Significance of buoyancy in turbulence closure for computational fluid dynamics modelling of ultraviolet disinfection in maturation ponds.

Buoyancy-driven turbulent dispersion in a maturation pond is studied using a combination of field measurements and computational fluid dynamics. Modelling flow in maturation ponds requires turbulent closure models because of the large physical size and the need to model on diurnal timescales. Simulation results are shown to be more sensitive to the inclusion of a buoyancy production term appearing in the turbulent transport equations than to the model choice. Comparisons with experimental thermal profiles show that without this term, thermal mixing is over-predicted. When including the term, stratification occurs but thermal mixing is under-predicted in the lower water column. In terms of pond performance, the effect of this term is to cause increased surface die-off of Escherichia coli during sunlight hours due to the generation of stratification. It is recommended that future modelling consider and implement this term.

Survival of Escherichia coli in two sewage treatment plants using UV irradiation and chlorination for disinfection.

We investigated the survival of Escherichia coli in two STPs utilising UV irradiation (STP-A) or chlorination (STP-B) for disinfection. In all, 370 E. coli strains isolated from raw influent sewage (IS), secondary treated effluent (STE) and effluent after the disinfection processes of both STPs were typed using a high resolution biochemical fingerprinting method and were grouped into common (C-) and single (S-) biochemical phenotypes (BPTs). In STP-A, 83 BPTs comprising 123 isolates were found in IS and STE, of which 7 BPTs survived UV irradiation. Isolates tested from the same sites of STP-B (n = 220) comprised 122 BPTs, however, only two BPTs were found post-chlorination. A representative isolate from each BPT from both STPs was tested for the presence of 11 virulence genes (VGs) associated with uropathogenic (UPEC) or intestinal pathogenic (IPEC) E. coli strains. Strains surviving UV irradiation were distributed among seven phylogenetic groups with five BPTs carrying VGs associated with either UPEC (4 BPTs) or IPEC (1 BPT). In contrast, E. coli strains found in STP-B carried no VGs. Strains from both STPs were resistant to up to 12 out of the 21 antibiotics tested but there was no significant difference between the numbers of antibiotics to which surviving strains were resistant to in these STPs. Our data suggests that some E. coli strains have a better ability to survive STPs utilising chlorination and UV irradiation for disinfection. However, strains that survive UV irradiation are more diverse and may carry more VGs than those surviving SPTs using chlorination.

Antibiotic resistant Staphylococcus aureus in hospital wastewaters and sewage treatment plants with special reference to methicillin-resistant Staphylococcus aureus (MRSA).

AIMS: To investigate the presence of methicillin-resistant Staphylococcus aureus (MRSA) in untreated hospital wastewaters (UHWW), their transmission into the receiving sewage treatment plant (STP) and survival through the STP treatment. METHODS AND RESULTS: Over eight consecutive weeks of sampling, we isolated 224 Staph. aureus strains from UHWW-1, UHWW-2 and its receiving STP inlet (SI) and post-treatment outlet (SO). These strains were typed using the PhP typing method and RAPD-PCR and tested for their antibiotic resistance patterns. Resistance to cefoxitin and the presence of mecA gene identified MRSA isolates. In all, 11 common (C) and 156 single (S) PhP-RAPD types were found among isolates, with two multidrug resistant (MDR) C-types found in H2, SI and SO. These C-type strains also showed resistance to cefoxitin and vancomycin. The mean number of antibiotics to which the strains from UHWW were resistant (5.14 ± 2) was significantly higher than the STP isolates (2.9 ± 1.9) (P < 0.0001). Among the 131 (68%) MRSA strains, 24 were also vancomycin resistant. MDR strains (including MRSA) were more prevalent in hospital wastewaters than in the STP. CONCLUSION: This study provides evidence of the survival of MRSA strains in UHWWs and their transit to the STP and then through to the final treated effluent and chlorination stage. SIGNIFICANCE AND IMPACT OF THE STUDY: This preliminary study identifies the need to further investigate the load of MRSA in hospitals' wastewaters and possible their survival in STPs. From a public health point of view, this potential route of hospital MRSA dissemination is of great importance.

Pathogenic Escherichia coli found in sewage treatment plants and environmental waters.

We previously demonstrated that some Escherichia coli strains with uropathogenic properties survived treatment stages of sewage treatment plants (STPs), suggesting that they may be released into the environment. We investigated the presence of such strains in the surrounding environmental waters of four STPs from which these persistent strains were isolated. In all, 264 E. coli isolates were collected from 129 receiving water sites in a 20-km radius surrounding STPs. We also included 93 E. coli strains collected from 18 animal species for comparison. Isolates were typed using a high-resolution biochemical fingerprinting method (the PhPlate system), and grouped into common (C) types. One hundred forty-seven (56%) environmental isolates were identical to strains found in STPs' final effluents. Of these, 140 (95%) carried virulence genes (VGs) associated with intestinal pathogenic E. coli (IPEC) or uropathogenic E. coli (UPEC) and were found in a variety of sites within areas sampled. Of the remaining 117 environmental strains not identical to STP strains, 105 belonged to 18 C types and 102 of them carried VGs found among IPEC or UPEC strains. These strains belonged mainly to phylogenetic groups A (A0 and A1) and B1 and to a lesser extent B2(2), B2(3), D1, and D2. Eight of 18 environmental C types, comprising 50 isolates, were also identical to bird strains. The presence of a high percentage of environmental E. coli in waters near STPs carrying VGs associated with IPEC and UPEC suggests that they may have derived from STP effluents and other nonpoint sources.

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.

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.

Cell surface hydrophobicity and mycolic acid composition of Rhodococcus strains isolated from activated sludge foam.

The bacteria causing foaming in activated sludge plants are considered to be hydrophobic, and their hydrophobicity is assumed to be a crucial factor in their foam-forming ability. This study showed no consistent relationship between cell surface hydrophobicity (CSH), as determined by microbial adherence to hydrocarbons, of three Rhodococcus spp. isolated from activated sludge foam and their ability to produce a stable foam. There also appeared to be no correlation between the mycolic acid composition of these strains, in terms of chain length or degree of unsaturation, and either CSH or foaming ability. Zeolite and bentonite successfully prevented foaming by a Rhodococcus sp. in pure culture, which suggests that cell surface charge may also play a role in foam stabilisation.

Three isolates of novel polyphosphate-accumulating gram-positive cocci, obtained from activated sludge, belong to a new genus, Tetrasphaera gen. nov., and description of two new species, Tetrasphaera japonica sp. nov. and Tetrasphaera australiensis sp. nov.

Two isolates of Gram-positive cocci (Ben 109T and Ben 110) which could accumulate polyphosphate and were microscopically similar in appearance to so-called 'G-bacteria', appearing as tetrads, were isolated from samples of activated sludge biomass by micromanipulation and grown in axenic culture. On the basis of their phenotypic and chemotaxonomic characters and 16S rDNA sequences, these isolates, together with strain T1-X7T isolated and described previously in Japan, belong to a new genus. These isolates are phylogenetically different from Tessaracoccus bendigoensis, Friedmanniella spumicola and Friedmanniella capsulata, Gram-positive cocci isolated previously in this laboratory. They are characterized by type A1 gamma peptidoglycan, with meso-diaminopimelic acid as the diagnostic diamino acid. The main cellular fatty acid of Ben 109T, Ben 110 and T1-X7T is 14-methylpentadecanoic acid (i-C16:0). The major menaquinones of Ben 109T are MK-8(H4), with MK-8(H2) and MK-8 in trace amounts. In Ben 110 MK-8(H4) and MK-6(H4) are the major menaquinones, while T1-X7T has MK-8(H4), MK-7(H4) and MK-6(H4) as its menaquinones. All three contain phosphatidylinositol, phosphatidylglycerol and diphosphatidylglycerol as their polar lipids. These properties, together with 16S rDNA sequence data, suggest that they all belong to a single new genus for which the name Tetrasphaera gen. nov. is proposed. However, the lipid, cellular fatty acid profiles and DNA-DNA similarity data suggest that Ben 109T and Ben 110 are sufficiently different from T1-X7T to represent a different species of the genus Tetrasphaera. Strain T1-X7T represents the type species Tetrasphaera japonica sp. nov. of this new genus, and strains Ben 109T and Ben 110 belong to the other species, Tetrasphaera australiensis sp. nov.

Analysis of the structural diversity of mycolic acids of Rhodococcus and Gordonia [correction of Gordonla] isolates from activated sludge foams by selective ion monitoring gas chromatography-mass spectrometry (SIM GC-MS)

A method using Selective Ion Monitoring (SIM) gas chromatography-mass spectrometry is described for analysis of mycolic acids which reveals a hitherto unrecognised chemical structural diversity among these in some members of the Mycolata. The structural interpretation of mass spectral data of mycolic acids from Rhodococcus spp and Gordonia [corrected] spp using SIM is discussed.

The filamentous morphotype Eikelboom type 1863 is not a single genetic entity.

Five isolates of a filamentous bacterial morphotype with the distinctive diagnostic microscopic features of Eikelboom Type 1863 were obtained from activated sludge sewage treatment plants in Victoria, Australia. On the basis of phenotypic evidence and 16S rDNA sequence data, these isolates proved to be polyphyletic. Two (Ben 06 and Ben 06C) are from the Chryseobacterium subgroup which is in the Cytophaga group, subdivision I of the Flexibacter-Cytophaga-Bacteroides phylum. Two (Ben 56 and Ben 59) belong to the genus Acinetobacter, and one (Ben 58) is a Moraxella sp., closest to Mor. osloensis. The significance of these findings to the reliance on microscopic features for identification of these filamentous bacteria in activated sludge is discussed.

The opportunistic pathogen Nocardia farcinica is a foam-producing bacterium in activated sludge plants.

A Gram-positive unicellular coccal-diphtheroid rod causing foam in an activated sludge plant was successfully isolated by micromanipulation. Phenotypic characterization and 16S rDNA sequencing identified it as Nocardia farcinica. This is the first report that this opportunistic pathogen is a foam-causing bacterium in activated sludge, and the clinical implications of these observations are discussed.