Biofilms: The Cooperative Research Centre For Water Quality and Treatment

Bioflms
Understanding the
Impact on Water
Quality and Water
Treatment Processes
Management Implications
from the Research Programs
of the Cooperative Research
Centre for Water Quality
and Treatment
The Cooperative Research Centre for
Water Quality and Treatment
Purpose
TheAustralianDrinkingWaterGuidelines2004,incorporatingtheFrameworkfortheManagementofDrinkingWater
Quality,hastakenarisk-basedapproachfromcatchmenttotaptoensuringhighqualitydrinkingwater.Theapproach
requires a thorough defnition and understanding of the risks to water quality, thus allowing the implementation of
appropriate control barriers and procedures. The risk-based approach to drinking water quality management is now
beingadoptedasbestpracticeinternationally,aswellasinotherareassuchasthemanagementofrecycledwaterand
recreationalwaters.
Much of the research into drinking water quality has
focusedoneffectivecatchmentmanagementpractices
and optimising treatment processes to ensure high
quality and safe drinking water. The risks present in
distribution systems, however, are less well defned
butmaycontributeappreciablytooverallwaterquality
risks. Indeed, the optimisation of treatment practices to produce high quality water now places more onus on the
effectivemanagementofdistributionsystemstoensurethatwaterqualitydegradationisminimised.
It is widely acknowledged that bioflms are an important
source of water quality degradation in distribution
systems.
These fact sheets highlight and summarise the research
outcomes of bioflm related projects conducted by the
AustralianCooperativeResearchCentre(CRC)forWater
Quality and Treatment through its Distribution Program.
The fact sheets also summarise bioflm studies undertaken
byAustralianresearchgroupsoutsideoftheCRCforWater
Quality and Treatment. The fact sheets are intended to
highlight outcomes from bioflm research rather than being
a detailed report. Further detail of the research fndings
foundinthefactsheetscanbesourcedthroughtheFurther
Informationsectionsattheendofeachfactsheet.
TABLE OF CONTENTS
Research Background Page 1
Introduction Page 2
BIOFILMS AND WATER QUALITY
FS1
Water Quality/Bioflm Interactions Page 5
FS2
Disinfectant Decay Page 8
FS3
Taste and Odour Page 10
FS4
Incorporation of Potential Pathogens Page 12
FS5
Bioflm Development on Other Surfaces in Drinking Water Systems Page 16
BIOFILM MITIGATION STRATEGIES
FS6
Organic Carbon Removal Page 19
FS7
Disinfection Page 21
FS8
Bioflm Removal Page 22
TOOLS FOR INVESTIGATING BIOFILMS
FS9
Bioflm Monitoring Hardware Page 26
FS10
Bioflm Analytical Methods Page 30
FS11
Determination of Biodegradable Organic Carbon Page 36
Acknowledgements Page 39
Relationship between Bioflm Research and Elements of the Framework for Drinking Water Quality Management Page 40
Page
Research Background
Bioflms and water quality FS 1, FS 2, FS 3, FS 4, FS 5
This set of fact sheets summarises research aimed at understanding the impact of bioflms on water
quality. Research topics include the contribution of bioflms to disinfectant decay, the contribution of
bioflms to the aesthetic quality of water, and the contribution of bioflms to public health risk. In
addition, the main water quality parameters that affect bioflm development are presented.
The scope of work covered by the fact sheets includes both laboratory-scale and feld-scale
investigations conducted through the CRC for Water Quality and Treatment as well as by external
research groups.
Outcomes from these studies will assist water quality managers to assess the contribution of bioflms
to water quality issues in their distribution systems. The identifcation of root causes of water quality
degradation will provide the basis for targeted mitigation strategies.
Bioflm mitigation strategies FS 6, FS 7, FS 8
Given that bioflms are ubiquitous in drinking water distribution systems and are often implicated in
water quality degradation events, the fact sheets present evidence of the effectiveness of the main
mitigation strategies available to operators to control bioflms. These strategies include disinfection,
removal of natural organic matter and physical cleaning. The fact sheets summarise the key fndings
from research conducted within and outside the CRC for Water Quality and Treatment on the
effciency of these mitigation techniques.
These fndings will assist distribution system managers in targeting water treatment and mains
cleaning strategies to reduce bioflm development.
Tools for investigating bioflms FS 9, FS 10, FS 11
The need to assess the effectiveness of system management processes, and to determine whether
bioflms contribute to water quality and public health risk, requires methods to assess bioflm
development in the feld. In addition, more fundamental research is often required to understand
bioflm development and the impact of bioflms on water quality, under controlled laboratory
conditions.
The fact sheets review the various tools that have been used by bioflm related projects conducted
within and outside the CRC for Water Quality and Treatment. The fact sheets are not intended to be
a comprehensive list of all techniques available to study bacterial regrowth but rather those techniques
that Australian researchers have the greatest experience using and hence are readily available
within Australia.
Armed with these tools, system operators will be in a position to assess improvements to water
treatment, the optimisation of disinfection strategies, mains cleaning procedures and address the
requirements of the Framework for the Management of Drinking Water Quality, based on bacterial
regrowth and bioflm formation.
Page 2
Bioflm defnition
Bioflms occur whenever water is in contact with a solid surface, such as a distribution system pipe.
Bioflms represent a build up of microorganisms attached to a surface and embedded in a matrix
of various organic polymers of microbial origin. Bioflms may also contain a high inorganic content
such as sediment, scale and corrosion deposits. This is particularly the case for bioflms containing
iron- and manganese- oxidising bacteria.
Structure of bioflms
Bioflms in distribution systems are not a continuous layer on pipe surfaces; bioflms consist of an
assortment of cells, microcolonies and polymeric substances excreted by the bioflm microorganisms
(bioflm matrix), which are variable in both time and space. Microbial concentrations in bioflms
are normally greater than those found in the overlying water. Bioflm bacteria exist and react to
conditions found in specialised environments at both the macro- and importantly, at the micro-level
within bioflms. Being comprised of microorganisms, bioflms are dynamic and continuously respond
to environmental factors such as nutrient availability, water temperature, water chemistry, hydraulics
and surface conditions. As a result, bioflms can vary over very small distances in thickness, tenacity,
surface distribution, microbial population and chemical composition.
Of relevance to the water industry, the polymeric matrix of bioflms provides protection to colonising
bacteria from chemical disinfection, while the detachment of cells accumulated at the surface
provides a source of microorganisms in the overlying water phase.
Formation of bioflms
A sequence of events leads to the formation of bioflms on a surface.
1. Any immersed surface instantaneously attracts organic and inorganic molecules from
the overlying water thus forming a conditioning flm. The formation of a conditioning flm
is particularly important in nutrient-depleted environments such as drinking water, where
the accumulation of organic molecules at a surface creates a relatively nutrient-rich local
environment.
2. Primary colonising bacteria adhere to pipe surfaces. Due to the presence of a nutrient-rich
conditioning flm, bacteria that fnd their way to a surface are at an advantage compared to
those in the nutrient-depleted aqueous phase. The primary colonising bacteria multiply and
can further condition the surface to favour colonisation by other bacteria or higher organisms
(eg protozoa and fungi) leading to bioflm maturation and population succession.
3. Shear forces exerted by fowing water impact on the mechanical stability of bioflms causing
continuous erosion of surface layers. Indeed hydraulic shear can limit bioflm thickness.
Another important phenomenon is sloughing, the occasional detachment of large portions
of bioflms upon reaching a critical density. Sloughing can be generated by a sudden change
in shear conditions, a change in disinfectant concentrations or by the bacteria themselves.
As a result, bioflm formation is a continual process of attachment, development, and loss
or detachment. Bioflm detachment provides the potential for detached microorganisms to
colonise clean downstream surfaces and hence propagate through a distribution system.
Bioflm detachment is the primary means by which bioflms contribute to an increase in health risk
from drinking water. This has implications for water quality management. Of importance, from a risk
assessment point of view, is the establishment of indicator bacteria, such as faecal coliforms, in
bioflms. Their subsequent detachment can contribute to compliance failures without any implication
of recent faecal contamination. This necessarily reduces the effectiveness of faecal coliforms as
indicators of faecal pollution in distributed water. In addition, the growth of bioflms can lead to an
increase in the concentration of heterotrophic plate count bacteria and the possible contribution of
potentially pathogenic microorganisms to the overlying water. This risk is presently considered to be
low based on current epidemiological evidence.
Introduction
Page 3
Factors affecting bioflm formation
The availability of nutrients is a key driver in the formation of bioflms. Bioflm bacteria predominantly
use biodegradable organic carbon as their main nutrient source. It has been suggested that the ratio
of 100 carbon:10 nitrogen:1 phosphorus is required for bacterial growth. Given that organic carbon is
usually required in greater quantities than other nutrients and is present in comparatively low levels
in drinking water, organic carbon is often considered as the limiting nutrient for bacterial growth in this
environment. Phosphorus has also been shown to limit bacterial growth in drinking waters containing
large quantities of organic carbon or extremely low levels of phosphorus. Reducing biodegradable
organic carbon concentrations in treated drinking water is clearly one way that bioflm development
can be controlled without the need for increased disinfection.
Bacteria sensitive to disinfectants are fully inactivated by disinfection, whereas resistant bacteria,
or those exposed to low concentrations of disinfectants may only sustain cell damage. In these
cases the cells may enter a viable but non-cultivable state. This has particular implications for
coliform indicator bacteria. While still viable, coliforms may be diffcult to resuscitate after exposure
to disinfectants, thus leading to lower indicator cell concentrations and an inappropriate measure
of risk. In addition, the application of disinfectants in a distribution system provides a selective
pressure for those microorganisms that are disinfectant resistant, such as mycobacteria. Importantly,
disinfectants applied to drinking water systems interact with the polymers that make up the bioflm
matrix leading to accelerated disinfectant decay, increased disinfectant demand and a reduced
impact of disinfectants on bioflm-associated microorganisms.
Though diffcult to control, temperature also has a large impact on bacterial activity in a distribution
system. High water temperatures (approximately 15C or above) promote bacterial metabolism
and growth, while accelerating disinfectant decay. Indeed, certain bacteria such as Legionella
and nitrifying bacteria are not active at lower temperatures. During warmer periods, the increased
metabolic activity of microorganisms coupled with a loss of disinfectant residual, due to temperature,
can lead to increased bioflm development.
Drinking water pipes are made of various materials and each impact on bioflm development either
through differences in surface roughness or hydrophobicity. For example, the colonisation of iron
pipes by iron oxidising bacteria can accelerate corrosion whereas the interaction between iron pipes
and disinfectant can promote disinfectant decay, further adding to the potential for regrowth.
Daily fuctuations in customer demand can result in signifcant changes to the hydraulic conditions in
distribution systems. The main impact on bioflms is the generation of shear stress at pipe surfaces.
Shear stress can impact on the tenacity and thickness of bioflms but can also challenge the
mechanical stability of bioflms leading to bioflm erosion and sloughing. These detachment processes
ultimately control bioflm accumulation on pipe surfaces but may also contribute to a degradation of
water quality or potential increase in risk through the release of bound microorganisms. Water fow
conditions also affect the transport of nutrients as well as disinfectants into bioflms. It as been shown
that bacterial regrowth and bioflm development is more likely to occur in low fow areas, such as
experienced in dead ends and service reservoirs, where the residence time of water is extended
resulting in disinfectant decay and low hydraulic shear.
Finally, it is important to note that no one factor impacts on bioflm development. Rather the extent
of bioflm development is dependent on a complex interaction of all of the factors discussed.
Furthermore, as one factor becomes more dominant over another, the bioflm will react to this change.
It is important, therefore, to understand how each factor interacts with the others in order to effciently
control bioflm formation in distribution systems.
Bioflms and risk management
To date much of the focus of water supply risk management has been on catchment management
and water treatment, particularly in the case of recycled water. While it is accepted that these are
key areas of risk reduction, in order to better defne the overall risks to water quality and hence
ensure that risk management strategies are appropriately focused, attention also needs to be paid to
distribution systems and in particular bioflms.
Page 4
Bioflms can also be responsible for a number of water quality issues that can negatively impact on
the quality of the distributed water product. A better understanding of bioflms in distribution systems
and their impact on water quality can assist in determining the need for and targeting of management
strategies to effciently reduce the deterioration of distributed water quality.
Further Information
Safe Piped Water: Managing Microbial Water Quality in Piped Distribution Systems. (2004) Ainsworth
R (ed) WHO/IWA Publishing, Essex, UK.
http://www.who.int/water_sanitation_health/dwq/924156251X/en/
Heterotrophic Plate Counts and Drinking-Water Safety: The Signifcance of HPCs for Water Quality
and the Human Health (2003) Bartram J, Cotruvo J, Exner M, Fricker C and Glasmacher A (eds)
WHO/IWA Publishing, Essex, UK.
http://www.who.int/water_sanitation_health/dwq/hpc/en/
Page 5
FS 1
Research Findings
Laboratory studies
A model of biodegradable organic carbon (BOC) utilisation by bioflms formed in bioflm reactors
fed acetate-C supplemented drinking water predicted a BOC saturation concentration above
which there was little increase in BOC uptake and little additional bioflm development.
For BOC concentrations below the saturation threshold, the model predicted a proportional
reduction in the BOC uptake rate per unit area, which implies reduced bioflm development.
An increase in bioflm development caused an increased release of bioflm cells to the aqueous
phase. Detached bioflm cells recolonised downstream surfaces and appeared to have the ability
to form bioflm biomass (for defnition see FS 10) more rapidly than aqueous phase bacteria.
The use of fuorescence in situ hybridisation (FISH) showed that bioflm bacteria populations
shifted in response to changes in BOC concentrations (through addition of acetate-C) and
disinfectant type.
Bioflm development in response to an increase in organic carbon was more rapid but yielded
less bioflm biomass in chlorinated water compared to chloraminated water for the same total
chlorine concentration.
Field studies
Algal cells were readily observed in bioflms formed in the unfltered Melbourne water supply,
particularly after treatment with chlorine. It is likely that chlorination altered the surface
characteristics of the algae, increasing their ability to attach to surfaces. Bacteria dominated
bioflms in fltered water supplies in Sydney; algal cells were not observed.
Bioflm development in ExoSamplers (see FS 9), measured as total bioflm cell numbers (see FS
10), was determined at four sites in each of two Sydney systems over one and a half years. Sites
were selected to represent the beginning middle and end of the systems. Bioflm parameters were
compared to BOC concentrations (measured as rapidly assimilable organic matter [RAOM]; see
FS 11), temperature and free chlorine residuals. It was assumed that the impact of environmental
parameters on bioflm development was not instantaneous. As a result, mean values for bioflm
and environmental parameters at each site were compared. This provided a comparison of water
quality changes and bioflm along the length of each system.
In both systems, there was a very large correlation between free chlorine and total bioflm cell
numbers (see table below); mean total bioflm cell numbers decreased with increasing mean free
chlorine concentrations.
RAOM
r
2
(p value)
Free Chlorine
r
2
(p value)
Temperature
r
2
(p value)
System 1
Total bioflm cell
numbers
0.71
(p < 0.2)
-0.74
(p < 0.2)
-0.90
(p < 0.1)
System 2
Total bioflm cell
numbers
-0.76
(p < 0.2)
-0.82
(p < 0.1)
0.55
(p < 0.3)
In both systems, there was a very large correlation between mean RAOM concentrations and
mean total bioflm cell numbers. The correlation between total bioflm cell numbers and RAOM
was positive in System 1. This was primarily due to the site at the end of the system where there
was a relatively large mean number of total bioflm cells associated with a very low mean free
chlorine residual, compared to the other sites in the system. It is not clear why mean RAOM
FS 1 Water Quality/Bioflm Interactions
FS 1
Page 6
concentrations were highest at this site. With the last site ignored, there is a negative correlation
between RAOM and total bioflm cell numbers across the other sites in the system ie RAOM
declined across the system with increasing mean total bioflm cell numbers. Temperature was
not considered a main driving force for bioflm development in System 1, even considering the
near perfect correlation (r
2
= -0.90). In this system there was little difference in temperatures
between sites across the system i.e. there were relatively large variations in mean total bioflm
cell numbers for only a small variation in temperature.
In the case of System 2, the correlation between mean RAOM values and mean total bioflm
cell numbers was negative. Similar to System 1, the greatest mean total bioflm cell numbers
was associated with the lowest mean free chlorine concentration. It is likely that the lowering of
mean chlorine residuals coupled with relatively higher temperatures at the sites at the end of the
system resulted in increased biological activity and a decrease in RAOM concentrations across
the system. In contrast to System 1, there were relatively large differences in temperature with
higher temperatures found at sites at the end of the system compared to those closer to the plant;
temperature, therefore, was seen to play a larger role in bioflm development in this system.
None of the above correlations were signifcant at the 95% level. This was primarily due to
the small number of sampling points (n = 4) and highlights the need to include more sampling
points in such studies to provide suffcient statistical robustness. The results indicated that at the
relatively high mean concentrations of RAOM found in the systems (> 150 g acetate-C.L
-1
), that
chlorine and to a lesser extent temperature were key drivers of bioflm development. The feld
studies highlighted the complex interactions between water quality parameters.
Bioflm development in modifed Robbins devices (CRC for Water Quality and Treatment/
University of New South Wales) deployed in the feld, whether measured as heterotrophic plate
counts, total bioflm cells or bioflm biomass, was greater under a low fow regime (0.42 L.s
-1
)
compared to a relatively high fow regime (1.1 L.s
-1
), for comparable water quality.
Implications
Management of BOC concentrations in drinking water through appropriate treatment is a way
that bioflm development can be controlled without increasing disinfectant residuals. By reducing
BOC concentrations, it may be possible to reduce disinfectant residuals in distribution systems.
It is not possible, however, to propose global set point values for BOC concentrations to control
bioflm development. Set point values will be system specifc and dependant on a range of
conditions including temperature and disinfectant concentration.
Field studies to investigate the impact of water quality parameters on bioflm development need
to represent sites from beginning to the end of a system to take into account variations in water
quality as it travels along a system. A suffcient number of sites are required (greater than 4 is
suggested) to ensure statistical robustness. It is essential that water quality parameters include
at least BOC, disinfectant residual and temperature.
Hydraulics impact on bioflm development with greater bioflm development under low fow
regimes.
Further Information
Angles ML, Chandy J, Kastl G, Jegatheesan V, Cox P and Fisher I (1999) Bioflms in drinking water:
infuence of organic carbon and disinfection. Water 26(4):30-33.
Angles ML, Kastl G, Chandy JP, Payyappat S, Sathasivan A, Fisher I and Stevens M (2003) Bioflm
devices to facilitate water quality investigations in distribution systems. Proceedings of the IWA
Leading Edge Conference: Drinking Water and Wastewater Treatment Technologies, Amsterdam,
The Netherlands.
Chandy JC and Angles ML (2004) Factors Infuencing the Development of Bioflms under Controlled
Conditions. CRC for Water Quality and Treatment Research Report No. 20.
FS 1
Page 7
Chandy JC and Angles ML (2005) Physical and Chemical Effects on Distribution System Bioflms and
Incorporated Pathogens. CRC for Water Quality and Treatment Research Report - in preparation.
Chandy J and Angles M (2001) Determination of nutrients limiting bioflm formation and the subsequent
impact on disinfectant decay. Water Research 35(11): 2677-2682.
Kastl G, Fisher I, Sathasivan A (2005) Optimisation of Chlorine Residual in a Distribution System
(Greenvale Sydenham, Melbourne). CRC for Water Quality and Treatment Research Report -
submitted.
Storey MV and Ashbolt N J (2002) A comparison of methods and models for the analysis of water
distribution pipe bioflms. Water Science and Technology: Water Supply 2(4):73-80.
Page 8
FS 2
Research Findings
Laboratory studies
Biodegradable organic carbon fractions, such as acetate, that do not readily react with disinfectants
are converted by bioflm bacteria into disinfectant reactive compounds, and therefore can have an
indirect impact on disinfectant demand. Addition of only 100 g.L
-1
acetate-C, to Sydney drinking
water produced an increase in bioflm biomass (for defnition see FS 10) and a 50% decrease in
average chloramine residuals (mediated by bioflms) for the chloramine concentrations used in
the study (average = 0.7 mg.L
-1
).
At concentrations less than 100 g acetate-C.L
-1
, bioflm development and the impact on
disinfectant decay was minimal, for the chloramine concentrations used in the study (average =
0.7 mg.L
-1
).
Removal of disinfectant by bioflms is a dynamic process dependent on the presence of
biodegradable organic carbon (BOC) ie bioflms need a constant supply of BOC to maintain
bioflm integrity and exert a disinfectant demand. Removal of BOC from a laboratory system
caused a decrease in established bioflm biomass and a loss of the ability of bioflms to impact
on disinfectant decay.
Field studies
A feld study, conducted in a Melbourne bulk water delivery system (pipe size greater than 600
mm diameter) using bioflm ExoSamplers, large diameter pipe coupon samplers (see FS 9) and
the CRC developed chorine decay model, showed that while it is likely that large diameter pipe
surfaces, including bioflms, contributed marginally to chlorine decay this effect was minor in
comparison with decay in the bulk water. This was likely due to the large diameter of pipe used
in the study.
A feld study, conducted in a Sydney reticulation system (pipe size down to 150 mm diameter)
using the bioflm ExoSamplers and the CRC chlorine decay model, identifed regions where pipe
surfaces, including bioflms, contributed substantially (approximately two fold) to chlorine decay
compared to the bulk reaction rate. This was likely due to the smaller surface area/volume ratio
in the smaller pipes used in this study, compared to the Melbourne study.
Implications
An increase in BOC concentration reduces the ability of a given disinfectant residual to control
bioflm regrowth and may promote disinfectant loss.
The potential of water to promote bioflm development and the subsequent impact of bioflms on
disinfectant decay should be considered in disinfection optimisation strategies. It is not possible
to determine the impact of bioflms on disinfectant decay from standard laboratory disinfectant
decay tests, which only assess the bulk water decay rate. The role of wall effects on disinfectant
decay, including those of bioflms, forms part of the CRC Distribution System Management Tools
model and enables system managers to focus either on reducing wall effects (including bioflm
management) or on managing bulk water disinfectant decay rates.
Effort needs to be applied to reduce organic carbon in drinking water through appropriate
treatment. This would have multiple effects including the limitation of bioflm development and
hence reducing the impact on disinfectant decay, reducing components directly responsible for
disinfectant decay and reducing the precursors of disinfectant by-product formation.
Investigation of pipe wall effects, including bioflms and pipe condition, are recommended in
areas where pipe wall effects on chlorine decay outweigh the bulk chlorine decay reaction rate.
FS 2 Disinfectant Decay
FS 2
Page 9
Further Information
Angles ML, Chandy J, Kastl G, Jegatheesan V, Cox P and Fisher I (1999) Bioflms in drinking water:
infuence of organic carbon and disinfection. Water 26(4):30-33.
Chandy J and Angles M (2001) Combined effects of limiting nutrient and disinfectant on drinking
water distribution systems bioflm formation. Water Research 35(11): 2677-2682.
Fisher I, Chen P, Kastl G (2005) Consolidation of Management Tools for Distribution Systems. CRC
for Water Quality and Treatment Research Report - in preparation.
submitted.
Jegatheesan V, Kastl G, Angles M, Chandy J, and I Fisher (2000) Modelling bioflm growth and
disinfectant decay in drinking water. Water Science and Technology 41(4):339-345.
Research Findings
The occurrence of a sporadic swampy odour in a Perth distribution system was attributed to
the production of dimethyl trisulphide (DMTS). It was identifed that precursors to DMTS were
polysulphides.
Sulphide/polysulphide concentrations were low in treated water due to oxidation during treatment;
inorganic polysulphides are extremely unstable in the presence of oxygen. Production of sulphides
therefore occurred in the distribution system.
Polysulphide-rich bioflms and sediments were widespread in the distribution system and occurred
on pipes constructed of different materials including asbestos cement, reinforced concrete,
copper and cement-lined steel. The occurrence of polysulphides was not dependant on the water
type; polysulphides were isolated from pipes fed either groundwater or surface water.
The presence of bioflm/sediment matrices appeared to retard the oxidation of polysulphides by
preventing their diffusion into the overlying oxygenated water or by protecting bioflm associated
polysulphides from the oxidative action of chlorine and dissolved oxygen. The results imply that
the bioflms and sediments contained anoxic zones.
Laboratory scale bioflm reactor studies showed that the presence of DMTS was insignifcant in
the absence of bioflms, compared to DMTS production in the presence of bioflms.
Processes that contributed to increased DMTS production in the bioflm reactors included:
methiolation by methane thio-containing compounds; abiotic methylation of oligosulphides in
bioflms by methylating agents; biological methylation of oligosulphides and H
2
S as part of a
detoxifcation process; stagnant conditions leading to oxygen depletion; and the stimulation of
bioflm formation and activity from increased biodegradable organic carbon concentrations.
Maintenance of a chlorine residual (0.7 mg.L
-1
at the start to 0.1 mg.L
-1
at the end of a system)
controls DMTS production in the distribution system. A disadvantage of increased chlorination,
however, is the possibility of elevated disinfectant by-product formation.
Current studies
A CRC for Water Quality and Treatment doctoral candidate at Griffth University is undertaking a
study investigating the triggers of taste and odour production in drinking water. The study will focus
on the production of geosmin and 2-methylisoborneol (MIB) by cyanobacteria and actinomycetes
in water, sediments and bioflms. Bioflms will be assessed using modifed Robbins devices
developed at Griffth University (see FS 9). The study will be completed by the end of 2006.
Implementation
The control of swampy odour in Perth distribution systems requires that chlorine residuals be
maintained to the end of the distribution system. This has been achieved by reducing dissolved
organic carbon concentrations using MIEX
(Magnetic Ion Exchange resin) and hence reducing

chlorine demand. Furthermore Water Corporation is investigating the use of biological fltration
post MIEX

to produce biologically stable water and further lower the bioflm formation potential
in the distribution system. This will have the effect of further reducing disinfectant demand. For
water utilities in Australia that employ coagulation in their water treatment process, an alternative
cost effective option for reducing DOC concentrations is by enhanced coagulation.
FS 3
Page 10
FS 3 Taste and Odour
FS 3
Page
Further Information
FS 6 Organic carbon removal Field studies
Franzmann PD, Heitz A, Zappia LR, Wajon JE and Xanthis K (2001) The formation of malodorous
dimethyl oligosulphides in treated groundwater: the role of bioflms and potential precursors. Water
Research 35:1730-1738.
Heitz A, Kagi RI and Alexander R (2000) Polysulfde sulfur in pipewall bioflms: its role in the formation
of swampy odour in distribution systems. Water Science and Technology 41(4-5):271-278.
Kastl G, Sathasivan, A, Fisher I and Van Leeuwen J (2004) Modeling DOC removal by enhanced
coagulation. Journal American Water Works Association 92(2):79-89.
Wajon JE, Alexander R, Kagi RI and Kavanagh B (1985) Dimethyltrisulphide and objectionable
odours in potable water. Chemosphere 14:8589.
Wajon JE and Heitz A (1995) The reactions of some sulfur compounds in water supplies in Perth,
Australia. Water Science and Technology 3(11):8792.
Wajon JE and Wilmot PD (1992) Sulfur compounds causing odour in water. Chem. Aust.
59:406408.
MIEX
resin http://www.miexresin.com/miexresin/index.asp
FS 4
Page 12
Research Findings
Cryptosporidium
Cryptosporidium oocysts were readily and rapidly incorporated into drinking water bioflms
formed either in bioflm reactors or a pipe rig constructed from exhumed cement-lined cast iron
drinking water pipes (approximately 70 years old; 100 mm ). In bioflm reactors, incorporation
was observed within 5 minutes post-inoculation under batch conditions and by 15 hours post-
inoculation under fow conditions.
The incorporation of Cryptosporidium oocysts into bioflm reactor bioflms was signifcantly
greater under a relatively high linear fow velocity (0.14 m.s
-1
; turbulent fow) and a diurnal fow
velocity regime (range 0.05 m.s
-1
to 0.16 m.s
-1
; laminar fow to turbulent fow) compared to oocyst
incorporation under a relatively low linear fow velocity (0.05 m.s
-1
; laminar fow). This indicates
that more oocysts were transported to the sampling slide surfaces under the turbulent (high linear
fow velocity) conditions compared to the laminar (low linear fow velocity), leading to greater
oocyst incorporation into bioflms.
There was no signifcant difference between oocyst incorporation in bioflms or oocyst attachment
to clean surfaces, suggesting that in the short-term the presence of bioflms is not a prerequisite
for oocyst attachment.
Incorporated oocysts persisted in bioflms for up to 225 days (32 weeks) in laboratory bioflm
reactors and up to 2 weeks in the exhumed pipe rig; after which time the experiments were
terminated. No assessment of oocyst viability was undertaken. The fact that detectable oocysts
were present after these time periods has implications for system monitoring. Current monitoring
for oocysts is based on oocyst detection without the assessment of oocyst viability.
Cryptosporidium oocysts were detected in the aqueous phase of bioflm reactors for 58 days
and in the aqueous phase of the exhumed pipe rig for 7 days, after a single inoculation event;
after which time the experiments were terminated. Both systems were run under relatively short
detention times. The results indicated that surface associated oocysts periodically detached from
the surfaces. This has implications for system management as oocysts may be detected well
after a contamination event.
Detachment of oocysts from surfaces in the bioflm reactors was primarily driven by a sudden
change from low fow velocity (0.05 m.s
-1
; laminar fow) to high fow velocity (0.14 m.s
-1
; turbulent
fow).
Detachment of oocysts from surfaces in the bioflm reactors was not affected by a sudden increase
in chloramine concentrations from 0 mg.L
-1
to those normally found in distribution systems (1.2
mg.L
-1
). Similarly, an increase in free chlorine concentrations from 0 mg.L
-1
to 2 mg.L
-1
had little
impact on oocyst detachment from surfaces in the pipe rig. In contrast, a shock dose of 20 mg.L
-1

chlorine resulted in a substantial release of oocysts from the pipe rig bioflms.
Detached oocysts were able to recolonise downstream surfaces. The linear fow velocity
signifcantly affected oocyst reattachment in the system; the greatest reattachment was under a
relatively high fow velocity regime (0.14 m.s
-1
; turbulent fow).
The detachment and subsequent reattachment of oocysts may be one way that oocysts persist
(by attaching to surfaces) and propagate (by detachment and reincorporation into downstream
bioflms) within a distribution system. This has implications for system management as oocysts
may be detected well after a contamination event.
Viruses
A single dose of model enteric viruses, bacteriophages B40-8 and MS2, added to bioflm reactors
resulted in approximately 1% of the viruses being incorporated into drinking water bioflms.
Bacteriophages are similar to human enteric viruses in structure, size, composition and morphology,
as well as behaviour in, and resistance to, conventional water treatment processes.
FS 4 Incorporation of Potential Pathogens
- The B40-8 bacteriophage persisted in bioflms for up to 30 days; after which time the
experiment was terminated. The bacteriophage MS2 was only detected in bioflms for 22
days.
- A bi-phasic model described an initial rapid loss followed by a more gradual loss of
bacteriophage from the bioflms. The data supported the presence of a sub-population
of bacteriophages (approximately 0.01%) that have the potential to persist over an
extended period of time, potentially in excess of 100 days for B40-8 bacteriophages and
even longer for MS2. The fact that MS2 was only detected in reactors for 22 days was
probably dependant on the level of sensitivity of sampling and detection.
- Bacteriophages incorporated and persisted in bioflms in the presence of 0.56 0.12
mg.L
-1
total chlorine, indicating that bioflms provide protection to viruses from the effect
of disinfectants.
- Both B40-8 and MS2 were shown to accumulate in bacterial microcolonies within
bioflms.
A continuous low dose of bacteriophage resulted in the surface accumulation of approximately
50% of the B40-8 from the adjacent bulk water but the surface accumulation of only 1% of MS2
from the adjacent water, over the frst 6 days.
- The concentrations of surface associated bacteriophage decreased with time until they
were not detected, despite continuous dosing to the reactors. The loss of bacteriophage
from the reactors was associated with an increase in chlorine concentrations.
- Total chlorine concentrations between 1.0 to 2.0 mg.L
-1
were shown to inactivate both
free and surface associated bacteriophages within the experimental system.
A quantitative microbial risk assessment (QMRA) using the maximum risk dose-response
relationship for a water distribution system was undertaken based on persistence studies of
bacteriophages, X174, MS2 and B40-8 in a laboratory scale modifed Robbins device fed
artifcial reuse water. The probability of infection (Pi) from ingesting water contaminated with
sloughed bioflm containing enteric virus particles was calculated based on the extent of
virus inclusion into bioflms under assumed normal operating conditions (0.01 virus particle.
100 mL
-1
), sub-optimal operating conditions (1 virus particle.L
-1
) and worst case conditions (10
virus particles.L
-1
). Probabilities of infection were calculated for single-hit events of ingestion
of 1 mL, 100 mL and 1L of water and an annual Pi calculated based on daily ingestion of 1 mL
of water. A range of bioflm thicknesses (100 m, 200 m and 300 m) and percent of bioflm
sloughed (10%, 50% and 90%) were used in the prediction of Pi. The USEPA considers the
tolerable annual level of risk to be one infection in 10,000 (ie 10
-4
) in the general population.
Bolded values in the table over page exceed this level of risk.
Bacteria
It was not possible to detect the incorporation of Escherichia coli into bioflms formed in bioflm
reactors by culture-based methods after 24 hours post inoculation. This was in the absence of
chlorine. In the aqueous phase of reactors, E. coli could be detected by molecular techniques
(polymerase chain reaction) but could not be detected by culture methods up to 60 days post
inoculation. The total cell count in the aqueous phase remained at a level commensurate with
the concentration of introduced E. coli indicating that E. coli was still present in the reactor for 60
days, albeit under non-fow conditions. Based on the results it is likely that E. coli had entered a
viable but non-cultivable state.
FS 4
Page 13
FS 4
Page 14
No. of viruses
.L
-
Bioflm
thickness
(m)
% of 1m
of bioflm
sloughed
P
i
(single dose) P
i
(annual)
1 mL 100 mL L 1 mL
0.01
100
10 5.2 x 10
-8
5.2 x 10
-6
5.2 x 10
-5
1.9 x 10
-5
50 2.6 x 10
-7
2.6 x 10
-5
2.6 x 10
-4
9.5 x 10
-5
90 4.7 x 10
-7
4.7 x 10
-5
4.7 x 10
-4
1.7 x 10
-4
200
10 1.0 x 10
-7
1.0 x 10
-5
1.0 x 10
-4
3.8 x 10
-5
50 5.2 x 10
-7
5.2 x 10
-5
5.2 x 10
-4
1.9 x 10
-4
90 9.3 x 10
-7
9.3 x 10
-5
9.3 x 10
-4
3.4 x 10
-4
300
10 1.6 x 10
-7
1.6 x 10
-5
1.6 x 10
-4
5.7 x 10
-5
50 7.8 x 10
-7
7.8 x 10
-5
7.8 x 10
-4
2.8 x 10
-4
90 1.4 x 10
-6
1.4 x 10
-4
1.4 x 10
-3
5.1 x 10
-4
1.0
100
10 5.2 x 10
-6
5.2 x 10
-4
5.2 x 10
-3
1.9 x 10
-3
50 2.6 x 10
-5
2.6 x 10
-3
0.026 9.4 x 10
-3
90 4.7 x 10
-5
4.7 x 10
-3
0.046 0.017
200
10 1.0 x 10
-5
1.0 x 10
-3
0.010 3.8 x 10
-3
50 5.2 x 10
-5
5.2 x 10
-3
0.051 0.019
90 9.3 x 10
-5
9.3 x 10
-3
0.089 0.034
300
10 1.6 x 10
-5
1.6 x 10
-3
0.015 5.7 x 10
-3
50 7.8 x 10
-5
7.8 x 10
-3
0.075 0.028
90 1.4 x 10
-4
0.014 0.13 0.050
10
100
10 5.2 x 10
-5
5.2 x 10
-3
0.051 0.019
50 2.6 x 10
-4
0.026 0.23 0.090
90 4.7 x 10
-4
0.046 0.37 0.16
200
10 1.0 x 10
-4
0.010 0.099 0.037
50 5.2 x 10
-4
0.051 0.40 0.17
90 9.3 x 10
-4
0.089 0.61 0.29
300
10 1.6 x 10
-4
0.015 0.14 0.055
50 7.8 x 10
-4
0.075 0.54 0.25
90 1.4 x 10
-3
0.13 0.75 0.40
(Table reproduced from Storey MV (2002) The Ecology of Enteric Viruses within Distribution Pipe
Bioflms. PhD Thesis. CRC for Water Quality and Treatment/University of New South Wales).
Implications
In order to fully address the Framework for Management of Drinking Water Quality the contribution
of risk from distribution systems should be defned.
The reliance on grab sampling to determine health risks needs to be reconsidered. This is due to
the variability between grab samples arising from the intermittent introduction of microorganisms to
a system and the sporadic release of microorganisms from bioflms. Establishment of acceptable
health risks should be by a process such as qualitative microbial risk assessment, taking into
account the results of effective catchment management and treatment.
Risk assessments of microorganisms in distribution systems, particularly in bioflms, needs to take
into account viability measurements. This is particularly relevant for Cryptosporidium oocysts,
which may persist in bioflms for extended time periods before detachment, enteric viruses of
which little is known of their occurrence or survival in drinking water, and bacteria, either indicator
or opportunistic pathogens, which may enter into a viable but non-cultivable state.
Further Information
Bomo AM, Storey MV and Ashbolt NJ. (2004) Detection, integration and persistence of aeromonads
in water distribution pipe bioflms. Journal of Water and Health, 2(2):83-96.
Conditions. CRC for Water Quality and Treatment Research Report No. 20, Sydney Australia.
Chandy JC and Angles ML (2005) Physical and Chemical Effects on Distribution System Bioflms and
Incorporated Pathogens. CRC for Water Quality and Treatment Research Report - in preparation.
Chandy JC, Warnecke M and Angles ML (2003) Interaction of Cryptosporidium oocysts with drinking
water bioflms. Proceedings IWA International Symposium on Health Related Water Microbiology,
Cape Town, South Africa.
Storey MV (2002) The Ecology of Enteric Viruses within Distribution Pipe Bioflms. PhD Thesis. CRC
for Water Quality and Treatment/University of New South Wales.
Storey MV and Ashbolt NJ (2001) Persistence of two model enteric viruses (B40-8 and MS-2
bacteriophages) in water distribution pipe bioflms. Water Science and Technology 43(12):133-138.
Storey MV and Ashbolt NJ (2003) Enteric virions and microbial bioflms--a secondary source of public
health concern? Water Science and Technology 48(3):97-104.
Storey MV and Ashbolt NJ (2003) A risk model for enteric virus accumulation and release
from recycled water distribution pipe bioflms. Water Science and Technology: Water Supply
3(3):93-100.
Warnecke M (2000) Cryptosporidium oocyst interactions with drinking water pipe bioflms. CRC for
Water Quality and Treatment Research Report No. 7.
FS 4
Page 15
FS 5
Page 16
FS 5 Bioflm Development on Other Surfaces in Drinking Water Systems
Research Findings
Floating membrane covers
Considerable amounts of bioflm were found to form on the underside of membrane covers in a
pilot-scale reservoir in Western Australia.
Warm temperatures (33C) on the underside of the foating membrane and the lack of mixing of
disinfectant-containing water through the whole water profle resulted in limited exposure of the
membrane to disinfectant. These factors were conducive to microbial growth.
Implications covered reservoirs
Submerged mixers should be installed in membrane-covered reservoirs to prevent thermal
stratifcation of the water column and ensure complete dispersion of disinfectant. The focus should
be on adequate disinfectant contact with the membrane surface to control bioflm development
and bacterial regrowth.
A period of shock high dose disinfection (up to 25 mg L
-1
chlorine for 7 days), ensuring adequate
contact with the underside of membrane covers, should be used to destroy bioflms that may
develop on the underside of membranes.
General considerations covered reservoirs
Sump pumps should be provided to remove rainwater from the surface of covers as this represents
a contamination source if the membrane is compromised.
Covers should be inspected weekly for perforations and bond failure.
Covers should be cleaned annually.
Disinfection levels in reservoirs should not fall below 0.1 mg L
-1
chlorine.
Monitoring of temperature, circulation, disinfectant levels, and standard microbiological quality
(including Pseudomonas aeruginosa) should be undertaken in covered reservoirs.
Re-disinfection may be required at the outlet of a membrane-covered reservoir.
For further information contact:
David Masters, Water Corporation
Email: david.masters@watercorporation.com.au
or
Peter Franzmann or Luke Zappia, CSIRO Land and Water
Email: peter.franzmann@csiro.au or luke.zappia@csiro.au
Biologically activated substrata and water quality improvements
Biologically mediated NOM removal
- Granular activated carbon (GAC) was installed in a Western Australian
treatment train to remove natural organic matter (NOM) and hence reduce
trihalomethane (THM) concentrations in the distributed water. The GAC flter
characteristics were: 4 m
3
total volume, comprising 1.26 m
3
of quartz gravel
(40 cm depth) overlaid with 0.6 mm sand (20 cm) and 1.6 m
3
GAC (50 cm depth).
The flter had an empty bed contact time of 8 minutes and was run at a hydraulic
velocity 3.2 m.h
-1
.
- After an initial three month flter maturing period, the effectiveness of dissolved organic
carbon (DOC) removal remained constant at about 20% for a further twelve months.
The relatively constant removal of low levels of DOC by the flter suggested that it was
operating in biological mode (ie when DOC removal is due to biodegradation rather than
adsorption to the GAC).
- The use of GAC for the removal of THM precursors resulted in a reduction of chlorine
decay by 15%.
- Across the flter, biodegradable organic carbon (BDOC) decreased by 15% and
assimilable organic carbon (AOC) showed no change, but AOC was already in low
concentration. After chlorination of the water from the GAC flter, the BDOC and AOC
concentrations increased by 29 and 750%, respectively.
- The average concentration of THMs in the distribution system decreased by 47% following
the installation of the GAC flter in combination with aeration and reduced retention times
in the distribution system.
or
Removal of cyanobacterial metabolites
- There was no indication that the removal of 2-methyl isoborneol (MIB) and geosmin by
spent GAC, or biologically activated carbon, was by biological processes. Due to the
low molecular weight and low concentration of MIB and geosmin, compared to NOM,
and the large surface area of GAC flters, it was likely that removal was primarily by
adsorption to GAC micropores not occupied by pre-adsorbed natural organic matter.
- There was evidence that the removal of MIB and geosmin by biologically activate sand
was by biological processes. Biological processes on biologically activate sand exposed
to MIB and geosmin for 78 days, under fow conditions, removed approximately 65-70%
of the metabolites. Degradation of MIB and geosmin occurred after a lag of 9 days.
- The removal of microcystin-LR and microcystin-LA was by biological processes on spent
or biologically activate GAC. Sterilisation of spent GAC decreased its ability to remove
these toxins. Microcystin LR and LA have a high molecular weight and hence compete
with NOM fractions for the same micropores in GAC. Degradation of microcystin-LR and
LA occurred after a lag of 16 days under fow conditions, and neither toxin was detected
in the flter effuent water.
- The removal of microcystin-LR and microcystin-LA was by biological processes on
biologically activated flter sand. Degradation of microcystin-LR and LA occurred after
a lag of 3 days under fow conditions, and neither toxin was detected in the flter effuent
water.
- A gene responsible for the degradation of microcystin, mlrA, was detected by polymerase
chain reaction in bioflms associated with the sand.
- The removal of the cyanobacterial toxin, cylindrospermopsin, was by biological processes
on biologically activated sand. Complete degradation of the toxin was achieved after 70
days under batch conditions. Degradation occurred after a lag of 8 days.
Page 17
FS 5
Page 18
FS 5
Implications biologically activated substrata
Assessment of the effective life of GAC should take into account the impact of bioflms colonising
GAC particles on the removal of organic carbon and cyanobacterial toxins.
Biologically activated sand is capable of removing cyanobacterial toxins and metabolites.
Microcystin was by far the easiest to degrade on biologically activated sand, compared to the
taste & odour compounds, geosmin and MIB.
Filter media can be screened for the mlrA gene to determine its capacity to remove microcystin.
Effective removal of cyanobacterial metabolites by either GAC or biologically activated sand
included a lag time of days before microorganisms degraded the compounds.
Removal of MIB and geosmin was increased by extending the time of exposure of the metabolites
to biologically activated flter sand. Hydraulic loading of the compounds affected removal
effciency.
It is possible that accumulation of pesticides on GAC may occur over time, with subsequent
release into the distributed water. It is recommended that pesticide levels in GAC flters be
monitored. Furthermore, the potential for biodegradation of target pesticides by the microbial
population on GAC flters should be investigated.
More Information
Newcombe G, Ho L, Hoefel D, Saint C, Slyman N and Oemcke D (2003) The Role of Biological
Filtration in the Removal of Algal Metabolites Using GAC. Proceedings of the Australian Water
Association Federal Convention, Ozwater. Perth, Australia.
Ho L, Wijesundara S, Shaw R, ODonohue M, Saint C and Newcombe G (2005) Biological fltration
processes for the removal of algal metabolites. Water 32(4):40-43.
Ho L, Meyn T, Keegan A, Brookes J, Saint C and Newcombe G (2005) Biological treatment of
microcystin toxins. Proceedings of the Australian Water Association Ozwater Convention and
Exhibition. Brisbane, Australia.
Gayle Newcombe or Lionel Ho, Australian Water Quality Centre/CRC for Water Quality and Treatment
(Algal metabolite removal)
Email: gayle.newcombe@sawater.com.au or lionel.ho@sawater.com.au
Peter Franzmann or Luke Zappia, CSIRO Land and Water (Pesticide accumulation)
FS 6 Organic carbon removal
Research Findings
Laboratory studies
Total organic carbon (TOC) removal by granular activated carbon (GAC) fltration in one Sydney
system produced highly stable water (TOC = 0.1 to 1.2 mg.L
-1
). No bioflm development was
detected in bioflm reactors after 60 days, for the free chlorine residual present in the system
(approximately 1 mg.L
-1
). The results indicated that TOC removal increased the availability of
chlorine to disinfect microorganisms, rather than removing organic carbon from developing
bioflms, leading to their starvation. This was based on the observation that no bioflm development
was detected in the chlorinated GAC treated water supplemented with biodegradable organic
nutrients.
A cessation of acetate-C addition to bioflm reactors caused a decrease in established bioflm
biomass, a loss of the ability of bioflms to impact on disinfectant decay and a reduction in the
number of bioflm cells released to the aqueous phase.
Bioflm development and the impact on disinfectant decay was minimal at rapidly assimilable
organic matter (RAOM) concentrations less than 100 g acetate-C equivalents.L
-1
, given the
levels of chloramine (0.6 mg.L
-1
to 0.8 mg.L
-1
), inorganic nutrients and hydraulic regime in the
bioflm reactor systems.
A range of laboratory studies has shown that removal of dissolved organic carbon by coagulation
with alum or a combination of alum/MIEX

generally results in an increase in bacterial regrowth
potential (BRP; for method see FS 11) in chlorine free water.
Limited work comparing alum coagulation and treatment with MIEX

showed that MIEX

treatment gave the greatest reduction in BRP following chlorination. This improvement appears
to be mitigated by low doses of alum.
Enhanced coagulation (at low pH) is an alternative option to remove DOC for Australian water
treatment plants that already use coagulation. Jar test studies showed that enhanced coagulation
with ferric chloride at pH 5 can remove greater than 50% DOC while enhanced coagulation with
alum at pH 5 can remove approximately 40% DOC.
Field studies
The use of MIEX

to treat water in Western and South Australia reduced DOC concentrations by
50%. Further treatment with low alum doses in SA Water reduced DOC by a further 10-20%.
Comparison of bioflm development in a Western Australian distribution system before and after
the implementation of MIEX

treatment showed that MIEX
reduced the potential for bioflm

formation by approximately 70%.
In a treatment plant in South Australia, biodegradable dissolved organic carbon (BDOC) was
signifcantly reduced after MIEX

treatment and further with alum coagulation. BRP was found to
increase following treatment with MIEX
but reduced to less than the raw water BRP following

alum treatment.
Implications
Future water quality optimisation strategies should include the removal of organic carbon,
particularly biodegradable fractions. This will limit bioflm production through limiting a primary
nutrient. Organic carbon removal will also render disinfectants more stable and biologically active
through the removal of competing organic carbon fractions. This will translate into a possible
reduction in disinfectant concentrations required to maintain water quality.
A model, mEnCo, has been developed by the CRC for Water Quality and Treatment that enables
the rapid determination of coagulant dose (either alum or ferric chloride) and pH control reagents
Page 19
FS 6
Page 20
FS 6
for enhanced coagulation. The model also allows prediction of the residual DOC concentration
in water over a wide range of coagulant doses and coagulation pH conditions from limited
jar test data.
Further Information
Chandy, JC and Angles, ML (2004) Factors Infuencing the Development of Bioflms under Controlled
Drikas M, Morran JY, Cook D and Bursill DB (2003) Operating the MIEX

Process with Microfltration
or Coagulation. Proceedings of the American Water Works Association Water Quality Technology
Conference, Philadelphia.
Drikas M, Chow C and Cook D (2003) The Impact of Recalcitrant Organic Character on Disinfection
Stability, Trihalomethane Formation and Bacterial Regrowth: An Evaluation of Magnetic Ion Exchange
Resin (MIEX
) and Alum Coagulation. Aqua 52(7):475-487.

Kastl G, Sathasivan, A, Fisher I and van Leeuwen J (2004) Modeling DOC removal by enhanced
coagulation. Journal American Water Works Association 92(2):79-89.
van Leeuwen J, Holmes M, Heidenreich C, Daly R, Fisher I, Kastl G, Sathasivan A and Bursill D
(2003) Modeling the application of inorganic coagulants and pH control reagents for removal of
organic matter from drinking waters. Proceedings of Modsim, Townsville.
For further information on the South Australian studies contact:
Mary Drikas or Jim Morran, Australian Water Quality Centre
Email: mary.drikas@sawater.com.au or jim.morran@sawater.com.au
For further information on the Western Australian studies contact:
or
FS 7 Disinfection
Research Findings
Laboratory studies
The concentration of biodegradable organic carbon (BOC) can determine the effectiveness of
disinfectants to control bioflm development. Neither chlorine nor chloramine, at the concentrations
used (mean = 0.5 mg.L
-1
and 0.6 mg.L
-1
, respectively), were able to prevent an increase in
bioflm development when the concentration of biodegradable organic carbon was increased by
400 g.L
-1
in bioflm reactors.
Concentrations of free chlorine (0.5 mg.L
-1
) and chloramine (0.6 mg.L
-1
) were suffcient to prevent
an increase in bioflm accumulation in a Sydney system for at least 90 days. The results indicated
that the optimum free chlorine concentration to control bioflm development was below 0.5 mg.L
-1

for the nutrient levels, temperature and hydraulic regime used in the study. Conversely a decrease
in chloramine concentrations to 0.41 mg.L
-1
in the same system produced an increase in bioflm
development and subsequent increase in chloramine decay.
There was a difference in the response of bioflms to an increase in biodegradable organic
carbon in bioflm reactors, under different disinfectant regimes. Bioflms developed more rapidly
in the chlorinated water but to a lesser extent than in a chloraminated system, under the nutrient
concentrations and hydraulic regime used in the study.
Field studies
Bioflm development in a Western Australian distribution system was greatly restricted at chlorine
concentrations above 0.3 mg.L
-1
.
Minimal bioflm development (greater than 14 pg ATP.cm
-2
; for method see FS 10) was detected
in a chlorinated Western Australian distribution system after 100 days (mean total chlorine
concentration 0.51 mg.L
-1
). Conversely considerable bioflm development (mean bioflm
2, 070 pg ATP.cm
-2
) was detected in dechlorinated water at the same site after 100 days (mean
total chlorine concentration 0.06 mg.L
-1
).
Implication
Disinfection management should consider the concentration of BOC and the subsequent impact
on bioflm development in the optimisation process.
Further Information
For further information on the Western Australian studies contact:
or
Page 21
FS 7
Page 22
FS 8
Research Findings
Swabbing was shown to be more effective at removing bioflm biomass (for defnition
see FS 10).
- Sequential fushing, air scouring and swabbing of a distribution main (300 mm )
showed that even after fushing (fow velocity 1 m.s
-1
to 1.5 m.s
-1
) and air scouring,
swabbing removed approximately four times the bioflm biomass (for method see FS
10) compared to the other techniques (Figure 1).
0
50
100
150
200
250
R
e
s
e
r
v
o
i
r
0

m
i
n

f
l
u
s
h
5

m
i
n

f
l
u
s
h
1
0

m
i
n

f
l
u
s
h
A
i
r

s
c
o
u
r
e
d
S
w
a
b
B
i
o
f
i
l
m

b
i
o
m
a
s
s

(
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)
Figure 1. Bioflm biomass in discharge water following fushing, air scouring and swabbing performed
in sequence in a Sydney distribution main.
- In another study, only a small amount of bioflm biomass (0.05 g.mL
-1
discharge
water) was removed by air scouring immediately following an initial round of main
swabbing (Figure 2a). This indicated that swabbing removed a large proportion
of the surface bioflm with the remainder virtually unaffected by the air scouring
technique.
- In another section of pipe, initial air scouring removed a detectable amount of
bioflm (Figure 2b). By the fourth air scouring run, very little bioflm biomass was
removed (0.05 g.mL
-1
discharge water;

Figure 2b). Nevertheless, subsequent
swabbing removed an additional and greater amount of bioflm overall (Figure 2b).
This supported the observation that mains swabbing was more effective at removing
bioflm than air scouring.
Flushing is the cheapest option for mains cleaning but not the most effective.
The table presented below shows the cost of various bioflm removal techniques used by Sydney
Water in 2003.
Flushing $0.30 - $1.00/m
Air scouring $4.00 - $6.00/m (min. $0.70/m)
Mains swabbing (<200 mm) $6.00 - $8.00/m
Mains swabbing (>200 mm) $8.00 - $30.00/m
FS 8 Bioflm Removal
B
i
o
f
l
m

b
i
o
m
a
s
s
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)

0
5
10
15
20
25
30
35
40
1
s
t

s
w
a
b
2
n
d

s
w
a
b
3
r
d

s
w
a
b
4
t
h

s
w
a
b
5
t
h

s
w
a
b
1
s
t

a
i
r

a
f
t
e
r

s
w
a
b
2
n
d

a
i
r

a
f
t
e
r

s
w
a
b
1
s
t

s
c
o
u
r
2
n
d

s
c
o
u
r
3
r
d

s
c
o
u
r
4
t
h

s
c
o
u
r
2
n
d

s
w
a
b

a
f
t
e
r

a
i
r
3
r
d

s
w
a
b

a
f
t
e
r

a
i
r
4
t
h

s
w
a
b

a
f
t
e
r

a
i
r
B
i
o
f
i
l
m

b
i
o
m
a
s
s

(
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)
Figure 2. Bioflm biomass in discharge waters after (a) swabbing followed by air scouring and (b) air
scouring followed by swabbing.
It should be noted that Sydney Water uses fushing primarily to deliver water containing an elevated
disinfectant residual into areas where water quality degradation is apparent, rather than to clean pipe
surfaces.
There was a strong and signifcant correlation (r = 0.9, p < 0.01) between bioflm biomass
concentration and turbidity in mains swabbing discharge waters in two Sydney distribution
systems (Figure 3a and b). Only on one occasion was elevated bioflm biomass detected with
only a small increase in turbidity (Figure 3b).
There was no evidence of adverse water quality impacts following swabbing. A survey of distributed
water from swabbing sites and the surrounding system showed no increase in turbidity or bioflm
biomass (for method see FS 10) in the bulk water post swabbing (Figure 3a and b). There was no
detectable increase in HPC bacteria post swabbing compared to before swabbing. Heterotrophic
plate count bacteria concentrations were typically greater in swabbing discharge waters, compared
to distributed water, confrming bioflm removal.
Hydroblasting (water pressure 2,500 psi) of a 750 mm main in a Sydney distribution system was
suffcient to remove intact bioflm from the pipe surface without observable adverse effects on
the cement lining. A comparison of bioflm biomass (for method see FS 10) scraped from the
pipe wall before and after hydroblasting showed that approximately 3% of bioflm remained after
the cleaning procedure. A previous trial of hydroblasting (water pressure 12,500 psi) resulted
in observable damage to the cement lining of the pipe. The hydroblasting procedure used
approximately 270 L.min
-1
at a cleaning head velocity of approximately 3-5 m.min
-1
. The cost is
approximately $150.m
-1
depending on pipe size.
Page 23
FS 8
B
i
o
f
l
m

b
i
o
m
a
s
s
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)

a)
b)
Page 24
FS 8
0
1000
2000
3000
4000
5000
6000
7000
8000
B
e
f
o
r
e

S
i
t
e

H
B
e
f
o
r
e

S
i
t
e

I
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

H
A
f
t
e
r

S
i
t
e

I
B
e
f
o
r
e

S
i
t
e

J
B
e
f
o
r
e

S
i
t
e

K
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
S
w
a
b

#
4
A
f
t
e
r

S
i
t
e

J
A
f
t
e
r

S
i
t
e

K
B
e
f
o
r
e

S
i
t
e

L
S
w
a
b

#
1
A
f
t
e
r

S
i
t
e

L
0
20
40
60
80
100
120
140
0
1000
2000
3000
4000
5000
6000
7000
8000
B
e
f
o
r
e

S
i
t
e

A
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
S
w
a
b

#
4
A
f
t
e
r

S
i
t
e

A
B
e
f
o
r
e

S
i
t
e

B
B
e
f
o
r
e

S
i
t
e

C
C
o
m
p
o
s
i
t
e
A
f
t
e
r

S
i
t
e

B
A
f
t
e
r

S
i
t
e

C
B
e
f
o
r
e

S
i
t
e

D
B
e
f
o
r
e

S
i
t
e

E
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

D
A
f
t
e
r

S
i
t
e

E
B
e
f
o
r
e

S
i
t
e

F
B
e
f
o
r
e

S
i
t
e

G
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

F
A
f
t
e
r

S
i
t
e

G
0
20
40
60
80
100
120
140
Turbidity Biofilm
a) b)
T
u
r
b
i
d
i
t
y

(
N
T
U
)
B
i
o
f
l
m

B
i
o
m
a
s
s

(
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)
T
u
r
b
i
d
i
t
y

(
N
T
U
)
B
i
o
f
l
m

B
i
o
m
a
s
s

(
g
/
m
L

d
i
s
c
h
a
r
g
e

w
a
t
e
r
)
0
1000
2000
3000
4000
5000
6000
7000
8000
B
e
f
o
r
e

S
i
t
e

H
B
e
f
o
r
e

S
i
t
e

I
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

H
A
f
t
e
r

S
i
t
e

I
B
e
f
o
r
e

S
i
t
e

J
B
e
f
o
r
e

S
i
t
e

K
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
S
w
a
b

#
4
A
f
t
e
r

S
i
t
e

J
A
f
t
e
r

S
i
t
e

K
B
e
f
o
r
e

S
i
t
e

L
S
w
a
b

#
1
A
f
t
e
r

S
i
t
e

L
0
20
40
60
80
100
120
140
0
1000
2000
3000
4000
5000
6000
7000
8000
B
e
f
o
r
e

S
i
t
e

A
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
S
w
a
b

#
4
A
f
t
e
r

S
i
t
e

A
B
e
f
o
r
e

S
i
t
e

B
B
e
f
o
r
e

S
i
t
e

C
C
o
m
p
o
s
i
t
e
A
f
t
e
r

S
i
t
e

B
A
f
t
e
r

S
i
t
e

C
B
e
f
o
r
e

S
i
t
e

D
B
e
f
o
r
e

S
i
t
e

E
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

D
A
f
t
e
r

S
i
t
e

E
B
e
f
o
r
e

S
i
t
e

F
B
e
f
o
r
e

S
i
t
e

G
S
w
a
b

#
1
S
w
a
b

#
2
S
w
a
b

#
3
A
f
t
e
r

S
i
t
e

F
A
f
t
e
r

S
i
t
e

G
0
20
40
60
80
100
120
140
Turbidity Biofilm
a) b)
Figure 3. Bioflm biomass and turbidity in distributed water before and after mains swabbing, and
in mains swabbing discharge waters at various sites, in two Sydney distribution systems (a and b).
Before and after water samples were collected from customer taps supplied water by the swabbed
pipe section. Swab water samples were collected from swab discharge waters. The swab sample
number denotes the number of swabs used on the pipe ie Swab number 1 refers to the sample taken
from swab discharge waters during the frst swab, swab number 2 refers to swab discharge waters
during the second swab etc. The composite swab sample refers to a sample of swab discharge
water taken as a composite of all swabs for this pipe section.
Bioflm
Bioflm
Implication
Bioflm biomass or turbidity measurements in discharge waters provide quantitation of mains
cleaning procedures. This allows a comparison of cleaning procedures as well as providing a
quantitative record that can be used to establish a frequency of cleaning or to correlate pipe
surface condition with water quality degradation.
Given that bioflm development is dependant on a number of factors including system hydraulics,
temperature, biodegradable organic carbon concentration, disinfectant concentration and pipe
material, it is diffcult to establish a frequency for mains cleaning in general. In lieu of having a
thorough understanding of bioflm development in all parts of a distribution system, the current
practice of conducting mains cleaning based on water quality degradation, such as loss of
disinfectant residual, increased concentrations of bacteria and customer complaints, appears
to be the most effcient. The use of wall reactivity estimates (equivalent diameter) derived from
modelling may be used to show when disinfectant residual loss due to interaction with a pipe wall
is excessive.
Further Information
Clive Copelin, Sydney Water
Email: clive.copelin@sydneywater.com.au
submitted.
Page 25
FS 8
Page 26
FS 9
Bioflm formation monitors
Bioflm reactor (Sydney Water/CRC for Water Quality and Treatment)
Used in Sydney and Adelaide, Australia and Sweden
Related Factsheets FS 1, FS 3, FS 4, FS 7
The bioflm reactor is a portable device suitable for laboratory or distribution
system studies to determine the ability of distributed water to promote
bioflm development, to assess water treatment strategies to minimise
bioflm development and to facilitate bioflm characterisation. Ideal for
conducting experiments under controlled conditions.
Inert materials within the reactor have little impact on water quality
parameters. The reactors are suitable for investigating subtle changes
in drinking water quality such as chlorine decay due to bioflms and the
impact of biodegradable organic carbon (BOC) on bioflm formation. The
reactors can also be used to optimise chlorine residuals based on limitation
of bioflm development.
Independent control of shear stress/linear fow velocity and residence time (determined by rate of
infow). This allows fow velocity in a pipe of given length to be simulated. A diurnal profle of fow
in a distribution system can also be simulated.
Contains 20 sampling slides (cut into three sections giving 60 sampling surfaces) with an
effective total sampling area of 360 cm
2
. Slides are made of stainless steel or uPVC, though it
should be noted that examination of uPVC slides by epifuorescence microscopy is limited by
autofuorescence. Sampling slides enable direct microscopic observation of bioflms on the slide
surface.
Bioflm reactors can only be run under atmospheric pressure.
Mark Angles, Sydney Water.
Email: mark.angles@sydneywater.com.au
Bioflm monitor (KIWA, The Netherlands)
Used in Western Australia
Related Fact sheets FS 6, FS 7
The KIWA bioflm monitor is a portable device suitable for laboratory or
distribution system studies to determine the ability of distributed water
to promote bioflm development, to assess water treatment strategies
to minimise bioflm development and to characterise bioflms.
KIWA bioflm monitors are constructed of glass and have minimal impact
on water quality parameters. The reactors are suitable to investigate
subtle changes in drinking water quality such as the impact of BOC on
bioflm formation and chlorine decay due to bioflm.
Water residence time and shear stress/linear fow velocity are adjustable
but cannot be controlled independently.
Monitors contain 40 glass rings that ft snugly into a glass tube. Effective total sampling area of
696 cm
2
. Water fowing through the tube passes over the inner surfaces of the rings, simulating
water fowing over the inner surface of a pipe. The glass rings prevent direct observation of the
bioflm on the ring surface. Bioflm development is assessed by removing bioflm from the ring
surface (for methods see FS 10).
KIWA bioflm monitors can be run up to 400 kPa pressure without pressure reduction.
FS 9 Bioflm Monitoring Hardware
Peter Franzmann or Luke Zappia, CSIRO Land and Water.
Bioflm ExoSampler (Sydney Water)
Used in Sydney and Melbourne, Australia
Related fact sheets FS 1, FS 4, FS 7
A portable device suitable for laboratory or distribution
system studies to determine the ability of distributed
water to promote bioflm development and water
treatment strategies to minimise bioflm development.
The units can also be used to characterise bioflms.
Inert materials of the ExoSampler (same materials as used for the bioflm reactor) have little
impact on water quality parameters.
Water residence time and shear stress/linear fow velocity are adjustable but cannot be controlled
independently.
Contains 24 sampling slides positioned on parallel plates. Effective total sampling area of 144 cm
2
.
Slides can be made of stainless steel or uPVC. Sampling slides are compatible/interchangeable
with the bioflm reactors and modifed Robbins device (UNSW/CRC for Water Quality and
Treatment). Sampling slides allow direct microscopic observation of bioflms on the slide surface,
though it should be noted that examination of uPVC slides by epifuorescence microscopy is
limited by autofuorescence.
Bioflm ExoSamplers can be run up to 1,200 kPa pressure.
Mark Angles or George Kastl, Sydney Water.
Email: mark.angles@sydneywater.com.au or george.kastl@sydneywater.com.au
Modifed Robbins Device (Griffth University)
Used in Gold Coast, Australia
Related fact sheets FS 2
The modifed Robbins Device (MRD) developed by Griffth University, Queensland is a
portable device that can be used to determine the ability of distributed water to promote bioflm
development, to assess water treatment strategies to minimise bioflm development and to
characterise bioflms.
The MRD (Griffth) is constructed of uPVC and has little
impact on water quality parameters.
Water residence time and shear stress/linear fow velocity
are adjustable but cannot be controlled independently.
The MRD (Griffth) contains 24 sampling coupons. The
coupons are made of uPVC, ft fush to the wall of the
device and hence minimise the disruption to hydraulic
conditions.
The MRD (Griffth) can be run at system pressure.
Helen Stratton, Griffth University.
Email: h.stratton@griffth.edu.au
Page 27
FS 9
Page 28
FS 9
In situ bioflm monitors
Pipe Coupon Samplers
Large diameter (Sydney Water/Melbourne Water/CRC for Water Quality and Treatment)
Used in Melbourne, Australia
Related fact sheets FS 1 and FS 2
Large diameter pipe coupon samplers are fxed devices suitable for distribution system studies of
bioflm formation to assess the impact of bioflms on water quality, determine the effcacy of water
treatment strategies to minimise bioflm
development, to assess the effectiveness of
distribution system management practices
and to characterise bioflms.
The large diameter pipe coupon sampler
is suitable for insertion of test surfaces into
large trunk mains (greater than 400 mm). A
separate sample space is required adjacent
to the pipe.
The pipe coupon sampler (large diameter)
is constructed of high-grade stainless
steel and hence has little impact on water
quality.
The design of the coupon sampler allows
the accurate placement of the sampling surfaces fush to the pipe wall such that water quality as
well as hydraulic effects can be examined.
The test surfaces comprise 3 sampling slides (approximately 28 cm
2
total sampling area), which
can be made of either stainless steel or uPVC. The sampling slides allow direct microscopic
observation of bioflm on the surface.
The unit can be installed and sampled under pressure and hence, without interruption to water
supply.
George Kastl, Sydney Water.
Email: george.kastl@sydneywater.com.au
Small diameter (Sydney Water)
Used in Sydney, Australia
The small diameter pipe coupon sampler is a fxed device suitable for distribution system
studies of bioflm formation including the impact of bioflms on water quality, the effectiveness
of water treatment strategies to minimise bioflm development, assessment of the effectiveness
of distribution system management practices and to
characterise distribution system bioflms.
The coupon sampler (small diameter) is suitable
for insertion of test surfaces into small distribution
mains (100 300 mm).
The test surfaces comprise either stainless steel
or cement coupons (3.8 cm
2
total sampling area).
Cement coupons are curved to maintain the shape
of the inner surface of a pipe and hence maintain
hydraulic conditions.
Small diameter pipe coupon samplers are ftted to
a new section of pipe, to ensure the coupons are
fush to the pipe wall, and the pipe section installed into the system. Sampling of the coupons can
be performed under pressure and hence without disruption to the water supply.
Clive Copelin, Sydney Water.
Email: clive.copelin@sydneywater.com.au
Modifed Robbins Device (University of New South Wales/CRC for Water Quality and
Treatment)
Used in Sydney, Australia
Related fact sheets FS 1, FS 4
The modifed Robbins Device (MRD) is a fxed device
which is suitable for distribution system or laboratory studies
to determine the ability of distributed water to promote
bioflm development, determine the impact of bioflms on
water quality, assess the effectiveness of water treatment
strategies to minimise bioflm development, and undertake
bioflm characterisation.
The MRD (University of New South Wales/CRC for Water
Quality and Treatment) is constructed of uPVC pipe (typically 150 mm x 2.0 metres).
The device contains 60 sampling slides (15 cm
2
each). Effective total sampling area of 900 cm
2
.
The sampling slides can be made of either uPVC or stainless steel. Slides are positioned in a
helical pattern down the length of the pipe by offsetting each slide by 36 from the previous slide.
This arrangement maintains hydraulic integrity in the system. Sampling slides are compatible/
interchangeable with the bioflm reactors and bioflm ExoSamplers. Sampling slides allow direct
microscopic observation of bioflms on the slide surface.
The MRD is constructed then ftted into a system. The MRD (University of New South Wales
/CRC for Water Quality and Treatment) needs to be isolated via two isolation valves during
sampling.
Modifed Robbins devices (University of New South Wales /CRC for Water Quality and Treatment)
are rated at 1,180 kPa.
Bioflms formed in bioflm reactors and bioflm ExoSamplers are representative of those formed
in the MRD.
Michael Storey, CSIRO Manufacturing and Infrastructure Technology
Email: michael.storey@csiro.au
Slidetrans
The Slidetrans allows sampling slides from the CRC for Water
Quality and Treatment/Sydney Water bioflm reactor, bioflm
ExoSampler and large diameter pipe coupon sampler to be
transported to the laboratory.
There is position for six slides. The unit is fully autoclavable.
It fts into a 250 mL sterile sample container and contains
a reservoir to maintain humid conditions thus preventing
desiccation of bioflm samples.
Page 29
FS 9
Page 30
FS 10
1) Bioflm Harvesting
A large majority of bioflm analytical methods outlined in this document require the removal of bioflm
from a test surface. It should be noted, however, that each bioflm harvesting method needs to be
validated in terms of the effectiveness of bioflm removal as well as the impact on the analyte being
tested. This is particularly the case when analysing bioflm microorganisms, which may be damaged
by some bioflm removal techniques.
Scraping and swabbing
Test surfaces can be scraped by a sterile scalpel blade or swabbed with a cotton swab. When the
inside of a large pipe is being sampled in situ, sterile paint scrapers can be used.
Removed bioflm is resuspended in an appropriate buffer solution eg strength Ringers solution,
PBS or sterile (chlorine-free) tapwater.
The scraping tool or swab should be rinsed into the bioflm sample suspension, taking into
account the volume used.
It is essential that the volume of resuspension buffer and area scraped/swabbed be known to
enable back calculation of the analyte concentration per unit of area.
A disadvantage of swabbing is the entrapment of microorganisms in the swab or the impact of
swab material on chemical analyses.
If possible, assessment of the surface after scraping/swabbing should be performed to evaluate
the effciency of removal. For example, in the case of bioflm cell enumeration on sampling slides,
the test surface can be stained to assess the extent of cells remaining on the surface.
Sonication
Sonication is the process of dispersing microorganisms by using sound-wave energy or ultrasound
eg releasing bacteria attached to surfaces.
The duration of sonication needs to be optimised to maximise the removal of bioflm microorganisms
without causing a reduction in recovery.
Assessment of the surface after sonication should be performed to evaluate the effciency of
removal. For example, in the case of bioflm cell enumeration on sampling slides, the test surface
can be stained to assess the extent of cells remaining on the surface.
Stomaching
Stomaching is the process of homogenising samples in a fashion similar to that found in the
stomach. Samples and diluent are placed in a plastic stomacher bag and homogenised by two
reciprocating paddles. The shear generated during stomaching is suffcient to harvest bioflm
cells from a surface.
This process may not be suitable for microorganisms that are susceptible to shear, for example
bacteriophages. In this case optimisation of stomaching time against loss of virus infectivity
needs to be assessed.
Assessment of the surface after stomaching should be performed to evaluate the effciency of
removal. For example, in the case of bioflm cell enumeration, the test surface can be stained to
assess the extent of cells remaining on the surface.
FS 10 Bioflm Analytical Methods
2) Bioflm Biomass
Drinking water bioflms predominantly contain carbohydrates (as polysaccharides) and proteins.
These are the key reactive bioflm components with disinfectants, in particular chlorine. Relative
values of bioflm biomass can be determined by measuring total carbohydrate and/or total protein.
Ideally both total carbohydrate and total protein should be assessed. This provides a better estimate
of bioflm biomass as often one component may be present in much lower concentrations than the
other. The bioflm biomass measurement includes all carbohydrate and protein components of the
bioflm, including those associated with cells. It should be noted that the determination of bioflm
biomass as total protein and total carbohydrate, provides a relative concentration of biomass for
comparative purposes. This method does not measure the entire biomass of a bioflm.
Joseph Chandy, Sydney Water
Email: joseph.chandy@sydneywater.co.au
or
Total protein
Total protein can be determined using the Bio-Rad Protein Assay (http://www.bio-rad.com) based
on the Bradford protein determination method (1976 Anal Biochem 72:248254). The assay
sensitivity is 1.25 g.mL
-1
protein. The method requires a spectrophotometer.
Total protein can also be determined using the NanoOrange
Protein Quantitation Kit. The

NanoOrange
Protein Quantitation assay is more sensitive than the Bradford method. The
NanoOrange
Protein Quantitation Kit is commercially available from Molecular Probes (www.

probes.com). The assay sensitivity is 10 ng.mL
-1
protein. The method requires a fuorometer.
Total carbohydrate
Total carbohydrate can be determined by the phenol-sulphuric acid method of Dubois (1956, Anal
Chem 28:350-356). This method detects simple sugars, oligosaccharides and polysaccharides,
including those found in bioflm exopolymers as described by Underwood et al. (1995, Wat.
Supply. 4:227-232). The test is simple to perform, highly sensitive and specifc. The method
requires a spectrophotometer.
3) Detection and Enumeration of Bioflm Microorganisms
Bacteria are responsible for the colonisation of surfaces and the production of the polymers that
constitute the bioflm matrix. Determination of the concentration of bacterial cells within a bioflm
indicates the extent of bacterial regrowth in a system and hence refects the capacity of water to
support regrowth.
Total bacterial cell enumeration
Total cell counts provide the concentration of both living and dead bacteria at a surface. Importantly
viable but non-cultivable cells can be enumerated using this method. Typically total cell counts
are orders-of-magnitude greater than heterotrophic plate counts. Total cell counts are usually
performed using fuorescent nucleic acid stains such as 4, 6-diamidino-2-phenylindole (DAPI),
acridine orange or Syto9 (Molecular Probes; www.probes.com). Stained cells are detected using
epifuorescence microscopy.
Total counts provide no information on whether bioflm bacteria are alive or dead. The Live/
Dead
BacLight stain (Molecular Probes) uses a combination of Syto9 (green) and propidium
Page 31
FS 10
Page 32
FS 10
iodide (red) to discriminate between live and dead bacteria. Stained cells are detected using
epifuorescence microscopy.
Total cell counts can be undertaken directly on bioflms when grown on a fat surface, such as
the sampling slides from the Bioflm reactor, bioflm ExoSampler, pipe coupon sampler (large
diameter) or modifed Robbins device (University of New South Wales/CRC for Water Quality
and Treatment). The beneft of direct bacterial counts is that the spatial organisation of the bioflm
can also be determined. A disadvantage of direct cell counts is the lack of resolution of individual
cells in densely populated bioflms. This can lead to inaccuracies in cell enumeration.
To circumvent the loss of resolution in densely populated bioflms, bioflm cells can be removed
from the surface (see FS 10 Bioflm Harvesting) and placed into suspension before staining
and enumeration. This process can disperse bioflm bacterial cells thus increasing the accuracy
of enumeration, provided that removal is optimised. A disadvantage of enumeration of cells in
suspension is that no information of bioflm structure can be gained. It should be noted that the
area harvested and volume of suspension medium needs to be known in order to calculate
concentration per unit of area.
or
Mark Angles, Sydney Water
Detection and enumeration of specifc bacteria
Fluorescence in situ hybridisation
- Cells from specifc bacterial groups, such as nitrifying bacteria or indicator bacteria, can
be identifed in intact bioflm using the fuorescence in situ hybridisation (FISH) technique.
The technique relies on the probing of specifc target sequences in the cells nucleic
acid with fuorescently tagged oligonucleotide probes (short segments of nucleic acid
complimentary to the cells nucleic acid target sequence).
- The technique is performed on whole cells, which can be visualised using epifuorescence
microscopy, either in intact bioflms or in suspension. The use of different coloured
fuorescent dyes allows more than one cell type to be visualised simultaneously.
- FISH on intact bioflms has the advantage of providing information on the spatial
distribution of cells within the bioflm.
- In the case of dense bioflms, specifc FISH-stained cells can be visualised using confocal
scanning laser microscopy. Confocal scanning laser microscopy has the advantage of
being able to generate a 3-D image of a bioflm thus identifying the position of cells within
the bioflm.
or
Polymerase chain reaction
- Polymerase chain reaction (PCR) enables the detection of particular genes or
microorganisms within a bioflm suspension, including bacteria, viruses and protozoa.
- DNA primers (short segments of DNA) specifc for a gene sequence of the target organism
are used to amplify the target gene up to approximately 2
30
times, thus making the PCR
product detectable.
- PCR provides no information on the spatial structure of the bioflm or cell concentration.
or
Heterotrophic plate count
Culturing bacteria on a solid, non-selective growth medium is a simple and widely accepted
method to isolate and enumerate bacteria from bioflms, as well as from the bulk water.
Bacteria from drinking water are typically cultured on R2A agar, which is formulated to grow
bacteria from oligotrophic (low nutrient) environments.
Even though R2A is considered to be non-selective, not all viable bacteria from the environment
can be cultured, as evidenced by the difference between live total counts and heterotrophic
plate count concentrations. In addition, viable but non-cultivable bacteria or fastidious bacteria
will not be detected on R2A culture media.
The advantage of culturing HPC bacteria is that it provides the opportunity to isolate bacteria
for typing.
As cells need to be harvested from the test surface in order to perform HPC counts, this technique
provides no information on bioflm spatial structure or organisation.
Detection of other microorganisms
Cryptosporidium oocysts and viruses can be detected in bioflms in situ or in bioflm suspension
using fuorescently tagged monoclonal or polyclonal antibodies.
Cryptosporidium oocysts can be isolated from bioflm suspensions using immunomagnetic
separation (IMS; Dynal) and then subsequently stained with a fuorescently tagged specifc
monoclonal antibody. Stained oocysts are visualised by epifuorescence microscopy.
Alternatively, Cryptosporidium oocysts can be visualised directly in bioflms.
Malcolm Warnecke or Mark Angles, Sydney Water
Email: malcolm.warnecke @sydneywater.com.au or mark.angles@sydneywater.com.au
Typing of bioflm microorganisms
The tests described in this section require the initial isolation of bacteria. This has limitations in terms
of selectivity, as described previously (see FS 10 Heterotrophic plate count).
Biochemical characterisation
- The simplest method for typing isolated HPC bacteria is by using API 20E or 20NE test
strips (www.biomerieux.com). The API 20E strips are used primarily to identify coliform
bacteria. API20NE are recommended for non-enteric bacteria.
Page 33
FS 10
Page 34
FS 10
- The advantage of the API identifcation systems is that the identity of isolates is provided
as well as a phenotypic code (biochemical profle). While usually ignored in a routine
sense, the identifcation code can be used to determine the similarity between isolates.
- A disadvantage of the API test strips is that not all environmental isolates can be identifed
using these systems. This is either because the test strips do not support the isolates
growth, contain an insuffcient range of tests to describe an isolate or an isolate is not
in the API identifcation database. Only Gram negative bacteria can be identifed by the
API systems.
Molecular typing
- In contrast to biochemical based characterisation tests, nucleic acid techniques (outlined
below) do not require gene expression.
Randomly amplifed polymorphic DNA
- Randomly amplifed polymorphic DNA (RAPD) relies on the random amplifcation of an
isolates nucleic acid using a set of defned but random primers.
- The band patterns or fngerprints of amplifed genomes can be compared to determine
the degree of relatedness or dissimilarity between isolates.
- There are a number of disadvantages with this technique. Only the presence of an
amplifcation product is compared between isolates but no weight is given to the absence
of a product. Furthermore, there is generally no determination if amplifcation products of
the same size are indeed of the same nucleic acid fragment.
DNA sequencing
- DNA sequencing provides the DNA base sequence of target sections of an organisms
nucleic acid. For example, comparisons between bacteria are usually based on the base
sequence for the 16S ribosomal sub-unit and for protozoa comparison is usually based
on the 18S ribosomal sub-unit.
- Comparison of DNA sequences is an accurate way to determine relatedness between
isolates.
- The identity of isolates can be determined by sequence comparison with known
sequences in a sequence database such as GenBank.
- Using current methods, DNA sequencing has a relatively fast turnaround time
(approximately 48 hours).
Other genetic based typing methods
- Though not used extensively in Australian bioflm projects, there is a large range of other
nucleic acid based techniques available to determine the relationships between isolates.
These include denaturing gradient gel electrophoresis (DGGE), restriction fragment
length polymorphism (RFLP), pulse-feld gel electrophoresis, ribotyping and repetitive
DNA sequences (Rep-PCR).
Brett Neilan, University of New South Wales
Email: b.neilan@unsw.edu.au
4) Determination of microbial activity
The majority of techniques used to detect and enumerate bioflm microorganisms do not provide
information on the metabolic state of these microorganisms in the environment. An exception is
the Live/Dead
BacLight stain, which gives an impression on the viable state of bioflm bacteria.
Nevertheless, apart from an increase in either bioflm cell concentrations or bioflm biomass,
microbial activity is a useful way to assess the metabolic state of bioflms or bacteria in a distribution
system. For example, the determination of microbial activity can be used to assess the effcacy of
disinfection in inhibiting bacterial regrowth or the effects of water treatment regimes on limiting bioflm
development.
ATP analysis
Adenosine triphosphate (ATP) is the major form of cellular energy. As such the concentration of
ATP refects both the concentration of cells in a bioflm as well as their metabolic state.
The analysis of ATP is based on the luciferase mediated reaction of ATP with luciferin to produce
light. The amount of light produced is proportional to the amount of ATP in the sample. The
method requires a luminometer to measure the light production.
The activity of cells can be calculated by normalising ATP against the number of viable cells in a
sample.
Cells need to be removed from the test surface in order to undertake the assay.
ATP measurement gives no indication of activity in situ.
Reducing dyes
The metabolic activity and hence viability of whole cells can be determined in situ by the reduction
of either 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) or 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-
phenyl tetrazolium chloride (INT).
The reduction of the INT dye produces an insoluble red-brown deposit in the cells. Cells can be
visualised and enumerated by light microscopy or measured by spectroscopy.
In the case of CTC, the reduction of the dye produces a fuorescent insoluble product in the
cells. Cells can be visualised and enumerated by epifuorescence microscopy or measured by
fuorometry.
Some disadvantages of the use of reducing dyes is the inability of certain bacteria to uptake
the dyes and reduce them, and the possibility of abiotic reduction of the dyes leading to
false positives.
Page 35
FS 10
Page 36
FS 11
As discussed previously (see FS 1, FS 4, FS 6 and FS 7), biodegradable organic carbon (BOC) is
a key contributing factor for bacterial regrowth and bioflm formation. As a result, treatment regimes
or the optimisation of disinfectant practices need to take into account the concentration of BOC.
There are two approaches to BOC determination. One approach involves the determination of BOC
based on the increase in bacterial cell biomass (biological assay) while the other relies on the direct
measurement of DOC reduction by biological processes. A drawback of each of the biodegradable
organic carbon methods is that turnaround time for results can be between 2 14 days depending
on the water type.
Assimilable Organic Carbon
The assimilable organic carbon (AOC) method is a biological assay and as such uses the growth
of bacteria to estimate the concentration of biodegradable organic carbon in a sample.
This method is based on an assay developed by van der Kooij (KIWA, The Netherlands) and
is currently the standard method as described in the Standard Methods for the Examination of
Water and Wastewater, 20th Edition (APHA, 1998).
The concentration of AOC is determined by comparison of growth of standard bacterial strains
in a pasteurised water sample with growth of the standard strains on a known concentration
of acetate. The proportion of growth in the sample compared to growth on acetate allows the
concentration of AOC, expressed as acetate-C equivalents, to be calculated. It is assumed that
acetate is an acceptable surrogate for biodegradable organic carbon in water.
Quantifcation of the standard microorganisms can be by plate count or ATP assay.
The method is best applied for assessing changes in biodegradability during treatment or passage
through a distribution system.
An advantage of the method is the ability to compare biodegradable organic carbon concentrations
in samples from different locations.
Some disadvantages of the method are that pasteurisation may change the nature of
biodegradable organic carbon in the sample, it is unclear if the standard strains are appropriate
for different water types, and inorganic nutrients (nitrogen and phosphate) may become limiting
at high biodegradable organic carbon concentrations. The test assumes that carbon is the limiting
nutrient but may not determine maximum AOC concentrations if an inorganic nutrient is limiting.
Bacterial Regrowth Potential
The bacterial regrowth potential (BRP) method is a biological assay and as such uses the growth
of bacteria to estimate the concentration of readily biodegradable organic carbon.
The method was developed in Germany by Werner and Hambsch (1986, Water Supply 4:227-232)
and modifed at AWQC by Withers and Drikas (1998, Proceedings AWA WaterTech Conference,
Brisbane).
The concentration of BOC is estimated from an increase in microbial growth in a sample
monitored by turbidity measurements (12 forward scatter). Originally BRP was expressed as
a function of growth rate and growth factor. Withers and Drikas modifed the method to provide
BRP expressed as acetate-C equivalents.
The method includes sample fltration to harvest the native bacterial population, which is
reinoculated into the fltered sample at a known concentration. The samples are supplemented
with inorganic nutrients to ensure carbon is the limiting nutrient. In this way the maximum BOC
concentration is determined.
FS 11 Determination of Biodegradable Organic Carbon
Advantages of the method include the use of microorganisms indigenous to the water being
tested (compared with standard bacteria in the AOC method). This provides information on the
ability of the water to support regrowth of the indigenous bacterial population.
The assay requires 500 mL of sample. Up to eight samples can be run at one time.
Mary Drikas, Australian Water Quality Centre, CRC for Water Quality and Treatment
Email: mary.drikas@sawater.com.au
Rapidly Assimilable Organic Matter
The rapidly assimilable organic matter (RAOM) method is a biological assay and as such uses
the growth of bacteria to estimate the concentration of readily biodegradable organic carbon.
The RAOM method was developed by Sydney Water through the CRC for Water Quality and
Treatment project Factors infuencing the development of bioflms under controlled conditions.
Concentration of BOC is determined by comparison of growth of indigenous bacteria in an
unsupplemented sample with growth of indigenous bacteria on a known concentration of acetate.
The proportion of growth in the sample compared to growth on acetate allows the calculation of
BOC in the sample expressed as acetate-C equivalents.
The method can be used to determine the nutrient, either organic or inorganic, that is limiting to
bacterial growth by comparing a sample supplemented with inorganic nutrients to a sample not
supplemented with inorganic nutrients.
The method uses ATP to assess growth of the indigenous population in 40 mL sample volumes.
Advantages of the method include the use of microorganisms indigenous to the water being tested.
This provides relevant information on the growth rates of the indigenous microbial population and
the ability of the water to support regrowth. The required sample volume is small which allows a
large amount of replication or many samples to be processed. There is no pre-treatment of the
sample and this reduces risk of sample alteration from fltration or pasteurisation.
Mark Angles, Sydney Water.
Biodegradable Dissolved Organic Carbon
There are two available methods to determine biodegradable dissolved organic carbon (BDOC); either
a batch method or a continuous fow method. Rather than indirectly deriving BOC concentrations
from bacterial growth (as is the case with AOC, BRP or RAOM), the determination of BDOC is based
on the direct measurement of the decrease of dissolved organic carbon (DOC) over time. When
compared with the indirect biological assays, the BDOC method typically yields higher values. This
indicates that the BDOC method allows quantifcation not only of the easily biodegradable organic
carbon but also of the carbon that will be degraded by bacteria more slowly during distribution.
Batch Method
- The batch method as described by Volk et al. (1994, Environ. Technol. 15:545-556)
involves the incubation of a water sample with biologically active sand. Biologically
active sand is usually sourced from sand flters in a water fltration plant. The DOC is
Page 37
FS 11
Page 38
FS 11
measured initially and then regularly until the DOC value reaches a plateau (typically 10
days). The BDOC in a sample is calculated as the difference between the fnal and initial
DOC values.
- A disadvantage with this technique is sourcing biologically active sand and achieving
reproducible results.
Continuous Flow Method
- The continuous fow method as described by Ribas et al ( 1991 J. Appl. Bacteriol. 71:371-
378) involves the continuous passage of water over a fxed biomass in glass columns.
The concentration of BDOC is calculated as the difference between DOC values in
the inlet water and DOC values in the outlet water. This method can provide BDOC
measurements within 2-5 hours.
- A disadvantage of this method is the time it takes to establish a bioflm on the support
material in the columns (months). Furthermore, some effort is required to maintain the
biomass in optimal condition to achieve consistency between samples if used in a grab
sample rather than continuous mode. It is also arguable whether biomass formed on
one water type can effectively measure BDOC for other water types. In effect it may be
necessary to have a set of columns for each water type being tested. It has been observed
that the columns are limited to the amount of DOC that can be utilised, indicating that
optimisation of the technique for different BDOC loads may be necessary.
- BDOC analysis is typically for the measurement of gross biodegradability of organic
carbon such as in raw waters or the outlet of a water fltration plant. Nevertheless the use
of a sensitive TOC machine can increase the sensitivity of the assay.
Mary Drikas, Australian Water Quality Centre
Email: mary.drikas@sawater.com.au
or
or
Contributing Research Projects
CRC for Water Quality and Treatment Projects
Factors Infuencing the Development of Bioflms Under Controlled Conditions
Physical and Chemical Effects on Distribution System Bioflms and Incorporated Pathogens
Optimisation of Chlorine Residual in a Distribution System (Greenvale Sydenham, Melbourne)
Consolidation of Management Tools for Distribution Systems
Cryptosporidium Oocyst Interactions with Drinking Water Pipe Bioflms
Developing Optimum Adsorption Processes Stage 2
Biological Filtration Processes for the Removal of Algal Metabolites
Development of Treatment Systems for Removal of Natural Organics
Modelling Coagulation to Maximise Removal of Organic Matter A Pilot Plant and Laboratory
Based Study
Development of Combined Treatment Process for the Removal of Recalcitrant Organic Matter
CRC for Water Quality and Treatment/University of New South Wales PhD project The Ecology of
Enteric Viruses within Distribution Pipe Bioflms
Other
Projects undertaken by CSIRO Land and Water, Curtin University and Water Corporation.
SA Water Corporation MIEX Application Project
Contributing Researchers
Soliman Allam, Brad Allpike, Mark Angles, Nicholas Ashbolt, Anthony Bodycoat, Joseph Chandy,
David Cooper, Clive Copelin, Mary Drikas, Ian Fisher, Peter Franzmann, Alice Gilyou, Tim Green,
Anna Heitz, Lionel Ho, Daniel Hoefel, Brad Hiller, Veeriah Jegatheesan, George Kastl, Laszlo Koska,
David Masters, Matthew Neesham, Brett Neilan, Gayle Newcombe, Norbet Okech, Bernie OLeary,
Alison Parr, Sudhi Payyappat, Bret Robinson, Chris Saint, Arumagam Sathasivan, Najwa Slyman,
Michael Storey, Helen Stratton, Michael Trefry, Heather Uwins, Dammika Vitanage, Eddie Wajon,
Malcolm Warnecke, Naomi Withers, Marek Wojnarowski, Kevin Xanthis, Hopi Yip, Luke Zappia.
Contributing Organisations
Australian Water Quality Centre, CSIRO Land and Water, Curtin University of Technology, Griffth
University, Melbourne Water, Micronix Pty. Ltd, Sydney Water, University of New South Wales, Water
Corporation.
Acknowledgements
Page 39
Disclaimer
The Cooperative Research Centre
for Water Quality and Treatment
and individual contributors are not
responsible for the outcomes of
any actions taken on the basis of
information in this document, nor
for any errors and omissions.
The Cooperative Research
Centre for Water Quality and
Treatment and individual
contributors disclaim all and any
liability to any person in respect of
anything, and the consequences
of anything, done or omitted to
be done by a person in reliance
upon the whole or any part of this
document.
The document does not purport
to be a comprehensive statement
and analysis of its subject matter,
and if further expert advice
is required, the services of a
competent professional should be
sought.
CRC for Water Quality and
Treatment 2005
Bioflms: Understanding the
Impact on Water Quality and
Water Treatment Processes.
Management implications
from the Research Programs
of the Cooperative Research
Centre for Water Quality and
Treatment.
ISBN 1876616458
Page 40
R
e
l
a
t
i
o
n
s
h
i
p

b
e
t
w
e
e
n

B
i
o
f
i
l
m

R
e
s
e
a
r
c
h

a
n
d

E
l
e
m
e
n
t
s

o
f

t
h
e

F
r
a
m
e
w
o
r
k

f
o
r

D
r
i
n
k
i
n
g

W
a
t
e
r

Q
u
a
l
i
t
y

M
a
n
a
g
e
m
e
n
t

F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

1
1

&

1
2

E
v
a
l
u
a
t
i
o
n

&

A
u
d
i
t
R
e
v
i
e
w

&

C
o
n
t
i
n
u
a
l
I
m
p
r
o
v
e
m
e
n
t
F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

2

H
a
z
a
r
d

I
d
e
n
t
i
f
i
c
a
t
i
o
n

a
n
d

R
i
s
k

A
s
s
e
s
s
m
e
n
t

A
s
s
e
s
s
m
e
n
t

o
f

W
a
t
e
r

Q
u
a
l
i
t
y

D
a
t
a

C
a
t
c
h
m
e
n
t
M
a
n
a
g
e
m
e
n
t
T
r
e
a
t
m
e
n
t

D
i
s
t
r
i
b
u
t
i
o
n
B
i
o
f
i
l
m
s
x
D
i
s
i
n
f
e
c
t
a
n
t

r
e
s
i
d
u
a
l

l
o
s
s

(
F
a
c
t

s
h
e
e
t
s

1

&

2
)
-
w
a
l
l

e
f
f
e
c
t
s

i
d
e
n
t
i
f
i
e
d

x
D
i
r
t
y

w
a
t
e
r

x
T
a
s
t
e

&

o
d
o
u
r

(
F
a
c
t

s
h
e
e
t

3
)
x
M
i
c
r
o
b
i
a
l

c
o
m
p
l
i
a
n
c
e
/
g
u
i
d
e
l
i
n
e

r
e
c
o
m
m
e
n
d
a
t
i
o
n

f
a
i
l
u
r
e
s

(
F
a
c
t

s
h
e
e
t

4
)
-
n
o
t

s
o
u
r
c
e

w
a
t
e
r
/
f
i
l
t
r
a
t
i
o
n

p
l
a
n
t

d
e
r
i
v
e
d

F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

2

W
a
t
e
r

S
u
p
p
l
y

S
y
s
t
e
m

A
n
a
l
y
s
i
s

B
u
l
k

W
a
t
e
r
F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

9

R
e
s
e
a
r
c
h

a
n
d

D
e
v
e
l
o
p
m
e
n
t

F
i
e
l
d

B
a
s
e
d

I
n
v
e
s
t
i
g
a
t
i
o
n
s
L
a
b
o
r
a
t
o
r
y

B
a
s
e
d

I
n
v
e
s
t
i
g
a
t
i
o
n
s
O
p
t
i
m
i
s
e
d

D
i
s
i
n
f
e
c
t
i
o
n
F
a
c
t

s
h
e
e
t

7

R
e
m
o
v
a
l

o
f

T
o
x
i
n
s

&

O
r
g
a
n
i
c

C
a
r
b
o
n

F
a
c
t

s
h
e
e
t
s

5

&

6

F
a
c
t

s
h
e
e
t

8

M
a
i
n
s

C
l
e
a
n
i
n
g

F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

4

O
p
e
r
a
t
i
o
n
a
l

P
r
o
c
e
d
u
r
e
s

a
n
d

P
r
o
c
e
s
s

C
o
n
t
r
o
l
F
r
a
m
e
w
o
r
k

E
l
e
m
e
n
t

4

V
e
r
i
f
i
c
a
t
i
o
n

o
f

d
r
i
n
k
i
n
g

w
a
t
e
r

q
u
a
l
i
t
y

A
s
s
e
s
s
m
e
n
t

o
f

B
i
o
d
e
g
r
a
d
a
b
l
e

O
r
g
a
n
i
c

C
a
r
b
o
n

F
a
c
t
s
h
e
e
t

1
1

B
i
o
f
i
l
m

M
o
n
i
t
o
r
i
n
g

D
e
v
i
c
e
s
F
a
c
t

s
h
e
e
t

9

B
i
o
f
i
l
m

A
s
s
e
s
s
m
e
n
t

T
e
c
h
n
i
q
u
e
s
F
a
c
t

s
h
e
e
t

1
0

InAustralia,drinkingwaterqualitymanagementisundertakeninthecontextoftheAustralianDrinkingWaterGuidelinesFramework.
In the table below the salient research fndings are presented within the Framework to aid in their implementation by the Australian
WaterIndustry.
ADWG Framework Elements Key research fndings and reference to Fact Sheet No.
Assessment
ofthe
Drinking
WaterSupply
System
Hazard
Identifcation and
RiskAssessment
FS 1 Algal cells were readily observed in unfltered water bioflms whereas fltered
water bioflms were predominantly bacterial.
FS 1 Biodegradable organic carbon can be the primary driver of bioflm regrowth in
chlorinatedsystems.
FS 2 Pipe surfaces, including bioflms, contribute greatest to disinfectant decay in
smalldiameterpipescomparedtolargediameterpipes.
FS4 Developmentofaquantitativemicrobialriskassessmentmodelforvirusexposure
fromdrinkingwater.
FS 5 Reservoirs with foating covers need adequate mixing to prevent bioflm growth
ontheundersideofcovers.
Preventative
Measures
forDrinking
WaterQuality
Management
Preventative
Measuresand
CriticalControl
Points
FS 1 & Removal of biodegradable organic carbon causes a decrease in both bioflm
FS 6 biomass and the impact of bioflms on disinfectant decay.
FS3 Swampyodour(dimethyltrisulphide)productionwaslimitedbyremovaloforganic
carbonusingMIEX
andeffectivesystemchlorination.
FS5 Periodic shock high dose disinfection should be applied to reservoirs with
foating covers to control bioflm development.
FS5 Granular activated carbon can reduce chlorine demand and THM formation
throughtheremovalofprecursors.
FS 5 The removal of cyanobacterial toxins was predominantly by bioflms growing on
spent granular activated carbon or flter sand.
FS 6 Bioflm development was reduced by 70% following organic carbon removal by
MIEX
.
FS 7 Disinfection management should account for bioflm development.
Operational
Procedures
andProcess
Control
Operational
Monitoring
FS5 Coveredreservoirsshouldbemonitoredfortemperature,circulation,disinfectant
levels(>0.1mg.L
-1
)andmicrobialquality.
FS 5 Assessment of the effective life of GAC needs to account for the impact of bioflms
onorganiccarbonremovalandcyanobacterialtoxinremoval.
FS 8 Turbidity can be used as a surrogate for bioflm removal during mains swabbing,
allowing quantifcation of the effectiveness of pipe cleaning.
Correctiveaction FS 8 Mains swabbing is the most effective routine bioflm removal method.
Researchand
Development
Investigative
Studiesand
Research
Monitoring
FS 1 & An increase in biodegradable organic carbon causes an increase in bioflm
FS 2 development, bioflm mediated disinfectant decay and release of cells to the
aqueousphase.
FS 1 Detached bioflm cells can recolonise downstream surfaces and form bioflms
fasterthanaqueousphasecells.
FS 1 & Bioflms develop more rapidly in chlorinated water but to a lesser extent than in
FS7 chloraminatedwater.
FS 2 Disinfection decay by bioflms is dependent on the presence of readily
biodegradableorganiccarbon.
FS 2 Bioflms convert non-reactive biodegradable organic carbon into fractions that
readilyreactwithdisinfectants.
FS 3 Dimethyl trisulphide production in bioflms is associated with swampy odour
FS4 Cryptosporidium oocystsarereadilyincorporatedinto,persistinandsporadically
detach from, pipe bioflms.
FS4 Cryptosporidium oocysts are incorporated into bioflms more under high or
diurnally varying fow velocities than under low fow velocities.
FS 4 Flow velocities are the most infuential factor promoting Cryptosporidium oocyst
detachment.
FS 4 Bacteriophages are incorporated into bioflms and persist in the presence of 0.6
mg.L
-1
chloramine.
FS4 Total chlorine concentration of 1 - 2 mg.L
-1
inactivates surface associated and
freebacteriophages.
FS 10 Methods to monitor bioflm development and activity developed.
FS11 Methodstoassessbiodegradableorganiccarbondeveloped.
Validationof
processes
FS 1 Bioflm monitoring should involve a multi parametric approach rather than relying
on a single bioflm analyte.
Designof
equipment
FS 9 Development of various bioflm monitoring devices to assess regrowth in pipes
andmonitorwaterqualitybasedonbacterialregrowth.
CRC for Water Quality and Treatment
Private Mail Bag 3
Salisbury SOUTH AUSTRALIA 5108
Tel: (08) 8259 0211
Fax: (08) 8259 0228
E-mail: crc@sawater.com.au
Web: www.waterquality.crc.org.au
The Cooperative Research Centre (CRC) for Water Quality and Treatment is Australias
national drinking water research centre. An unincorporated joint venture between
29 different organisations from the Australian water industry, major universities,
CSIRO, and local and state governments, the CRC combines expertise in water
quality and public health.
The CRC for Water Quality and Treatment is established and supported under the
Federal Governments Cooperative Research Centres Program.
The Cooperative Research Centre for Water
Quality and Treatment is an unincorporated
joint venture between:
ACTEW Corporation
Australian Water Quality Centre
Australian Water Services Pty Ltd
Brisbane City Council
Centre for Appropriate Technology Inc
City West Water Ltd
CSIRO
Curtin University of Technology
Department of Human Services Victoria
Griffth University
Melbourne Water Corporation
Monash University
Orica Australia Pty Ltd
Power and Water Corporation
Queensland Health Pathology & Scientifc
Services
RMIT University
South Australian Water Corporation
South East Water Ltd
Sydney Catchment Authority
Sydney Water Corporation
The University of Adelaide
The University of New South Wales
The University of Queensland
United Water International Pty Ltd
University of South Australia
University of Technology, Sydney
Water Corporation
Water Services Association of Australia
Yarra Valley Water Ltd

Biofilms: The Cooperative Research Centre For Water Quality and Treatment

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Biofilms: The Cooperative Research Centre For Water Quality and Treatment

Uploaded by

Copyright:

Available Formats

Bioflms

(Magnetic Ion Exchange resin) and hence reducing

reduced the potential for bioflm

but reduced to less than the raw water BRP following

) and Alum Coagulation. Aqua 52(7):475-487.

Protein Quantitation Kit. The

Protein Quantitation Kit is commercially available from Molecular Probes (www.

You might also like