Has it really taken WEF 25 years to discover the studies on viable, but
non-culturable bacteria?

Finds Fecal Coliforms Appear to Reactivate in Centrifuge Dewatered Solids At
Four of Seven Facilities Tested.


WEF has [claimed it] guided technological development in water quality since it was founded in Illinois in
1928 through a grant from the Chemical Foundation as the Federation of Sewage Works Associations. The
name was changed to the Federation of Sewage and Industrial Wastes Associations in 1950 and to the
Water Pollution Control Federation in 1960. To reflect an expanded focus that includes nonpoint, as well as
point, sources of pollution, the name was changed to Water Environment Federation in 1991.  

The WERF is the research arm of the Water Environment Federation (WEF). Both organizations are located
in Alexandria, Virginia. Their webpages can be found at www.wef.org and www.werf.org.

This outdated (June 2006) study confirms that the Sludge Treatment Processes don't work!
With all of the government research by WEF/WERF and it's scientific partners, they would like the public to believe the
"viable, but non-culturable" phenomenon is new to them and EPA. In the  June 2006 Viable, but non-culturable disease
causing organisms in sludge study, WEF scientists now admit  "The issue of viable but non-culturable (VBNC) bacteria
was advanced in the 1980s, and gained significant interest in medicine, the food industry, and many other fields."

For the past twenty-five years public health, our lives, as well as the quality of our air, water and food has been firmly in
the hands of waste disposal people within EPA and WEF. After 25 years of putting the public health, air, water and food
chain at risk by promoting sewage sludge as a safe fertilizer and garden soil amendment free of disease causing
organisms -- Twelve years of running a government funded public relations program to discredit people who have been
exposed to the disease causing organisms and suffered sickness, and even death --Ten years of funding scientific
studies to assure the public disease causing organisms in sludge are eliminated or killed -- Yet, these waste disposal
scientists now want us to give them time to study the problem and how to fix it. But, WEF only plans to do more research
on one non-disease causing organism as an indicator for the 1,407 human pathogen species that may be in sludge and
there is no fix to kill a spore forming bacteria or fungi.

Are these waste disposal scientists really saying they have never completed any research on disease
causing organisms in the sludge (they playfully call biosolids) to verify the treatment processes?

In 2004, The FDA said for the purpose of its regulations, spore-forming microorganisms include both the spore and
vegetative cells. Furthermore, "Bacteria produce spores as a means to survive adverse environmental conditions, while
some fungi use them as a form of reproduction. Spores show great resistance to high temperature, freezing, dryness,
antibacterial agents, radiation, and toxic chemicals. Under favorable conditions, spores can germinate into actively
growing bacteria and fungi. Many of these spore-forming microorganisms are pathogenic to humans and have been
implicated in causing morbidity and mortality. To ensure the safety of a biological product manufactured in a facility in
which spore-forming microorganisms are present, these microorganisms must be kept under tight control to avoid the
release of spores into the manufacturing atmosphere and potential contamination of other products."

Could it be that the waste disposal scientists at WEF/WERF and EPA have never completed a simple a
literature review?

February 2006:  Dr. Edo McGowan asked,  "If Staphylococcus aureus are found dead, does that mean that the
problem is solved?" The corollary: Are they dead, or merely in the viable but nonculturable state, in a starvation
arrested state, or killed from a starvation but otherwise in a recoverable state by sudden nutrient excess in the culture?

James D. Oliver, Department of Biology, University of North Carolina at Charlotte, 2005, notes, "Since the original 1982
paper from the laboratory of Rita Colwell (Xu et al., 1982), over 400 papers have appeared which describe various
aspects of the phenomenon most commonly referred to as the " but nonculturable (VBNC) state" (48)

James D. Oliver,Department of Biology, University of North Carolina at Charlotte, 2005.
Furthermore, he said, "The number of species described to enter the VBNC state constantly increases, with
approximately 60 now reported to demonstrate this physiological response. Included are a large number of human
pathogens, including Campylobacter spp., E. coli (including EHEC strains), Francisella tularensis, Helicobacter pylori,
Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, several
Salmonella and Shigella spp. and Vibrio cholerae, V. Parahaemolyticus, and V. Vulnificus." (48)

The 2001 Stefano Dumontet et al., study " The Importance of Pathogenic Organisms in Sewage and Sewage Sludge"
reported in the J. Air & Waste Manage. Assoc., "Bacterial pathogens in sewage sludge contribute significantly to health
problems, locally and globally."  "Technical limitations for detection and isolation procedures can be considered the main
difficulties in monitoring sludge pathogens. In addition, pathogenic bacteria  introduced into a hostile environment may
become viable but not culturable121 without losing their virulence factors. Note also that sludge may contain several
xenobiotic compounds, and composted sludge could facilitate inhospitalities between pathogenic bacteria." (29)

In his 2004 paper,  Evaluation of bacteriological indicators of disinfection for alkaline treated biosolids, EPA's Mark
Meckes,  cautioned, "Again, it can be stated that lime treatment did not significantly reduce the number of spore forming
bacteria." (4)

"Straub, Pepper and Gerba say that the list of pathogens are not constant but keep changing: As advances in analytical
techniques and changes in society have occurred, new pathogens are recognized and the  significance of well-known
ones change. Microorganisms are subject to mutation and evolution, allowing for adaptation  to changes in the
environment. In addition, many pathogens are viable but nonculturable by current techniques (Rozak  and Colwell
1987), and actual concentrations in sludge are probably underestimated.(p. 58)"

In 1986, Gerba does not mention viable but nondetectable bacteria, but he does note the potential for regrowth. He
said, "In the case of indicator bacteria such as coliforms and fecal coliforms, regrowth may occur in buried sludges
(Donnelly and Scarpino, 1984)." (55)

In their 1973 study, Airborne Stability of Simian Virus 40 , T. G. AKERS, et al.,    found viruses could also be viable
(infectious), but non-cultureable (VBNC). They said, "Previous studies, which have revealed that infectious viral nucleic  
acids were not damaged with respect to biological activity either by atomization, aerosol storage, or collection (186)

In  1973,  John Walker, retired head of EPA sludge program, who was with the USDA at that time, explained to EPA that
non-detection of Salmonella did not mean the bacteria were dead.  USDA research showed that liming sludge, or liming
the soil, only inactivated Salmonella for about 30 days. Then there was regrowth to the original levels.. EPA also noted
in the 1989 proposed 503 sludge regulation that there could be explosive regrowth of Salmonella.
(National Association of State Universities and Land Grant Colleges, 1973)".

How much sludge has been illegally applied to the land since 1992?

In 1992-2003 EPA said, "If improperly treated sewage sludge was illegally applied to land or placed on a surface
disposal site, humans and animals could be exposed to pathogens directly by coming into contact with the sewage
sludge, or indirectly by consuming drinking water or food contaminated by sewage sludge pathogens. Insects, birds,
rodents, and even farm workers could contribute to these exposure routes by transporting sewage sludge and sewage
sludge pathogens away from the site.

However, there no one to say its illegal since part 503 is self-implementing and the EPA Office of Inspector General
found the EPA office of Enforcement and Compliance Assurance (OECA) provides none  [people], even though it has
program responsibilities. As we explained in our prior report, that office has disinvested from the biosolids program.


Water Environment Research Foundation   (WERF)

June 2006
list of associated documents

Based on the data collected in this study, researchers determined that more coliforms may have actually
been present after anaerobic digestion but were not picked up by the standard culturing method.
Researchers suspect that certain configurations of anaerobic digestion processes lead to conditions which
inhibit the ability of the coliforms to grow and be measured by SCMs. In other words, indicator organisms
may "hibernate" and become "non-culturable." This phenomenon is referred to as "viable but
non-culturable". The issue of viable but non-culturable (VBNC) bacteria was advanced in the 1980s, and
gained significant interest in medicine, the food industry, and many other fields.

Study Finds Fecal Conforms Appear to Reactivate in Centrifuge Dewatered
Solids At Four of Seven Facilities Tested

In a recent study of anaerobically digested solids from seven wastewater treatment facilities, counts of fecal coliform
bacteria increased after dewatering at four of the facilities tested. Immediately after centrifugation, fecal coliform counts
increased from very low or nondetectable levels, often by as much as several orders of magnitude, at the four facilities
where increases were observed. This study only looked at seven facilities and only facilities using anaerobic digestion
and high-solids centrifugation for dewatering; numerous other stabilization and dewatering processes are also widely

The Water Environment Research Foundation (WERF) and its research partners initially undertook this study in
response to reports of increases of fecal coliform counts following anaerobic digestion and dewatering at a few
wastewater treatment facilities. A number of utilities (such as our utility research partner in this study) are also in the
process of spending millions of dollars to upgrade or build new digestion and dewatering facilities to produce biosolids
using the types of processes studied here. These processes include both mesophilic and thermophilic digestion for
stabilization and high-solids centrifugation for dewatering. This research, and ongoing work to further address this issue,
demonstrates the leadership of the nation's wastewater community as supported by WERF in continuing to ensure
scientifically sound and environmentally safe biosolids management practices.

The study identifies some potential control methods and mitigation strategies that utilities could use to reduce coliform
levels. Developing specific information on mechanisms for reactivation and regrowth, as well as control methods and
operational changes, is the focus of further research which is already underway.

Why Is This Observed Increase in Coliforms an Important Issue?

Regulations developed to protect public health rely on indicator organisms such as fecal coliform bacteria to estimate
the levels of pathogens in wastewater and solids. Reliably estimating the levels of pathogens in wastewater solids by
testing for indicator organisms is one means of measuring compliance with these regulations. The concentration of fecal
coliform bacteria in wastewater and wastewater solids also is used to indicate the effectiveness of treatment processes
used to reduce levels of pathogens.

What Processes Are Used to Reduce Pathogens and Moisture Content in Biosolids?
Anaerobic digestion processes are widely used at wastewater treatment plants to stabilize sludges and reduce the
number of pathogens, often in order to meet Federal and other regulatory requirements. Anaerobic digestion processes
are generally classified as either mesophilic or thermophilic, depending on the temperature at which they operate.
Mesophilic processes operate at 30-40°C (86-104°F), while thermophilic processes typically operate at 53-60°C (127-
140°F). Temperature-phased anaerobic digestion (TPAD) combines both a mesophilic and a thermophilic process in
separate stages.

Dewatering processes are often used after digestion to reduce the moisture content of the sludge. A high-solids
centrifuge is a type of dewatering device which produces lower moisture sludges by achieving greater centrifugal forces
in the unit. Anaerobic digestion followed by high-solids centrifugation is one combination of treatment processes used to
produce biosolids for land application.

What Was Learned from this Research?

Of the seven facilities tested during this study, researchers observed large increases in fecal coliforms at four facilities
after anaerobically digested solids were processed using high-solids centrifuges. In some cases these increases were
as much as several orders of magnitude. Two of these facilities used mesophilic digestion, one used thermophilic
digestion, and one used TPAD. The other three facilities (one mesophilic, one thermophilic, and one TPAD) did not
show an increase in fecal coliforms following high-solids centrifugation.

Researchers used two methods to estimate coliforms, including E. coli (a non-pathogenic coliform used to indicate the
presence of pathogens). The standard culturing method (SCM), sometimes used to measure regulatory compliance,
showed decreased coliform levels after digestion, followed by increases after dewatering at four facilities.
A second microbial measurement method, quantitative polymerase chain reaction (PCR) was also used. PCR is a
method that measures the number of copies of DNA present, rather than counting the number of organisms that are
actively growing. PCR is not currently used for regulatory purposes at wastewater facilities; however it is commonly used
as a microbial detection tool in other fields, including the food and medical industries.

Measurements using PCR consistently showed less reduction in the concentration of coliform bacteria during digestion
compared to decreases measured by SCMs. The differences observed by these two methods may be explained by two
related hypotheses: creation of a "viable, but non-culturable" condition for indicator organisms in
digestion followed by a "reactivation" of these organisms during dewatering.

Why Is This Happening?

Based on the data collected in this study, researchers determined that more coliforms may have actually been present
after anaerobic digestion but were not picked up by the standard culturing method. Researchers suspect that certain
configurations of anaerobic digestion processes lead to conditions which inhibit the ability of the coliforms to grow and
be measured by SCMs. In other words, indicator organisms may "hibernate" and become "non-culturable." This
phenomenon is referred to as "viable but non-culturable". The issue of viable but non-culturable (VBNC) bacteria was
advanced in the 1980s, and gained significant interest in medicine, the food industry, and many other fields.

Researchers further theorize that after centrifuge dewatering, conditions change so that these VBNC organisms come
out of "hibernation", begin to grow, and can be enumerated by SCMs. The process by which bacteria become culturable
after the VBNC state is called resuscitation or reactivation. Under ideal conditions, coliforms take 20-30 minutes to
double in numbers as a result of cell division (regrowth). Reactivation is suspected here since the solids residence time
in the centrifuge, typically 20-30 minutes, is too short for regrowth to account for the several orders of magnitude
increase in fecal coliforms observed at the four facilities.

The mechanisms that result in reactivation are currently unknown. Researchers think possible explanations for
reactivation include changes in environmental conditions, removal of growth inhibitors, or shear forces during
centrifugation. The completed study did not evaluate how widespread this occurrence is or the potential role of different
digestion and dewatering processes. It did however show that not all facilities using anaerobic digestion with high-solids
centrifugation demonstrate this phenomenon.

How Can Utilities Use the Results of this Study?

The fall results are available in the study report, Examination of Reactivation and Regrowth of Fecal Coliforms in
Centrifuge De-watered, Anaerobically Digested Sludges. The report provides information on the following:

• Procedures and test methods that can be applied to assure measures for pathogen reduction are being achieved. k.
Potential mitigation options (e.g., changes to digester hydraulics, dewatering chemical additions, or longer-term storage)
that might be considered by a facility to achieve desired reductions in both pathogenic and indicator organisms.

What Are the Next Steps?

A few of public wastewater utilities are conducting additional testing to determine if there is an increase in measured
fecal coliforms following dewatering. Where this is occurring, additional test procedures and operational changes are
being evaluated and implemented on a case-by-case basis. WERF is planning and conducting additional research to
better understand what is happening and to provide more specific information to wastewater utilities.

Follow-on studies by WERF and its research partners are already underway and include five additional wastewater
facilities. These studies are addressing the following questions that will help to better define the conditions under which
these increases in coliforms are likely to occur, the extent of this phenomenon, and options for wastewater treatment
facilities to consider if it is observed:

« What combinations of wastewater process technologies lead to this observed increase in fecal coliforms?

• What specific process designs and operating conditions contribute to this increase in fecal coliforms?

m What are the mechanisms for reactivation? m What conditions contribute to regrowth?

WERF will continue to report on the results from these follow-on studies; they are expected to be completed in late
2007. WERF is committed to ensuring that the science around this issue is fully addressed. Please contact WERF using
the information provided below if your facility would like to participate in this research.

For More Information Contact:
Daniel M. Weltering Director of Research
703-684-2470, ext. 2447
[email protected]

Alan Hais
Program Director,
Solids Treatment, Residuals & Reuse
703-684-2470, ext. 7146
[email protected]
Amit Pramanik
Senior Program Director,
Wastewater Treatment & Reuse
703-684-2470, ext. 7228
[email protected]

Elizabeth Striano Director of Communications
703-68402470, ext. 7908 [email protected]