome sequence is 98% unique at the DNA level and contains 57 putative genes. The genome can be divided into two components, where the first is modular and encodes phage structural proteins and lysis genes. The second is not modular, and the genes harbored there are involved in DNA replication, repair, and metabolism. Some have no known function. GTE2 shows promising results in controlling stable foam production by its host bacteria under laboratory conditions, suggesting that it may prove useful in the field as a biocontrol agent.

The filamentous bacterium Eikelboom morphotype type 0914 responsible for bulking in activated sludge plants is identified here for the first time as a member of the phylum Chloroflexi subgroup 1. Two FISH probes, CFX67a and CFX67b, targeting the 16S rRNA sequences of this filament morphotype were designed, validated and used successfully for its in situ identification. A survey of plants in eastern Australia with the CFX67a probe showed it targeted only the type 0914 morphotype that was common especially in long sludge age plants designed to remove phosphorus and nitrogen microbiologically, although being in very low abundance in many samples. Filaments responding to the CFX67b probe also exhibited the type 0914 morphology but were less frequent, although again occurring in similarly configured plants. All these filaments showed an uneven FISH signal suggesting their ribosomes are localized at the ends of their cells. Furthermore, some generated distinctive FISH signals in all biomass samples containing them, where only certain cells within any single trichome fluoresced with probes designed against different target sites. Helper probes for each of these were required before all cells fluoresced above the visual detection limits of FISH., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Screening pure cultures of 65 mycolic acid producing bacteria (Mycolata) isolated mainly from activated sludge with a laboratory based foaming test revealed that not all foamed under the conditions used. However, for most, the data were generally consistent with the flotation theory as an explanation for foaming. Thus a stable foam required three components, air bubbles, surfactants and hydrophobic cells. With non-hydrophobic cells, an unstable foam was generated, and in the absence of surfactants, cells formed a greasy surface scum. Addition of surfactant converted a scumming population into one forming a stable foam. The ability to generate a foam depended on a threshold cell number, which varied between individual isolates and reduced markedly in the presence of surfactant. Consequently, the concept of a universal threshold applicable to all foaming Mycolata is not supported by these data. The role of surfactants in foaming is poorly understood, but evidence is presented for the first time that surfactin synthesised by Bacillus subtilis may be important., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The formation of stable foam in activated sludge plants is a global problem for which control is difficult. These foams are often stabilized by hydrophobic mycolic acid-synthesizing Actinobacteria, among which are Tsukamurella spp. This paper describes the isolation from activated sludge of the novel double-stranded DNA phage TPA2. This polyvalent Siphoviridae family phage is lytic for most Tsukamurella species. Whole-genome sequencing reveals that the TPA2 genome is circularly permuted (61,440 bp) and that 70% of its sequence is novel. We have identified 78 putative open reading frames, 95 pairs of inverted repeats, and 6 palindromes. The TPA2 genome has a modular gene structure that shares some similarity to those of Mycobacterium phages. A number of the genes display a mosaic architecture, suggesting that the TPA2 genome has evolved at least in part from genetic recombination events. The genome sequence reveals many novel genes that should inform any future discussion on Tsukamurella phage evolution.

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non-target rRNA sequences. We show here that probe target sites containing single-base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non-target sites resulting from single-base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)