Waterborne Pathogens
AWWA Manual M48 - First edition 1999

Biofilms are patches or masses of living and/or dead microorganisms that accumulate inside water storage reservoirs
or pipelines. Because treated water is not sterile, some degree of biofilms exists in all systems. Other types of
organisms or material can be "sandwiched" between layers in the biofilm, including nematodes, algae, bacteria,
pathogens, fungi, and mineral deposits. See chapter 2 of this manual for more information on biofilms.

New research indicates that biodegradable organic carbon (BDOC) and assimilable organic carbon (AOC) may provide
nutrients for many biofilm organisms. Systems with more than 100 ug/L BDOC or AOC tend to have more
bacteria than those systems with lower levels. Limiting the amount of BDOC and AOC by optimizing water treatment
processes is key to reducing this problem. Temperatures greater than 59° F (15° C) may also increase the growth of
some bacteria. Plants using ozone and biologically active filters should monitor the BDOC and AOC levels leaving the
treatment plant and throughout the distribution system and establish internal operating goals.

The microbiological quality of treated drinking water as it leaves the treatment plant is the highest it will ever be. As
water travels through the distribution pipe system to the consumer, its microbiological quality degrades at a variable
rate. The degradation process involves several factors, including loss of disinfectant residual; temperature changes;
flow velocity changes; biofilm sloughing and stirred-up pipe sediments caused by rapid changes in flow, or even flow
reversals; pipe breaks and replacements, and other maintenance activities; intrusions of contaminants into the pipe
network from pressure drops and cross-connections;
regrowth of bacteria that survived the treatment
and growth of bacteria in biofilms on the pipe walls and surfaces in storage reservoirs and tanks.

Some treatment processes, such as preoxidant treatment (ozone or chlorine) of the source water or coagulated
(settled) water before filtration, causes an increase in easily assimilable organic carbon (AOC) that can stimulate
bacterial growth or regrowth in the distribution system. Heterotrophic bacterial growth or regrowth usually occurs when
the disinfectant residual drops below 0.2 mg/L, the water temperature is greater than 40° F (10° C), and AOC is
greater than 50 ug/L.
Biological filtration, if practiced, can reduce AOC in filtered, distributed water. This may
reduce, but not necessarily eliminate, bacterial regrowth in the distribution system.

Ultimately, the maintenance of an adequate disinfection residual controls bacterial regrowth in the treated distribution
water. The SWTR specifies a minimum disinfectant residual of 0.2 mg/L for chlorine or 0.4 mg/L for chloramine in the
distribution system to control microbial growth. These disinfectant concentrations may control bacterial growth in the
bulk water,
but may be inadequate to control biofilm bacterial growth. Thus, the bacterial quality of bulk water
leaving the clearwell is usually in the range of less than 1 to 10 cfu/niL measured as heterotrophic plate count (HPC)
bacteria. On the other hand, water from other areas of the distribution system (distal areas of the system, dead ends,
storage tanks, and reservoirs) may have bacterial densities ranging from 10 cfu/mL to greater than 1 x 10 cfu/mL.
Dead-end areas of distribution systems often have high HPC levels along with colored water problems because of
corrosion processes, the long residence time of the water, and loss of disinfectant residual.

Elevated storage tanks often have relatively high HPC levels because of loss of disinfectant residual and, during warm
weather, high water temperature. Open storage reservoirs may have high HPC levels due to loss of disinfectant
residual and contamination from birds, feral and domestic animals, and the surrounding land.
Practical experience indicates that treated water leaving the clearwell must have a disinfectant residual considerably
higher than 0.2 to 0.4 mg/L in order to provide the minimum 0.2 to 0.4 mg/L residual at the ends of the system. Just
how much disinfectant residual must be applied may vary widely based on a variety of factors, including the total
organic carbon (TOG) present in the finished water that will exert a disinfectant demand; water temperature; age and
condition of the distribution system piping; extent of biofilm development; residence time of the water; duration of off-
line storage in standpipes or reservoirs; type of storage, i.e., covered versus uncovered reservoir; and incidence of
posttreatment contamination events that contribute additional disinfectant demand in the form of chemical and
microbial contaminants.

Biofilm Effects on Water Quality

Many heterotrophic bacteria that survive or pass through the treatment process are able to adapt to the low nutrient
(oligotrophic) conditions of treated drinking water. Once in the distribution system, their ability to associate, temporarily
or by colonization (biofilm growth), with surfaces confers survival advantages for the bacteria. Nutrients available to
bacteria in the bulk water are usually present at very low levels. This, coupled with the presence of a disinfectant
residual, means that bacteria freely suspended in the water are inhibited from growing. However, bacteria associated
with surfaces, such as pipe walls and sediments, are exposed to a continuous or even accumulating supply of nutrients
from flowing water.

Establishment of a biofilm involves transport of bacteria and nutrients to the pipe wall surface, bacterial adhesion and
multiplication, and buildup of microbial growth products. These products include various exopolymeric materials that
assist in binding cells to the pipe wall and forming a matrix within which cells are protected from disinfectant. Attachment
of bacteria to the pipe wall and other surfaces, or in sediments, provides a 150- to 3,000-fold increase in disinfection
resistance, depending on the surface material. Bacteria adapted to growth under low nutrient conditions have been
shown to be more resistant to disinfection than bacteria grown under rich nutrient conditions. Effectively disinfecting
biofilms in distribution systems is difficult because of the two factors just discussed. Additionally, the transport of the
disinfectant into the biofilm is limited by diffusion kinetics. Reactions of the disinfectant with extracellular matrix material,
nutrients from the water that diffuse along with disinfectant, and corrosion products from the pipe itself result in a
reduced disinfectant residual available to react with the bacterial cell.

The higher the disinfectant residual present in the bulk phase water, the more likely it is that sufficient disinfectant can
diffuse into the biofilm to inactivate bacteria found there. There is evidence that a chloramine residual can exert better
control of biofilm bacterial growth than does free chlorine. However, once a bacterial biofilm becomes established in a
distribution system, it is virtually impossible to get rid of using chlorine or chloramine. Most distribution biofilms appear
to be thin and patchy (discontinuous) rather than uniform and continuous. Maintaining an adequate disinfectant
residual undoubtedly limits the extent of development of a biofilm, but the disinfectant residual necessary to do so
varies with changes in source water quality and with the performance of treatment processes in removing particulates,
nutrients, and microorganisms.

Bacterial growth and regrowth in water distribution systems occurs in biofilms, and biofilm bacteria may enter the bulk
phase water by release from the biofilm or by sloughing of biofilm particles from hydraulic shearing. Bacterial densities
in the biofilm typically range from about 10 to 10 cfu/cm , measured by culture techniques. Maximum biofilm bacterial
densities occur when disinfectant residual is low or nonexistent, while lower biofilm densities occur when disinfectant
residuals in the bulk water are as high as 1.6 to 1.8 mg/L.
Under some relatively specific conditions, a water utility using a surface water source may experience intermittent or
continuous low-level occurrences of coliforms characterized as coliform biofilm, or as growth or regrowth of coliforms in
the distribution system. Utilities with such problems are primarily in the northeastern United States, where distribution
system age may be part of the problem. Additionally, such coliform biofilm problems have been characterized by
•     the inability to maintain a disinfectant residual greater than 0.2 mg/L
•     conditioning  of the   distribution   system  by  exposure   to   treated water containing relatively high levels of AOC
(> 100 ug/L) and TOC (> 2 mg/L)
•    warm water temperatures (T > 15° C) occurring from late spring to late fall
•     a significant percentage of iron pipe in service for more than 75 years
•     lack of a regular flushing program for dead-end areas
Although treatment processes may be producing water that meets microbiological standards, low levels of coliform
bacteria do pass through treatment and may survive to colonize the biofilm in some areas of the system. When nutrient
levels, disinfectant concentration, and temperature conditions are right, these organisms are able to grow, perhaps to
levels sufficient that they are released from the biofilm and appear in the bulk water during sample collection.


A water utility's ability to produce and maintain high quality drinking water is a major challenge. The pressure to
produce ever better quality water, driven by both current and proposed regulations, means that water utilities must
evaluate their current ability to meet regulatory goals, forecast and plan for treatment technology changes that may
become necessary, and balance the costs and benefits of new technologies against the need to increase costs to

To maintain or improve the microbial quality of drinking water, utilities must address issues such as watershed
management and source water protection; maintain good treatment and disinfection practices, including process
optimization; develop a thorough understanding of the hydraulics of their distribution system; be aggressive in
managing and operating the distribution system, including active programs for cross-connection control and scheduled
hydrant and dead-end flushing; develop a proactive microbial monitoring and data collection program; and make active
use of data analysis and information stemming from that analysis to implement better treatment and distribution
management practices.

Chapter 10
Heterotrophic Bacteria


The heterotrophic plate count (HPC) enumerates aerobic and facultative aerobic bacteria found in water. The bacteria
must be capable of growing on simple organic compounds (primarily carbohydrates, amino acids, and peptides) found
in the culture medium, and under specified incubation time and temperature. HPC bacteria can be any bacteria that
meet this definition-by-method; they can be different species at different geographic locations and at different times.
Populations detected also differ according to the culture medium used, incubation conditions, and temperature.

The number of HPC bacteria in drinking water can be as high as 10,000/mL in the absence of a free chlorine residual,
high turbidity, and the warm water temperatures of summer. HPC bacteria indicate a deterioration in drinking water

The predominant HPC bacterial genera are Alcaligenes, Acinetobacter, Flavobac-terium, and Pseudomonas. The
types of bacteria present on pipe surfaces are similar to those reported in drinking water. Pigmented bacteria can be
found in water and form a significant percentage of the HPC population of treated water. Other bacteria of potential
medical importance, such as Staphylococcus, Mycobacterium, and Serratia, have been found in water. HPC bacteria
are also found in large numbers in point-of-use and point-of-entry devices; they grow readily in the carbon filter of
these devices unless a bacteriostatic agent has been added to prevent regrowth. However, some bacteria grow even in
the presence of a bacteriostatic agent.


HPC bacteria are not normally used as an indicator of disease, and bacteria in this group are usually not directly
associated with a specific illness or disease. However, bacteria within the HPC can cause disease, both as primary
pathogens and as opportunistic pathogens, including pathogenic Escherichia coli (gastrointestinal illnesses), and the
opportunistic Pseudomonas (skin or lung infections) and Aeromo-nas (possibly gastrointestinal illnesses). Because the
media used for HPC are not designed to be selective and do not include differential indicators for specific bacterial
groups, all these bacteria remain as unspecified bacterial species.

The relationship of HPC to gastrointestinal disease has been reported as part of an epidemiological study of the
gastrointestinal health effects of drinking water. An apparent relationship between the HPC counts on medium R2A at
35° C and gastrointestinal illness was reported for consumers of water produced by reverse osmosis units. Very high
bacterial counts were observed in the reservoir containing the treated water, and bacteria with virulence characteristics
could have been responsible for this observation. Immunologically compromised people (due to microbial infections,
age, cancer treatments, etc.) are generally considered to be susceptible to any infections, and opportunistic pathogens
could play a role in such infections. In a second epidemiological study, fully treated tap water bottled immediately after
production was found to also support high HPC numbers but to have no health effect.