Pipe Biofilm Research Articles

A risk model for enteric virus accumulation and release from recycled water distribution pipe biofilms

Screening-level quantitative microbial risk assessments (QMRA) undertaken to assess health risks associated with the reticulation of recycled water have identified distribution pipe biofilms and their ability to accumulate enteric and opportunistic pathogens as potential sources of public health concern. A simplistic model to assess human health risks associated with enteric virions (extracellular viruses) present in recycled water for domestic use was therefore developed. Recycled water biofilms formed on glass and stainless steel coupons in a laboratory-scale distribution system were challenged with model enteric virions (B40-8, MS-2 and FX174 bacteriophages). Approximately 1% of bacteriophages present in the adjacent bulk water was incorporated into 3 month-old biofilms and a persistent sub-population (0.01%) of the model enteric virions remained infectious in biofilms throughout an experimental period of 30 days. Significant potential for virions to accumulate within biofilms was therefore demonstrated, and indeed biofilm uptake could mask treatment failures assessed through analysis of the bulk water (infectious virions would not be detected after 1.5 km even if 10.L-1 virions present in treated effluent). Subsequent sloughing of biofilm into the bulk water phase could therefore release infective virions within mobilized aggregates of biofilm and infect susceptible consumers. During normal operating conditions (one virion in 100 L of water) sufficient virions may accumulate within distribution pipe biofilms that a 50% sloughing event could present a Pi (annual) of 1.9 × 10-4 for the consumption of 1 mL of water, exceeding the US-EPA benchmark of 10-4.

A comparison of methods and models for the analysis of water distribution pipe biofilms

The suitability of three experimental devices: biofilm reactors™ (BR), biofilm exosamplers™ (BE), and modified robbins devices (MRD), for the analysis of water distribution pipe biofilms was examined in situ within an urban water distribution system (Rouse Hill Development Area, New South Wales). Stainless steel (ss) and unplasticized polyvinyl chloride (uPVC) coupons were conditioned with biofilm in each device for a period of 70 days. Biofilm removal techniques (sonication and stomaching) were evaluated and optimized for this study. A multiparametric quantification of biofilm biomass using total protein (NanoOrange™ protein determination) and carbohydrate (phenol-sulfuric assay) content, total number of bacterial cells (BacLight™ Live/Dead® Bacterial Viability Kit) and total number of heterotrophic bacteria (R2A plate counts) is proposed. The presence of biofilm-associated faecal indicator organisms (Enterococci, E. coli, somatic, F-RNA and B40-8 bacteriophages) was assayed for each biofilm homogenate. Variability both within and between biofilm devices was observed. Notwithstanding the shortcomings of the inherent heterogeneity observed with biofilm quantification, the relatively inexpensive biofilm devices were shown to yield reliable and comparable information on biofilm growth within an urban water distribution system. Furthermore, the multiparametric measurement of biofilm biomass was shown to provide a reliable and holistic quantification of distribution pipe biofilms.

Persistence of two model enteric viruses (B40-8 and MS-2 bacteriophages) in water distribution pipe biofilms

The persistence of two model enteric virions (Bacteroides fragilis phage B40-8 and coliphage MS-2) within pipe biofilms was investigated in situ in an urban distribution system. Biofilms were allowed to develop on uPVC and stainless steel (SS) coupons in a modified Robbins' device for 70 d within a 150 mm uPVC reticulation main. Coupons were then placed in annular reactors and slug dosed with B40-8 and MS-2 phages (10(8) pfu/mL). Pipe water velocity, pH and free chlorine were recorded during the experimental period. Biofilms on uPVC were generally more abundant (based on total bacterial counts, HPCs, total protein and total carbohydrate). Both B40-8 and MS-2 were incorporated into biofilms formed on uPVC and SS coupons (> 10(4) and > 10(3) pfu/microgram protein respectively) and persisted for > 30 d and 6 d respectively, reflecting biofilm biomass on the two pipe surfaces. Virion loss/inactivation from biofilm followed an initial rapid phase, followed by a very slow phase representing approximately 0.01% of the original virion population. Virions, therefore, have the potential to accumulate within distribution biofilm and problems could arise when clusters of biofilm-associated enteric virions become detached from the substrata by hydrodynamic forces or sudden changes in disinfection regime.

Development of equipment for in situ studies of biofilm in hot water systems

New equipment was developed for in situ studies of biofilms in hot water tanks and hot water pipes under normal operation and pressure. Sampling ports were installed in the wall of a hot water tank and through these operating shafts were inserted with a test plug in the end. The surface of the test plugs was made of the same material as used in the hot water system and the test plugs were flush with the inner surface of the tank. When the operating shaft was removed from the tank, biofilm could be collected. In the distribution system, biofilm samples were collected from test plugs inserted in sampling ports in a by‐pass. Heterotrophic plate counts (HPC) revealed 104‐106 CFU cm‐2 on the test plugs in the hot water system after an exposure period of 7 d. The number of bacteria was not influenced by the location of the plug within each cluster of plugs in the distribution system, or at different horizontal levels in the hot water tank after an exposure period of 7 d.

The relationship between pipe material and biofilm formation in a laboratory model system

The aim of this study was to compare biofilm accumulation and heterotrophic bacterial diversity on three pipe materials-cast iron, medium density polyethylene (MDPE), and unplasticised polyvinyl chloride (uPVC) - using a laboratory model system run over a short period (21 d) and a longer period (7 months). Newly Modified Robbins Devices (nMRD) were run in parallel, each containing 25 discs of one material with cold tap water flowing through the devices at 3 ml min(-1) (Reynolds Number 9.05) for 21 d. The numbers of bacteria on each material increased exponentially between 0 and 11 d when the biofilm viable count remained constant. The mean doubling times of the heterotrophic population on the materials during the exponential phase was 13.2 h for cast iron and 15.6 h for MDPE and uPVC. The same experiment was repeated under different environmental conditions with a lower temperature, higher free chlorine and lower number of organisms ml(-1) of incoming water. The exponential phase lengthened to 16 d but the steady state count remained the same. The mean viable count after 21 d and after 7 months was on average 97% higher on cast iron than on the other materials. Very few different colony types were isolated from each material with the largest number (nine) recovered from cast iron. The numbers of planktonic bacteria in the effluent water leaving each of the nMRDs directly correlated with the numbers in the biofilm phase on each of the materials. In addition the distribution and thickness of the biofilms on the MDPE and uPVC were observed using confocal scanning laser microscopy. In conclusion, MDPE and uPVC support the lowest numbers of bacteria in a steady state biofilm in the short term (21 d) and over a longer term (7 months). The diversity of heterotrophic bacteria was greatest on cast iron.

Biofilms, mains water and stainless steel

There seems to be a general shift in the U.K. and the rest of Europe away from copper to the use of stainless steel piping for carrying particular “problem” waters in buildings where corrosion has been found to cause failures in copper. As stainless steel is a relatively new plumbing system few reports are available of its short term and long term performance in potable water environments and of differences between grades. Results of the development of biofilms on stainless steel grades 304 and 316, as an appendage to a large buildings plumbing distribution system, after 24 months exposure to potable water are presented. The viable cell counts on stainless steel grade 304 pipe after 24 months averaged 1.9×10 3 cfu cm −2 compared to 3.2×10 2 cfu cm −2 on 316 piping. The viable cell and total cell count on matt (2D) finished stainless steel were not significantly higher ( p<0.05) when compared to smooth (2B) stainless steel. Total carbohydrate levels and biomass dry weight levels were slightly higher on 304 stainless steel than 316 stainless steel, but were not significantly ( p<0.05) different. A mixture of biofilm bacteria attached to stainless steel were evident including, Pseudomonas spp., Alcaligenes sp. and Corynebacterium/Arthrobacter spp. Metal ions were evident in both 304 and 316 pipe biofilms. Scanning electron microscopy provided evidence of microcolony formation of rod-shaped bacteria and diatoms. Results of the development of biofilms on stainless steel grades 304 and 316, as an appendage to a large buildings plumbing distribution system, after 5 months exposure to mains water at watervelocities of 0.32, 0.96 and 1.75 m s −1 are also presented.

Direct measurement of the adhesive strength of biofilms in pipes by micromanipulation

A novel micromanipulation technique has been developed to measure directly the adhesive strength of biofilms formed in pipe flows. A T-shaped probe was specially designed to pull Pseudomonas fluorescens biofilms away from the inner surface of a pipe to which they were attached. The adhesive strength between biofilms and the substratum was defined as the work required to remove the biofilms per unit area from the substratum. Results showed that the adhesive strength increased with the fluid velocity in which the biofilms were grown, with a typical value of 0.05∼0.2 J/m2. © Rapid Science Ltd. 1998

BIOFILM DEVELOPMENT ON STAINLESS STEEL IN MAINS WATER

Biofilm development on stainless steel in mains water has been poorly documented to date. Results are presented of the development of potable-water biofilms over 12 months on stainless-steel grades 304 and 316, used as appendages to a large building's plumbing distribution system. The viable cell counts on grade 304 pipe after 12 months averaged 2.8 × 10 3 cfu cm −2, compared to 3.6 × 10 2 cfu cm −2 on grade 316 pipe. The viable cell and total cell count on matt (2D) stainless steel remained significantly higher ( p < 0.05) when compared to smooth (2B) stainless steel after 4-months and 8-months biofilm development, but at month 12 the viable cell counts on the two steel finishes were not significantly different. Total carbohydrate levels and biomass dry weight levels were slightly but not significantly higher on grade 304 than on grade 316. A mixture of biofilm bacteria attached to stainless steel were evident, including Pseudomonas spp., Methylobacterium spp., Acinetobacter spp., Corynebacterium/Arthrobacter spp. and Micrococcus spp. Inductively coupled plasma spectrophotometer analysis of biofilms showed an accumulation of metal ions in both grades 304 and 316 pipe biofilms. Molybdenum (0.04 mg litre −1) was found to be associated with biofilms isolated from grade 316 after 4 months, and 0.05 mg litre −1 was found in the biofilms after 12-months exposure to mains water. Scanning electron microscopy provided evidence of microcolony formation of rod-shaped and coccoid-shaped bacteria and diatoms. © 1998 Elsevier Science Ltd. All rights reserved

Ammonia-oxidizing bacteria in a chloraminated distribution system: seasonal occurrence, distribution and disinfection resistance

Nitrification in chloraminated drinking water can have a number of adverse effects on water quality, including a loss of total chlorine and ammonia-N and an increase in the concentration of heterotrophic plate count bacteria and nitrite. To understand how nitrification develops, a study was conducted to examine the factors that influence the occurrence of ammonia-oxidizing bacteria (AOB) in a chloraminated distribution system. Samples were collected over an 18-month period from a raw-water source, a conventional treatment plant effluent, and two covered, finished-water reservoirs that previously experienced nitrification episodes. Sediment and biofilm samples were collected from the interior wall surfaces of two finished-water pipelines and one of the covered reservoirs. The AOB were enumerated by a most-probable-number technique, and isolates were isolated and identified. The resistance of naturally occurring AOB to chloramines and free chlorine was also examined. The results of the monitoring program indicated that the levels of AOB, identified as members of the genus Nitrosomonas, were seasonally dependent in both source and finished waters, with the highest levels observed in the warm summer months. The concentrations of AOB in the two reservoirs, both of which have floating covers made of synthetic rubber (Hypalon; E.I. du Pont de Nemours & Co., Inc., Wilmington, Del.), had most probable numbers that ranged from less than 0.2 to greater than 300/ml and correlated significantly with temperature and levels of heterotrophic plate count bacteria. No AOB were detected in the chloraminated reservoirs when the water temperature was below 16 to 18 degrees C. The study indicated that nitrifiers occur throughout the chloraminated distribution system. Higher concentrations of AOB were found in the reservoir and pipe sediment materials than in the pipe biofilm samples. The AOB were approximately 13 times more resistant to monochloramine than to free chlorine. After 33 min of exposure to 1.0 mg of monochloramine per liter (pH 8.2, 23 degrees C), 99% of an AOB culture was inactivated. The amounts of this disinfectant that are currently used (1.5 mg/liter at a 3:1 ratio of chlorine to ammonia-N) may be inadequate to control the growth of these organisms in the distribution system.