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Determining the composition of the complex microbial communities within biofilms can aid us in understanding the potential for biodegradation on different permeable reactive barrier materials. Microbial communities were examined under Antarctic conditions across laboratory and field studies using amplicon pyrosequencing, and the reaction of these communities to petroleum hydrocarbon contamination and NH4+ supplementation was assessed. Putative hydrocarbon degrading bacteria in the orders Actinomycetales and Burkholderiales were identified in laboratory flow cells and within the upper PRB and lower PRB at Casey Station, Antarctica. The release of NH4+ from ammonium exchanged zeolite was shown to reduce the microbial diversity of biofilms, when compared to natural zeolite. Bacteria from the orders Sphingomonadales, Rhodocyclales, Pseudomonadales and Xanthomonadales were also observed across the Zeopro™/granular activated carbon permeable reactive barrier mixtures. The bacterial species were observed within a uniform microbial biofilm layer, embedded within extracellular polymeric substances on the surface of the materials. This study demonstrates that petroleum hydrocarbon contamination can contribute to the enrichment of certain hydrocarbon degrading species on permeable reactive barrier materials.

The increase in global warming has favored growth of a range of opportunistic environmental bacteria and allowed some of these to become more pathogenic to humans. Aeromonas hydrophila is one such organism. Surviving in moist conditions in temperate climates, these bacteria have been associated with a range of diseases in humans, and in systemic infections can cause mortality in up to 46% of cases. Their capacity to form biofilms, carry antibiotic resistance mechanisms, and survive disinfection, has meant that they are not easily treated with traditional methods. Bacteriophage offer a possible alternative approach for controlling their growth. This study is the first to report the isolation and characterization of bacteriophages lytic against clinical strains of A. hydrophila which carry intrinsic antibiotic resistance genes. Functionally, these novel bacteriophages were shown to be capable of disrupting biofilms caused by clinical isolates of A. hydrophila. The potential exists for these to be tested in clinical and environmental settings.

Fusobacterium nucleatum is an important oral bacterium that has been linked to the development of chronic diseases such as periodontitis and colorectal cancer. In periodontal disease, F. nucleatum forms the backbone of the polymicrobial biofilm and in colorectal cancer is implicated in aetiology, metastasis and chemotherapy resistance. The control of this bacteria may be important in assisting treatment of these diseases. With increased rates of antibiotic resistance globally, there is need for development of alternatives such as bacteriophages, which may complement existing therapies. Here we describe the morphology, genomics and functional characteristics of FNU1, a novel bacteriophage lytic against F. nucleatum. Transmission electron microscopy revealed FNU1 to be a large Siphoviridae virus with capsid diameter of 88 nm and tail of approximately 310 nm in length. Its genome was 130914 bp, with six tRNAs, and 8% of its ORFs encoding putative defence genes. FNU1 was able to kill cells within and significantly reduce F. nucleatum biofilm mass. The identification and characterisation of this bacteriophage will enable new possibilities for the treatment and prevention of F. nucleatum associated diseases to be explored.

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.

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.

Summary 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.

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.

Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi . Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-β-hydroxyalkanoates or polyphosphate.

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.