One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

https://aem.asm.org/content/aem/73/16/5111.full.pdf

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

Rainwater harvesting, quality assessment and utilization in Kefalonia Island, Greece

Roofing as a source of nonpoint water pollution.

http://cfile208.uf.daum.net/attach/112D594250121DC7145E52

Quality of roof-harvested rainwater – Comparison of different roofing materials

Understanding the physical processes of pollutant build-up and wash-off on roof surfaces.

Thermal inactivation of water-borne pathogenic and indicator bacteria at sub-boiling temperatures

A research program into the quality of water supply from rainwater tanks over the last decade at the University of Newcastle in Australia has provided insights into water quality processes operating in rainwater tanks. A rainwater treatment train has been identified that includes processes in first flush devices, rainwater tanks and hot water services including the action of biofilms and heat death of bacteria in hot water services. The need for continuing scientific endeavour to improve understanding of this important water source is highlighted.

https://greywateraction.org/wp-content/uploads/2014/11/SIA_Coombes1.pdf

Key Messages from a Decade of Water Quality Research into Roof Collected Rainwater Supplies

Rainwater tanks are fast becoming accepted as a best practice device in urban water cycle management due to their ability to reduce mains water demand and to reduce stormwater runoff. However, concerns over health issues and bacterial contamination of hotwater systems have thus far hindered widespread recommendation of rainwater use in hotwater systems when an alternative mains water supply exists. While extensive research has been undertaken to determine heat tolerance ranges for pathogens under pasteurisation conditions in perishable goods, little data exists for thermal resistance in a freshwater medium. Laboratory experiments were conducted using a variety of water related bacteria to determine Dvalues within a water medium. D-values are defined as the time required to reduce a bacterial population by 90% at a given temperature. The bacteria were exposed to temperatures of E C, 60 C, and 5 C for varying lengths of time. The results showed that water related bacteria rapidly die off in temperatures relevant for domestic hot water systems. The Dvalues at 65 C and 60 C for E. coli were 3secs and 62secs respectively, while at 55 C E. coli displayed a biphasic death curve, with an initial Dvalue of 2lmins followed by 4mins. For Pseudomonas aeruginosa, the Dvalues at 65 C 60 C, and 55 C were 5secs, 49secs, and Smins, for Salmonella typhimurium, <2secs, 4secs, and 77secs, and for Klebsiella pneumoniae, <2secs, <2secs, and 35secs, respectively. The results indicate that after fifteen minutes at 60 C, E. coli concentrations will have been reduced by 15-log reductions, while the other pathogens experienced similar or even greater reductions. This is in agreement with water samples taken from domestic rainwater tanks and hot water systems which indicated almost complete kill off of bacteria through the hot water system. In view of Australian standards requiring temperature at or above 60 C to control L. pneumophila, these preliminary results suggest that it is highly improbable that human pathogens, if present in raintank water, will survive through a hot water system.

Thermal Destruction Analyses of Water Related Pathogens at Domestic Hot Water System Temperatures

Two small rainwater tanks and a 4A rated water efficient washing machine have been installed at a typically small house in Carrington an inner city suburb of Newcastle in New South Wales. Monitoring of the performance of the dual water supply scheme (rainwater and mains water) during a drought has revealed a 43% reduction in mains water demand. Analysis based on the monitoring results predicted that the combination of the rainwater tanks and 4A rated washing machine will reduce average annual mains water demand by 62% (104 kL). The cost of rainwater supply was calculated to be $0.28/kL and the benefits of the combined water management strategy were $3.24/kL of mains water saved. The microbial and chemical quality of rainwater was observed to improve throughout the rainwater treatment train. In addition the design and installation of the dual water supply scheme is presented.

Performance of rainwater tanks at an inner city house in Carrington NSW during a drought

Although the collection of rainwater for potable purposes is an ancient practice and is widespread in many parts of the world today, rainwater harvesting systems have largely been viewed as a black box. Research has neglected to investigate many important facets of rainwater harvesting including the critical area of process understanding with regard to water quality changes and treatment mechanisms. This paper overviews the possible treatment train and highlights gaps in our understanding. Exposure to UV, heat, and desiccation on the roof top destroy many bacteria. Within the tank, it is proposed that biofilms actively remove heavy metals and organics from the water, thus increasing the difficulty for planktonic organisms to survive. While many bacteria conglomerate in a micro-layer on the water surface, many of the heavy metals and other contaminants precipitate out of the water column and settle at the bottom of the tank. The precipitation of suspended solids and bacteria may also be possible during a rain event if re-suspension of the sediment occurs and acts as a coagulation-precipitation agent. Outlet taps on tanks are always located above the sediment layer, and in a trickle top up system the water surface layer is always above the outlet, effectively preventing these contaminants from being present in the available water supply. Tank water must also pass through a pump and possibly through a hotwater system, which impose sudden stresses on bacteria, disrupting cell structure and integrity. Hotwater systems maintained at 60 C in accordance with Australian Standards (AS3500.4.2) should not pose serious health risks as Legionella pneumophila, the main bacterial species of concern, are destroyed at temperatures above 50 C in water. Heat inactivation data are yet to be published for most other pathogenic species in a water medium. Rain harvesting in the urban environment produces valuable yields of water and research into treatment processes needs to be undertaken to ensure that in the future, this resource can be fully utilised.

Water Quality Treatment Processes in Domestic Rainwater Harvesting Systems

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