Biological control of problematic bacterial populations causing foaming in activated sludge wastewater treatment plants-phage therapy and beyond.

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.

Eikelboom filamentous morphotypes 0675 and 0041 embrace members of the Chloroflexi: resolving their phylogeny, and design of fluorescence in situ hybridisation probes for their identification.

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.

Radioisotopes produced by neutron irradiation of food.

The use of neutrons for cargo interrogation has the potential to drastically improve threat detection. Previous research has focussed on the production of (24)Na, based on the isotopes produced in pharmaceuticals and medical devices. For both the total activity and the ingestion dose we show that a variety of isotopes contribute and that (24)Na is only dominant under certain conditions. The composition of the foods has a strong influence on the resulting activity and ingestion dose suggesting that the pharmaceuticals and medical devices considered initially are not a viable analogue for foodstuffs. There is an energy dependence to the isotopes produced due to the cross-sections of different reactions varying with neutron energy. We show that this results in different isotopes dominating the ingestion dose at different energies, which has not been considered in the previous literature.

Isolation and characterization of bacteriophage SPI1, which infects the activated-sludge-foaming bacterium Skermania piniformis.

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.

Radiological risks of neutron interrogation of food.

In recent years there has been growing interest in the use of neutron scanning techniques for security. Neutron techniques with a range of energy spectra including thermal, white and fast neutrons have been shown to work in different scenarios. As international interest in neutron scanning increases the risk of activating cargo, especially foodstuffs must be considered. There has been a limited amount of research into the activation of foods by neutron beams and we have sought to improve the amount of information available. In this paper we show that for three important metrics; activity, ingestion dose and Time to Background there is a strong dependence on the food being irradiated and a weak dependence on the energy of irradiation. Previous studies into activation used results based on irradiation of pharmaceuticals as the basis for research into activation of food. The earlier work reports that (24)Na production is the dominant threat which motivated the search for (24)Na(n,γ)(24)Na in highly salted foods. We show that (42)K can be more significant than (24)Na in low sodium foods such as Bananas and Potatoes.

The impact of horizontal gene transfer on targeting the internal transcribed spacer region (ITS) to identify Acinetobacter junii strains.

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.

Fluorescence in situ hybridization probing of protozoal Entodinium spp. and their methanogenic colonizers in the rumen of cattle fed alfalfa hay or triticale straw.

AIMS: To develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. METHODS AND RESULTS: New FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. CONCLUSIONS: Simultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans.

AIMS: To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans. METHODS AND RESULTS: In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0.13 g l(-1) N) initial NaNO(3) or (NH(4))(2)SO(4) levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0.78 g l(-1) N) initial NaNO(3) levels. EPS produced by CSTR grown cultures with high (NH(4))(2)SO(4) levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO(3) levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units. CONCLUSIONS: While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.

Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process.

AIMS: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. METHODS AND RESULTS: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. CONCLUSIONS: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.

Isolates of 'Candidatus Nostocoida limicola' Blackall et al. 2000 should be described as three novel species of the genus Tetrasphaera, as Tetrasphaera jenkinsii sp. nov., Tetrasphaera vanveenii sp. nov. and Tetrasphaera veronensis sp. nov.

Despite differences in their morphologies, comparative analyses of 16S rRNA gene sequences revealed high levels of similarity (>94 %) between strains of the filamentous bacterium 'Candidatus Nostocoida limicola' and the cocci Tetrasphaera australiensis and Tetrasphaera japonica and the rod Tetrasphaera elongata, all isolated from activated sludge. These sequence data and their chemotaxonomic characters, including cell wall, menaquinone and lipid compositions and fingerprints of their 16S-23S rRNA intergenic regions, support the proposition that these isolates should be combined into a single genus containing six species, in the family Intrasporangiaceae in the Actinobacteria. This suggestion receives additional support from DNA-DNA hybridization data and when partial sequences of the rpoC1 gene are compared between these strains. Even though few phenotypic characterization data were obtained for these slowly growing isolates, it is proposed, on the basis of the extensive chemotaxonomic and molecular evidence presented here, that 'Candidatus N. limicola' strains Ben 17, Ben 18, Ben 67, Ben 68 and Ben 74 all be placed into the species Tetrasphaera jenkinsii sp. nov. (type strain Ben 74(T)=DSM 17519(T)=NCIMB 14128(T)), 'Candidatus N. limicola' strain Ben 70 into Tetrasphaera vanveenii sp. nov. (type strain Ben 70(T)=DSM 17518(T)=NCIMB 14127(T)) and 'Candidatus N. limicola' strains Ver 1 and Ver 2 into Tetrasphaera veronensis sp. nov. (type strain Ver 1(T)=DSM 17520(T)=NCIMB 14129(T)).

Substrate uptake by Gordonia amarae in activated sludge foams by FISH-MAR.

Gordonia amarae is a right-angled branching filament belonging to the mycolic acid-containing Actinobacteria which is commonly found in many foaming activated sludge wastewater treatment plants. Although studies on different substrates as sole carbon sources by pure cultures of G. amarae have been carried out, none have examined substrate uptake by this organism in situ. Uptake of several hydrophilic and hydrophobic substrates by G. amarae was evaluated in situ using a combination of fluorescence in situ hybridization and microautoradiography. G. amarae could assimilate a range of both hydrophilic and hydrophobic substrates. From the data, G. amarae appears to be physiologically active under aerobic, anaerobic and anoxic condition (NO2 and NO3) for some substrates. This might explain why attempts to control foaming caused by G. amarae using anoxic and anaerobic selectors have been unsuccessful. This study emphasizes that bacteria can behave differently in situ to pure cultures and that it is important to evaluate the in situ physiology of these bacteria if we are to better understand their role in the wastewater treatment process.

The in situ physiology of "Nostocoida limicola" II, a filamentous bacterial morphotype in bulking activated sludge, using fluorescence in situ hybridization and microautoradiography.

The in situ physiology of the actinobacterial bulking and foaming filamentous bacterium "Nostocoida limicola" II was studied by fluorescence in situ hybridization/microautoradiography. Substrate assimilation patterns of pure cultures of this bacterium were different to those seen in activated sludge biomass samples. There was no evidence to suggest that "N. limicola" II preferred hydrophobic substrates, but evidence was produced to support the view that it is metabolically active under anaerobic conditions in activated sludge.

Gene cassette-associated sequences from phosphorus and non-phosphorus removing microbial communities in aerobic:anaerobic sequencing batch reactors.

Mobile gene elements associated with integrons, including as gene cassettes, have been proposed to play an important role in bacterial evolution by providing an extensive genetic resource. This study hypothesized that critical genes for enzymes involved in EBPR systems, including those involved in polyphosphate, PHA and glycogen synthesis, may be present in mobile gene cassettes. Although no such genes were identified in any of the functional and deteriorated enhanced biological phosphorus removal (EBPR) laboratory-scale SBR systems examined here, many of the open reading frames (ORFs) remained unidentified because of the incompleteness of publicly available databases. An ORF of unknown function (SBR6-2) was encountered in deteriorated EBPR system with an unexpectedly high frequency, comprising 35% of the gene cassette-associated sequences for that system.

Which are the polyphosphate accumulating organisms in full-scale activated sludge enhanced biological phosphate removal systems in Australia?

AIMS: To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. METHODS: Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4'-6-diamidino-2-phenylindole) staining for polyP. RESULTS: Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. CONCLUSIONS: The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances.

Defluvicoccus vanus gen. nov., sp. nov., a novel Gram-negative coccus/coccobacillus in the 'Alphaproteobacteria' from activated sludge.

A novel Gram-negative coccus/coccobacillus, strain Ben 114(T), growing in tetrads, clusters or aggregates, was isolated from activated sludge by micromanipulation. 16S rRNA gene sequence analysis revealed that it belonged to the 'Alphaproteobacteria', with no close relatives among cultured bacterial isolates. On the basis of phylogenetic data, this organism is considered to belong to a new genus, Defluvicoccus, represented by the species Defluvicoccus vanus sp. nov., a name chosen because of the distinctive staining properties of this organism; only the cell wall stained strongly with a wide range of stains, giving the cell a hollow and empty appearance. No intracellular polyphosphate granules could be detected after staining, but poly-beta-hydroxyalkanoate inclusions were detected using Nile blue A staining. Because of its taxonomic distance from its closest relatives among the 'Alphaproteobacteria', namely members of the genera Azospirillum, Phaeospirillum, Rhodospirillum, Rhodocista, Magnetospirillum and Rhodospira, D. vanus is considered to represent a new phylogenetic lineage within subgroup 1 of the 'Alphaproteobacteria', the D. vanus subgroup. The type strain is Ben 114(T) (=NCIMB 13612(T)=CIP 107350(T)).

The large PAO cells in full-scale EBPR biomass samples are not yeast spores but possibly novel members of the beta-Proteobacteria.

Large, homogenous clusters of coccobacilli were found to be abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. The identity and the in situ physiology of these dominant microorganisms were investigated in this study. These large clustered cells were revealed to be neither Gram positive nor Gram negative bacteria and contain polyP granules. Cells with similar features were also observed in some enhanced biological phosphate removal (EBPR) systems and reported as yeast spores and Rhodocyclus-related polyphosphate accumulating organisms (PAOs). In this study, fluorescent in situ hybridization (FISH) probing showed these cells were prokaryotic and members of the beta-Proteobacteria. However, these large clustered cells did not respond to the PAO mix FISH probes. The in situ physiology of these large cells was studied with FISH in combination with microautoradiography (MAR) in order to understand their substrate assimilation abilities under different conditions as well as their phosphate uptake ability. These cells were able to take up acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions. Nile Blue A staining in combination with MAR showed that cells incubated under anaerobic conditions contained polyhydroxyalkanoates (PHA) granules. In addition, MAR showed aerobic 33Pi assimilation with all these substrates, consistent with them supporting an EBPR capacity in these large cells. As well as raising doubts about a role for yeasts in EBPR, this study suggests that much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems, and emphasizes the benefits of using techniques like FISH/MAR and PHA staining/MAR to resolve the in situ physiology of the populations of interest there.

The rapid identification of Acinetobacter species using Fourier transform infrared spectroscopy.

AIMS: Fourier transform infrared (FT-IR) was used to analyse a selection of Acinetobacter isolates in order to determine if this approach could discriminate readily between the known genomic species of this genus and environmental isolates from activated sludge. METHODS AND RESULTS: FT-IR spectroscopy is a rapid whole-organism fingerprinting method, typically taking only 10 s per sample, and generates 'holistic' biochemical profiles (or 'fingerprints') from biological materials. The cluster analysis produced by FT-IR was compared with previous polyphasic taxonomic studies on these isolates and with 16S-23S rDNA intergenic spacer region (ISR) fingerprinting presented in this paper. FT-IR and 16S-23S rDNA ISR analyses together indicate that some of the Acinetobacter genomic species are particularly heterogeneous and poorly defined, making characterization of the unknown environmental isolates with the genomic species difficult. CONCLUSIONS: Whilst the characterization of the isolates from activated sludge revealed by FT-IR and 16S-23S rDNA ISR were not directly comparable, the dendrogram produced from FT-IR data did correlate well with the outcomes of the other polyphasic taxonomic work. SIGNIFICANCE AND IMPACT OF THE STUDY: We believe it would be advantageous to pursue this approach further and establish a comprehensive database of taxonomically well-defined Acinetobacter species to aid the identification of unknown strains. In this instance, FT-IR may provide the rapid identification method eagerly sought for the routine identification of Acinetobacter isolates from a wide range of environmental sources.

Effects of culture conditions on the mycolic acid composition of isolates of Rhodococcus spp. from activated sludgefoams.

The influence of two different carbon sources and three incubation temperatures on the mycolic acid compositions of three Rhodococcus isolates from activated sludge was examined using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry (GC-MS). Considerable qualitative and quantitative differences were detected in the mycolic acid compositions of the three very closely related isolates grown under the same conditions. Culture age also affected both the chain lengths and proportions of saturated mycolic acids detected in cell extracts, but not in the same manner for each isolate. Mycolic acids generally were of shorter chain lengths in cells grown with Tween 80 compared to glucose grown cells in strain 11R but the opposite situation occurred with strains A7 and D5. In all three, the proportion of unsaturated mycolic acids decreased with increasing growth temperatures. The taxonomic relevance of these observations and possible explanations for the observed changes in mycolic acid composition under various culture conditions are discussed.

Environmental factors contributing to the "G bacteria" population in full-scale EBPR plants.

A survey of several enhanced biological phosphorus removal (EBPR) plants within Australia has demonstrated that a group of bacteria known as the "G" bacteria are able to proliferate under a broad range of plant configurations. The diverse designs and operational parameters of these plants did not permit definitive determination of the factor(s) contributing to the proliferation of G bacteria. Two plants were monitored over time to assess the G bacteria and phosphorus accumulating organisms (PAO) populations in relation to key operational parameters. The mixed liquor biomass and operational parameters were compared to other plants successfully and unsuccessfully reducing phosphorus from the wastewater. Two critical factors recognised in this study were the dissolved oxygen concentration in the aerobic zone and the type and amount of carbon source in the anaerobic zone.