Clemson University

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5-2007

The microbiological analysis of composting

Marion Shepherd
Clemson University, marions@clemson.edu

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THE MICROBIOLOGICAL ANALYSIS OF COMPOSTING
__________________________________________________

A Thesis
Presented to
the Graduate School of
Clemson University
__________________________________________________

In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Microbiology
__________________________________________________

by
Marion W. Shepherd, Jr.
May 2007
__________________________________________________

Accepted by:
Dr. Xiuping Jiang, Committee Chair
Dr. Annel K. Greene
Dr. Thomas A. Hughes

1
 ABSTRACT

Animal manures contain valuable nutrients which can be utilized for crop growth.

Consequently, the wastes are often spread across field where produce is grown without

treatment prior to land application. This practice is a potential threat to the environment

and human health, as pathogens contained in the manures may have extended survival in

soil, and could contaminate produce harvested for human consumption. Composting is a

process that has been often implemented on-farm to inactivate pathogens resident in

animal wastes. The objectives of this study were to: 1) perform a survey of South

Carolina poultry farms to determine if the methods implemented resulted in the

destruction of foodborne pathogens, and 2) determine the survival of E. coli O157:H7 in

a dairy manure-based compost performed in uncontrolled environmental conditions.

In the survey of poultry farms, nine compost heaps at different stages in the

composting process were analyzed on four poultry farms in upstate South Carolina. Both

the materials used and composting methods differed among the farms surveyed. In the

surveyed heaps, 71% of all internal samples contained moisture contents of less than

40%, which is considered as the minimum necessary for active composting. Ninety-one

(91) of 141 compost samples analyzed were positive for coliform populations ranging

from 1.00 to 6.00 log10 CFU/g. Approximately 94% of the surface samples analyzed

were positive for coliforms, compared to less than 50% of the internal samples. Seventy-

six percent of the surface samples were positive for presumptive Salmonella spp. Among

all internal samples, ca. 26% and 19% were positive for presumptive Salmonella and

2
Listeria spp, respectively. Both E. coli O157:H7 and L. monocytogenes was not

detectedin any of the samples. Among finished compost samples (n=21), ca. 62%, 33%,

and 14% were positive for coliforms, presumptive Salmonella, and presumptive Listeria,

respectively.

In the investigation of the survival of E. coli O157:H7, two trials were performed

involving duplicate compost heaps constructed on an outdoor, fenced site. The compost

heaps were comprised of dairy manure, old hay, feed waste, a sawdust-calf feces mixture,

and fresh hay. Samples of the composting mixture were inoculated with stx-negative E.

coli O157:H7 B6914 at initial cell numbers of ca. 107 and 105 CFU/g for Trial 1 and Trial

2, respectively. Individual sample bags were placed on the surface and at three locations

(top, center, and bottom) within each heap. In Trial 1, E. coli O157:H7 was detected by

enrichment through 14 days within the heaps. When inoculated with 105 CFU/g in Trial

2, E. coli O157:H7 was detected only through days 2, 2, and 5 at the top, center, and

bottom locations, respectively. For both trials, the pathogen survived at the heap's

surface for up to 4 months. The indicator microorganism, E. coli, was inactivated at a

rate similar to that of E. coli O157:H7.

Our studies demonstrated that foodborne pathogens may persist for extended

periods of time in the compost surface. This is important because it suggests that

improperly compost manures may serve as vectors in the dissemination of foodborne

pathogens on food products intended for human consumption.

3iv
 DEDICATION

I would like to say thank you, to my Lord and Savior, Jesus Christ, for giving me

the knowledge and strength to complete my thesis. I would like to dedicate this work to

my mother, Vertrell B. Shepherd, my father, Marion W Shepherd, Sr., and my sister,

Rashida J. B. Shepherd. Without their love, support, and encouragement this could not

have been possible.

2
 ACKNOWLEDGEMENTS

I would like to sincerely thank my advisor, Dr. Xiuping Jiang, for her guidance,

encouragement, and patience. I would like to thank Dr. Annel K. Greene and Dr.

Thomas A. Hughes for serving on my thesis committee. I would also like to thank all

past and current lab members I have worked with while completing my Master’s for their

friendship and assistance.

3
4
 TABLE OF CONTENTS

Page

TITLE PAGE ....................................................................................................... i

ABSTRACT......................................................................................................... iii

DEDICATION..................................................................................................... v

ACKNOWLEDGEMENTS................................................................................. vii

LIST OF TABLES............................................................................................... xi

LIST OF FIGURES ............................................................................................. xiii

CHAPTER

1. LITERATURE REVIEW ............................................................................... 1

Current agricultural practices................................................................... 1

Listeria monocytogenes ........................................................................... 2
Salmonella spp ......................................................................................... 3
E. coli O157:H7 ....................................................................................... 4
Pathogen survival in soil.......................................................................... 8
Microbiology of composting.................................................................... 11
Methods of composting............................................................................ 15
Composting of poultry wastes ................................................................. 17
Composting of cattle wastes .................................................................... 19
Inactivation of viruses and protozoans through composting ................... 21
Pathogen contamination of vegetables..................................................... 22
Summary .......................................................................................... 25
References .......................................................................................... 26

2. MICROBIOLOGICAL SURVEY OF COMPOSTING

OPERATIONS ON SOUTH CAROLINA POULTRY FARMS................... 33

Abstract .................................................................................................... 33
Introduction.............................................................................................. 34
Materials and Methods............................................................................. 36
Results .......................................................................................... 40
Discussion .......................................................................................... 44

5
Table of Contents (continued)

Conclusions .......................................................................................... 51
Acknowledgements.................................................................................. 52
References .......................................................................................... 53
Figure legends.......................................................................................... 56
Tables and figures .................................................................................... 57

3. FATE OF Escherichia coli O157:H7 DURING ON-FARM

DAIRY MANURE-BASED COMPOSTING ................................................ 65

Abstract .................................................................................................... 65
Introduction .......................................................................................... 66
Materials and Methods............................................................................. 67
Results .......................................................................................... 73
Discussion .......................................................................................... 77
Conclusions .......................................................................................... 82
Acknowledgements.................................................................................. 82
References .......................................................................................... 83
Figure legends.......................................................................................... 86
Tables and figures .................................................................................... 87

4. CONCLUSION............................................................................................. 93

x
 LIST OF TABLES

Table Page

1 Time necessary for composting depending on implemented

methods and substates used ..................................................................... 16

2.1 Compost heap setup and composting practices on

South Carolina poultry farms................................................................... 57

2.2 Summary of surveyed compost heaps during active

composting............................................................................................... 58

2.3 Prevalence of coliforms, E. coli, and presumptive

pathogens in compost undergoing first heating phase ............................. 59

2.4 Prevalence of coliforms, E. coli, and presumptive

pathogens in compostundergoing second heating phase ......................... 60

2.5 Abiotic and biotic analysis of finished compost samples ........................ 61

3.1 Composition, and biotic and abiotic analyses of

experimental compost heaps at the beginning of each trial ..................... 87

3.2 pH values and moisture content of Trial 1 compost

samples..................................................................................................... 88

3.3 Fate of E. coli and coliforms at different locations

of heaps during composting (Trial 1)....................................................... 89

3.4 Fate of E. coli and coliforms at different locations

of heaps during composting (Trial 2)....................................................... 90
2
 LIST OF FIGURES

Figure Page

1 Phase changes during the composting process ........................................ 12

2.1 Temperature profile of Farm S heaps ...................................................... 62

2.2 Impact of adding new material on coliform populations

in the initial compost heap (Farm G ) ...................................................... 63

2.3 Impact of adding of new material on coliform populations

in Bin #1 compost heap (Farm M)........................................................... 64

3.1 Temperature profiles of the composting heap for Trial 1 ........................ 91

3.2 Temperature profiles of the composting heap for Trial 2 ........................ 91

3.3 Fate of E. coli O157:H7 at different locations within the

heaps during composting of Trial 1 ......................................................... 92

3.4 Fate of E. coli O157:H7 at different locations within the

heaps during composting of Trial 2 ......................................................... 92

3
 CHAPTER ONE

LITERATURE REVIEW

Current agricultural practices

Food products, such as meats and vegetables, maybe contaminated with

pathogens during the growing and processing stages of food production. Most foodborne

pathogens are commonly shed in the feces of healthy animals, which serve as a major

source of contamination. Ensuring the safety of the food supply is critically important in

protecting the health of consumers. Many measures have been proposed in order to

control the spread of foodborne pathogens from the farm to the table (Coleman, 1995;

Cohen, 1998; Hafez, 1999; Curran, 2001, Crump et al., 2002; Scholthof, 2003). As a

reaction to the outbreaks of E. coli O157:H7 in 2006 linked to lettuce and spinach, the

legislature of the state of California approved three bills, in March 2007, designed to

improve the safety of fresh produce. Included in the bills were provisions that would:

regulate the use of water, fertilizer, and toilet facilities in the field; implement a system to

track produce so that contaminated products would be easily identified in lieu of an

outbreak; and mandate that leafy green vegetable growers be licensed by the state

(Associated Press, 2007).

To limit the risks of contaminating fresh produce before harvesting occurs, many

farms implement plans that ensure that “Good Agricultural Practices” (GAPs) are being

followed. The GAPs include assisting workers in maintaining

1
good hygiene through the availability of toilet facilities, the use of manure that has been

decontaminated of pathogens, the use of pathogen-free water for field irrigation, and the

creation of barriers to limit the entrance of wildlife and insects into areas where produce

is growing (Delazari et al., 2006). Adherence to GAPs is critically important, as the

breakdown at any point could result in the contamination of produce. Introduction of

untreated human or animal wastes, runoff associated with those materials, and insects

may all serve as sources or vectors of pathogen contamination.

Procedures are also followed on the farms to control the spread of enteric

pathogens among food animals. In an effort to decrease the exposure to limit infections

the following has been suggested: slaughtering and/or quarantining animals, disinfecting

areas where slaughtering has occurred, and screening animals for infection (Delazari et

al., 2006). Undertaking these measures will serve to limit the exposure of animals to

pathogens, which will in turn decrease pathogen concentrations in manures. Effective

implementation should result in a decrease in the incidence of foodborne diseases caused

by Listeria monocytogenes, non-typhoidal Salmonella spp., and Escherichia coli

O157:H7.

Listeria monocytogenes

L. monocytogenes is a short, facultatively anaerobic, Gram-positive, non-

sporeforming capable of growth over a wide range of temperatures. This organism

causes approximately 2,500 illnesses and is responsible for 27.6% of all foodborne deaths

each year in the United States (Mead et al., 1999). This pathogen is found across many

2
environmental sources, and has been detected in poultry, pig, and cattle manure (Pagotto

et al., 2006).

Infection with L. monocytogenes may cause stillbirths, miscarriages, meningitis,

or septicemia (Altekruse et al., 1997). The type of illness caused varies depending on the

infected individual though healthy adults usually experience symptoms typical of

gastrointestinal disorders. L. monocytogenes infection is caused due to the organism’s

ability to evade the host immune system and invade intestinal cells (Pagotto et al., 2006).

Once the organism travels through the lymphatic system and enters the bloodstream,

virulence factors such as hemolysin and internalin allow entrance and replication inside

host cells (Pagotto et al., 2006).

Salmonella spp.

Salmonella spp. are Gram-negative, rod shaped, facultatively anaerobic members

of the family Enterobacteriaceae. Some Salmonella serotypes are highly adapted to

causing human disease, but the focus of this study will be on serotypes that are not

characterized by causing enteric fever (non-Typhi serotypes). There are over 2,500

different non-typhoidal Salmonella serotypes and they are commonly associated with

mammals, insects, birds, and reptiles (Mølbak et al., 2006). Infections with salmonellae

are second only to Campylobacter spp. as the most reported cause of bacterial foodborne

illnesses with approximately 1.4 million illnesses and 580 deaths caused in the United

States each year (Mead et al., 1999). Though there are numerous Salmonella serotypes,

the four most common causes of human salmonellosis are, Typhimurium, Enteritidis,

Heidelberg, and Newport (Finke et al., 2002).

3
 Salmonella infections commonly occur through the fecal-oral route of

transmission. As few as 10 salmonellae cells have been associated with causing disease;

however, the actual number necessary may depend on the serotype and the function of

the host’s immune system (Møbak et al., 2006). Depending on the infectious dose,

gastrointestinal symptoms may develop anywhere from 6 hours up to 10 days after

infection. Most symptoms include diarrhea, vomiting, and abdominal and joint pain;

though septicemia may occur without intestinal complications (Mølbak et al., 2006). The

virulence factors associated with Salmonella induced diarrhea is thought to be caused by

an enterotoxin, inflammation occurs due to activated genes on the Salmonella

pathogenicity island 1, while Salmonella plasmid virulence genes are responsible for

systemic infections (Mølbak et al., 2006).

E. coli O157:H7

E. coli O157:H7 is responsible for approximately 73,500 and 60 deaths each year

in the United States (Mead et al., 1999). It is a Gram-negative, rod-shaped, facultative

anaerobic organism that is a member of the family Enterobacteriaceae. The pathogen is

usually associated with ruminant animals predominately, but has also been isolated from

animals such as pigeons, raccoons, and flies (Shere et al., 1998). Cattle infected with the

organism usually are asymptomatic carriers and shed the pathogen through their feces.

In a survey of fecal samples from cattle of different ages, E. coli O157:H7 was

detected at populations less than 102 CFU/g (detected after enrichment culture) up to 105

CFU/g (Zhao et al., 1995). Shere et al. (1998) reported that dairy heifers shed E. coli

O157:H7 at populations of ca. 2 – 5 log10 CFU/g for up to 16 weeks. It has been reported

4
that shedding of the pathogen is discontinuous. Grauke et al. (2002) reported that when

inoculated orally with E. coli O157:H7, cattle shed the pathogen in three intervals:

approximately 1 week, approximately 1 month, or 2 or more months. In this study, 50%

of the cattle observed shed the pathogen for 29 to 33 days.

Though discontinuous, E. coli O157:H7 shedding is thought to vary based on

cattle age, cattle diet, and seasonal variations. Zhao et al. (1995) report that weaned

calves were more likely to shed E. coli O157:H7 in feces than pre-weaned calves. A

study of fecal samples from 900 weaning calves across 15 different herds showed that

6.9% of calves tested shed E. coli O157:H7 in the feces (Laegreid et al., 1999). In that

study, analysis of the serum of the calves revealed that 63.3 to 100% of all individuals in

each heard contained antibodies to O157 antigens. These researchers conclude that

calves are infected with E. coli O157:H7 before weaning. Additionally, Heuvelink et al.

(1998) report that 21.7% of cattle between 4 and 12 months in age tested positive for E.

coli O157, the highest rate among all ages of cattle sampled. Grauke et al. (2002)

suggests that E. coli O157:H7 is more commonly isolated from the feces of younger

cattle because the rumen microflora is still developing, as the environment in a mature

rumen would limit the survival of the pathogen.

In regard to dietary impacts on the shedding of E. coli O157:H7, Dargatz et al.

(1997) reported that cattle fed with soybean meal were less likely to shed the pathogen in

their feces. Barley consumption and cattle on feed less than 20 days were more likely to

shed E. coli O157:H7. Heuvelink et al. (1998) suggested that diets high in fiber and low

in nutrients raise the pH in the rumen, creating conditions conducive for pathogen

survival.

5
 Chapman et al. (1997) reported that E. coli O157:H7 was isolated in cattle at a

higher rate in the spring compared to the winter. Additionally, Heuvelink et al. (1998)

indicated that cattle shed the pathogen more between the months of July through

September than over other intervals studied. These researchers suggest that incidence of

infection is greater during warmer months due to an increase in grazing resulting in

manure-contaminated pastures, and environmental conditions favorable for pathogen

survival.

Understanding E. coli O157:H7 shedding is important because this pathogen can

result in severe illness. It is reported that the infectious dose of E. coli O157:H7 for

humans is approximately 50 organisms (Fratamico and Smith, 2006). The incubation

period of infection can range from 1 to 8 days, depending on the infectious dose and

various host factors. In some cases, infection is asymptomatic, but is usually

characterized by diarrhea. Expression of two Shiga-like toxins (stx1 and stx2), the

predominate virulence factors of the pathogen, can result in bloody diarrhea, Hemolytic

Uremic Syndrome – a condition which impairs kidney function and possibly death,

especially in children under twelve years of age (Fratamico and Smith, 2006).

Developing strategies to limit L. monocytogenes, Salmonella spp. and E. coli

O157:H7 infections are important to food safety. Since three classes of pathogens

combine to cause approximately 1.5 million illnesses and 60% of all deaths related to

foodborne illnesses (Mead et al., 1999), procedures must be taken on the farm to treat

animal wastes, materials harboring these pathogens, to limit the risks of contaminating

commercially viable food products.

6
 Animal mortalities, especially poultry carcasses, are often contaminated with

Listeria and Salmonella, and, consequently, environmentally safe disposal methods are

needed. It is estimated that a flock of 50,000 broiler chickens grown for 49 days will

produce 2.2 metric tons (2,200 kg) of carcasses if the total mortality of the flock is 4.9%

(Blake et al., 1990). Methods of poultry carcass disposal have been burial, incineration

and rendering. Burial is considered as the most convenient method, but produces

unpleasant odors due to anaerobic degradation and may affect ground water quality

(Blake and Donald, 1992). Incineration is considered the safest disposal method for

carcass management; however, it is slow, labor intensive, and expensive (Blake and

Donald, 1992; CAST, 1996). In regard to rendering, while it is effective in carcass

treatment, it is labor intensive and expensive, much like incineration (Sander et al., 2002).

However, with rendering, a utilizable product is formed which may be sold for feeds or

other uses to cover expenses and generate a profit. Implementation of practical,

environmentally safe methods for the treatment of animal wastes, especially animal

mortalities, is a great concern.

In agriculture, it is a common practice to spread raw animal manure onto land

used for crop growth. The Council for Agricultural Science and Technology (1996)

stated that “manure decreases soil loss by improving soil physical characteristics

including structure, infiltration rate, permeability, bulk density and water holding

capacity” (CAST, 1996). Manure is also an excellent source of nitrogen, phosphorus,

and potassium – nutrients beneficial for crop growth. Dairy manure contains 5 – 16, 2 –

16, and 2 – 31 pounds per ton of nitrogen, phosphorus (in the form of P2O5), and

potassium (in the form of K2O) in non-liquid forms, respectively (Bates and Gagon,

7
1981). Poultry manure contains 4 – 111 pounds of nitrogen per ton, 1 – 96 pounds of

phosphorus (in the form of P2O5) per ton, and 2 – 55 pounds of potassium (in the form of

K2O) per ton, in non-liquid forms. Importantly, many of the nutrients are present in

forms that are readily available to growing plants.

Although manure contains resources that are valuable to the growth of crops, it

also harbors pathogenic microorganisms that could cause sickness in humans. This is of

great concern, as precipitation on fields covered with raw manure could create runoff,

introducing pathogenic microorganisms and soluble minerals in area waters. Water

supplies (rivers, lakes, and streams) are routinely monitored for fecal coliforms, as

elevated levels indicate that fecal contamination, usually attributed to livestock, has

occurred. As a consequence, bacterial, fungal, viral, and parasitic diseases may all be

transmitted through food or water contaminated with fecal matter from animals (Diesch,

1969). It is suggested that runoff from fields that had been recently incorporated with

manure contains nutrient concentrations and coliform counts comparable to fields that do

not contain manure (CAST, 1996). While the immediate incorporation of manure is

addressed, there still remains the possibility of runoff polluting water reservoirs used for

food and recreation if raw manure is not incorporated.

Pathogen Survival in Soil

Most foodborne pathogens are enteric in nature, but may survive for extended

periods in the environment. Lau and Ingham (2001) demonstrated that E. coli survived

longer than enterococci in loamy sand and silty clay loam soils when incorporated with

bovine manure. In this study, soils were exposed to environmental conditions similar to

8
what would be experienced in Wisconsin in late spring to early summer. Studies were

conducted in the laboratory to investigate the survival of E. coli O157:H7 (Jiang et al.,

2002) and L. monocytogenes (Jiang et al., 2004) in autoclaved and unautoclaved sandy

loam soils mixed with contaminated bovine manure. In both studies, contaminated

manures were mixed with soils at various ratios, and stored at 5, 15, and 21oC. In the

study of E. coli O157:H7, the pathogen survived for at least 35 days across all mixing

ratios and storage temperatures in autoclaved soils. Pathogen detection was occurred for

at least 138 days to greater than 226 days at 15oC in manure mixed in autoclaved soils,

depending on the manure to soil ratio. However, when manure was mixed with

unautoclaved soils, detection occurred for 103 to 193 days over various manure to soil

ratios at 21oC. For L. monocytogenes, when held at of 5 and 15oC, the pathogen was

detected through 43 days and 21 days in manure amended autoclaved and unautoclaved

soils, respectively. Additionally, at 21oC the pathogen survived for 14 days in manure-

amended autoclaved soil, compared to 21 days in manure-amended unautoclaved soil.

Researchers have also performed field-based studies to determine how pathogens

will survive in soil. Avery et al. (2004) inoculated both bovine slurry and ovine stomach

contents with E. coli O157:H7, and applied those materials to the surface and subsurface

(ca. 25 mm in depth) into soil cores. At the completion of the 8 week study, analysis of

soils samples revealed that E. coli O157:H7 was detectable in both surface and

subsurface soils samples, even though there was an evident decrease in populations

during the study. In an investigation of how bovine manure affects the bacteriological

quality of organic lettuce, sandy loam soil contaminated with manure was analyzed to

measure the persistence of several foodborne pathogens (Johannessen et al., 2004). In

9
this investigation, manures were not inoculated with any pathogenic microorganisms. In

two trials, commensal E. coli and thermotolerant coliforms detected in soils treated with

compost, firm manure, and manure slurry one week after fertilization. E. coli O157 was

detected in soil 1 week after firm manure and manure slurry fertilization. Importantly, in

trial 2, E. coli and thermotolerant coliforms were detected in soil 41 weeks after

fertilization. You et al. (2006), in a study comparing the survival of multi-drug resistant

(MDR) and drug susceptible (DS) Salmonella Newport in dairy manure, and soil

contaminated with dairy manure, found that both strains had similar survival profiles.

Detection of both pathogen strains occurred through direct plating for 107 and 158 days

in manure mixed with non-sterilized soil and manure mixed with sterilized soil,

respectively.

The studies described above illustrate the extended survival for both indicator and

pathogenic bacteria in soil. Clearly, the soil type and resident microbial populations can

affect pathogen survivability. Other environmental conditions such as pH and moisture

content of the soil, along with the affect of ultraviolet light, all have some effect on

pathogen survival. Knowing the affect of these factors could further the understanding of

what contributes to the decline, or supports the persistence, in soils.

Treatment of the animal wastes before incorporation into the soil through proper

composting will reduce and/or eliminate the likelihood of introducing pathogenic bacteria

into the environment where survival can occur over extended time periods.

10
 Microbiology of Composting

Composting is a method that can be used to effectively treat animal manures and

carcasses. It is defined by Glanville and Trampel (1997) as a method where “biological

degradation of organic residues under aerobic conditions…[result in] end products

consist[ing] largely of water, carbon dioxide, ammonia, heat, and a humus-like material

consisting of microorganisms, non-biodegradable inorganics, and organic compounds

that are resistant to rapid biodegradation.” Properly composted wastes are effective in the

destruction of pathogens, viruses, weed seeds, and nematodes (Misra et al., 2003).

Though composting may be conducted using a variety of substrates, there are

guidelines for ensuring successful composting. One major parameter of compost that

should be monitored is the carbon to nitrogen (C:N) ratio. It is stated that the C:N ratio

of 20:1 – 40:1 is acceptable for composting, while 25:1 – 30:1 is preferred (Sherman,

2005). If the right balance of these materials is not achieved, composting may be

hindered, as microbes use carbon for energy and growth, while nitrogen stores are used

for protein synthesis and reproduction.

Another important parameter of composting is the moisture content of composting

materials. Moisture contents in the range of 40 – 65% are acceptable, while 50 – 60% is

preferred (Sherman, 2005). Composts with too little moisture do not have enough

available water for the microorganisms to effectively metabolize nutrients, a function

essential for material decomposition. Conversely, material too saturated with moisture

could operate under anaerobic conditions due to the lack of oxygen entrance into the

heap.

11
 Other factors that will influence the composting process are pH and heap size.

The optimal range for pH in composting is 6.5 – 8.0. Numerous microorganisms

involved in composting allow for continuation of the process without disruption due to

pH fluctuations (Cochran and Carney, 2006). However, low pH may retard composts

from entering into the thermophilic phase (Sundberg et al., 2004). Heap size is an

important factor in composting, though it is dependent on the amount, and types, of

material used and particle sizes of the composted materials. Heaps that are too small in

size will not retain heat. As a consequence, the thermophilic phase cannot be reached

resulting in pathogen persistence in compost materials.

While the substrates used in composting may vary, depending on the nitrogenous

sources and bulking materials used, successful composting is usually characterized by the

following three phases: mesophilic, thermophilic, and cooling/maturation. The

composting phases are characterized by the changes of dominant microbial communities

and soluble nutrients utilized during the composting process (Smith, 1992).

Figure 1. Phase changes during the composting process (Smith, 1992)

12
 Many studies have been conducted to identify the organisms responsible for the

breakdown of organic material that occurs in composting. Studying the changes in the

microbial community when composting garbage, Ishii et al. (2000) analyzed the microbes

present in the compost using Density Gradient Gel Electrophoresis (DGGE) of the 16s

subunits of rRNA. Results indicated that 87% of the microorganisms in the thermophilic

phase of composting were identified as belonging to the genus Bacillus. Further analysis

showed that during the cooling phase, new organisms emerged that could metabolize

complex nutrient substrates. Furthermore, it was reported that changes in temperature

and available nutrients resulted in a final community much different than that from the

onset of composting. Another study observed the changes in the 16s rDNA restriction

fragment length polymorphism (RFLP) patterns in a compost held at 60oC for 14 days

(Nakasaki et al., 2005). In this study, dog food was used as a substitute for organic

substrates in the compost. Incubation of the compost at 60oC allowed the researchers to

study microbial succession in compost over a shorter time period. In the study, RFLP

patterns changed from day 0 through 4, days 5 through 7, and remained stable after day 9.

Analysis of RFLP bands when compost temperatures were highest revealed that Bacillus

species were dominant.

While analyses of 16s genetic subunits are very effective in the use of identifying

microbes involved in composting, analysis of the phospholipids fatty acid content of

microbes using Gas Chromatography – Fatty Acid Methyl Ester (GC-Fame) has also

been a useful technique in organism determination. Ryckeboer et al. (2003) found that

the genus Bacillus was most prevalent throughout the composting of vegetable, fruit, and

garden wastes, especially in the thermophilic phase. Additionally, there was in inverse

13
relationship between temperature and microbial diversity. Steger et al. (2005) reported

that when composting organic household waste and wheat straw under aerobic conditions

(16% oxygen), the temperature increase in the compost could be attributed to a

thermophilic bacterial community comprised of members of the genera Bacillus and

Thermus. This determination was made as the two genera have similar fatty acid

compositions. Bacterial communities of poultry manure, composted with either rice husk

or rice bran, were also tracked using PFLA analysis (Kato et al., 2005). Though the

temperature evolution of the composts varied, the establishment of a Gram-positive

bacterial community during the thermophilic phase was evident using FAME analysis.

The researchers, using data from past studies, inferred that Bacillus species, and possibly

actinomycetes, were the organisms detected in the Gram-positive community that was

detected from the thermophilic phase through the end of composting. Ishii et al. (2000)

and Ryckeboer et al. (2003) reported that microbial diversity increases after the

thermophilic phase to include members of the Bacillus taxon, various Gram-positive and

Gram-negative organisms, and fungal species.

From the data in the presented studies, it can be concluded that Gram-positive

communities, specifically the genus Bacillus, are the microorganisms that drive the

thermophilic phase of composting. This is not surprising, as Bacilli are organisms that

are found over a wide ranged of environmental niches, and can withstand elevated

temperatures. The importance of this genus of bacteria cannot be understated, as the

thermophilic composting phase is characterized the elevation of temperatures needed for

pathogen inactivation.

14
 Methods of Composting

The most common methods for composting include passive heaps, static heaps,

windrows, or in-vessel composting systems. Passive and static heaps are similar in that

they both involve the stacking of composting materials into mounds, referred to as heaps.

Passive heaps, however, are managed very little, while static heaps are maintained

through forced aeration or frequent mechanical turning (Sherman, 2005). In windrow

composting, composted materials are mixed and formed into long and narrow heaps,

which are subjected to regular agitation (Sherman, 2005). In-vessel composting is

characterized as a group of composting methods that use a container or enclosed staging

area to perform composting (Cochran and Carney, 2006). Using an in-vessel composting

system can also include mechanical or forced aeration of compost material. Table 1

describes composting times typically experienced based on material and methods

implemented (Rynk, 1992).

15
Table 1. Time necessary for composting depending on the implemented methods and

substrates used (Rynk, 1992).

While manures and other wastes have been composted using all varieties of

techniques, chicken mortalities have been primarily treated using bin composting, which

is considered an in-vessel system (Murphy et al., 1988). Gonzalez and Sanchez (2005)

suggested that static heaps could successfully be used to compost straw, hen manure, and

poultry mortalities.

To ensure that composts do not pose a risk to contaminate the environment and

the food supplies with pathogens, governmental agencies of the United States have

formulated the guidelines in which composting should be performed. When maintained

under certain conditions, composting is known as a “Process to Further Reduce

Pathogens”, or PFRP, by the United States Environmental Protection Agency (EPA)

16
(EPA, 1994). EPA regulations state that for Class A designation, which indicates that

pathogens are below detectable levels, static aerated compost heaps containing biosolids

should be maintained at temperatures of 55oC higher for 3 days (EPA, 1994). Similarly,

USDA recommends to organic growers that composting operations must maintain a

temperature in the range of 55-77oC for a minimum of 3 days for static aerated pile

system and 15 days with 5 turns for windrow system (Misra et al., 2003). If guidelines

are followed, the materials processed through composting may provide a pathogen-free

soil amendment, which allows for the slow release of nitrogen and phosphorus into the

soil.

Composting of Poultry Wastes

It is estimated that broiler chicken produce in between 12 and 23 billion

kilograms of wastes each year in the United States (Nachman et al., 2005), which

includes manure and carcasses. Poultry carcasses are commonly treated through

rendering, burial, and incineration. Rendering is relatively expensive, and burial and

incineration are detrimental to air and water quality (Gonzalez and Sanchez, 2005).

Composting poultry wastes is an inexpensive process in comparison to rendering, and

functions to improve soil conditions.

In a study by Chaudry et al. (1998) broiler poultry litter with moisture contents of

15, 25, and 35% were deepstacked (composted), and populations of indicator bacteria

were monitored. Results of the study indicate that total and fecal coliforms can be

eliminated after one week of deepstacking, regardless of the moisture content. It was

found that the moisture content did influence temperature evolution in the heap; however,

17
as litter deepstacked with 35% moisture held higher temperatures at monitored locations

than the other treatments. In a study investigating the survival of enteric bacteria with

and without aeration, poultry litter was deepstacked (Kwak et al., 2005). This group

revealed that enteric bacteria were inactivated between days 2 and 4 of litter deep

stacking. However, this group also reported that in litter that was not deepstacked,

Salmonella, Shigella, and E. coli were not detectable up to 4, 4, and 8 days post

inoculation, respectively. This data indicates that the composting of poultry litter results

in rapid elimination of pathogens.

Blake et al. (1994) conducted a field survey where 12 mini-composters were

monitored for the presence of indicator and pathogenic microbes. Throughout the survey,

temperatures in all heaps were above the 55oC threshold and Salmonella, Campylobacter

jejuni, and L. monocytogenes were not detected in any of the samples. Lawson and

Keeling (1999) composted poultry litter and carcasses using the methods suggested by

the USDA. Even at low ambient temperatures, the compost reached a peak temperature

of 71oC, inactivating Salmonella.

The composting of poultry carcasses is especially important, as “bacteria

breakdown the carcass[es], leaving only feathers and bones” (Sander et al., 2002). This

method of disposal provides a better option for poultry waste treatment over rendering

and burial, techniques that are expensive and may attract scavenger animals, respectively.

Studies have demonstrated that composting poultry wastes (litter and carcasses)

can result in the elimination of pathogenic bacteria. In addition to the increased

temperatures that occur in stacked litter, Turnbull and Snoeyenbos (1973) suggested that

ammonia production may result in Salmonella inactivation. However, this occurrence is

18
usually a result of composting poultry litter at below suggested C:N ratios (Elwell et al.,

1998). Further studies composting litter under various initial pH ranges should be

investigated for pathogen inactivation. Additionally, an investigation of microbial

populations present in poultry litter may result in the discovery of microorganisms that

release antimicrobial compounds into the surrounding environment, resulting in pathogen

death.

Composting of Cattle Wastes

It is estimated that 43.3 million tons of dry cattle manure (including beef, feeder,

and dairy cattle) are produced each year (Midwest Plan Service, 1985). Composting has

been used as a practical way to treat bovine wastes. Lung et al. (2001) studied the

inactivation of Salmonella and E. coli O157:H7 composting a fresh cow manure and

sawdust mixture. In their study, bioreactors with forced aeration where held at external

temperatures of 25 and 45oC and samples were enumerated for pathogen detection. They

reported that E. coli O157:H7 and Salmonella populations fell below the detection limits

3 and 2 days after the onset of composting incubated at 45oC, respectively. When

composted at 25oC, bacterial populations remained somewhat constant. In an

investigation of the fate of high levels (ca. 107 CFU/g) of E. coli O157:H7, bovine

manure was composted at 21 and 50oC (Jiang et al., 2003). The researchers reported that

external temperatures were maintained at 50oC, E. coli O157:H7 was detected at all

sampling locations through 7, but not 14, days of composting. In contrast, the pathogen

was detected at all sampling locations through 36 days of composting, though

inactivation occurred more rapidly at the bottom location, when the bioreactors were held

19
at 21oC. Hess et al. (2004) composted bovine manure inoculated with bovine-derived

and laboratory-grown E. coli O157:H7 strains in an effort to compare the inactivation

between the bacterial strains during composting. Results from the study indicate that

laboratory-grown strains were more resistant to heat than those derived from the animal,

as 300 degree-days were required for inactivation of the laboratory strain, while the

bovine-derived strain could be eliminated in manures from infected cattle in 180 degree-

days.

Laboratory-based studies, under well-controlled conditions, demonstrated that

contaminated wastes can be effectively treated through composting. However, heating in

the bioreactors must be induced through forced aeration or incubation at high external

temperatures. However, the controlled conditions used in laboratory studies do not

represent how composting may proceed in field conditions where the materials may be

exposed to varying ambient temperatures and other environmental factors.

In a field study, Larney et al. (2003) composted beef cattle manure with two types

of carbonaceous sources (barley straw and wood chips) and monitored total coliform and

E. coli populations over two summers (1998 and 1999). The researchers reported that ca.

3 log10 CFU/g of the target bacteria were eliminated during the mesophilic phase of

composting, where temperatures ranged between 33.5 to 41.5oC. The carbon source used

for composting was reported not to have a significant effect on bacterial survival of total

coliforms or E. coli. Total coliforms were detected through 94 composting days in 1998,

whereas E. coli populations fell below the minimum detection limit after 45 and 7 days of

composting in 1998 and 1999, respectively.

20
 Nicholson et al. (2005) analyzed the survival of three foodborne pathogens

inoculated into dairy farmyard manure. In the study, inoculated manure heaps were

either maintained as static or static aerated heaps. Results of the study indicated that in

unturned heaps, E. coli O157:H7 survived for 8 days, while Salmonella and Listeria both

survived 4 days. These researchers also reported that there were no significant

differences in survival of monitored foodborne pathogens in aerated and non-aerated

heaps.

Field studies, presented above, demonstrated that composting may result in the

inactivation of indicator and pathogenic bacteria. However, the constituents of and

methods implemented in, composting appears to influence the rate of pathogen

inactivation. Moreover, published studies of composting cattle waste under field

conditions do not address the issue of pathogen inactivation rates at different locations

within the compost. Similarly, though the compost surface is not subjected to the

elevated temperatures that occur within the heaps, studies do not address how pathogens

survive on undisturbed compost surfaces.

Inactivation of Viruses and Protozoans through Composting

In addition to bacterial pathogens, animal wastes are loaded with high numbers of

viruses and parasites that may cause illnesses in humans. Composting, when conducted

properly, can allow temperatures to reach thresholds high enough to inactivate viruses

and parasites in addition to harmful bacteria. Pourcher et al. (2005) reported that

infectious enteroviruses were inactivated after one month of composting a sewage sludge

– straw mixture. Virus inactivation was determined after incubation of a virus loaded

21
solution in Buffalo Green Monkey cells. The researchers also stated that other genomes

of enteroviruses were detected through 3 months of composting using PCR; however

these were deemed as non-infectious as they were not detected after culturing in an active

cell line. This study suggests that infectious enteroviruses can be effectively inactivated

through composting.

Rimhanen-Finne et al. (2004) investigated the inactivation of Cryptosporidium

parvum and Giardia intestinalis, two parasitic protozoans, and indicator bacteria

remaining after sewage sludge disinfection at wastewater treatment facilities in Finland.

Wastewater sludge, containing no animal manure inputs, were treated using the following

techniques: windrow composting, mesophilic anaerobic digestion and windrow

composting, drum and windrow composting, and tunnel and windrow composting. The

researchers reported that after 30 weeks, the protozoan pathogens were not detected in

sludge treated by any composting method.

The studies presented above have demonstrated that infectious viruses and

parasites can be inactivated by composting wastewater sludge. However, further studies

need to be performed to verify that viruses and parasites present in animals wastes may

be inactivated through composting.

Pathogen Contamination of Vegetables

The inactivation of pathogens in manure and compost is important not only to

decrease the transmission into the environment, but also to decrease the risk of

contaminating fresh produce. This is especially important in organic farming, as manure

22
or manure based fertilizers are commonly used in lieu of synthetic fertilities, which are

restricted by organic production guidelines.

Mukherjee et al. (2004) performed a study to determine the presence of indicator

and pathogenic organisms on produce from organic and conventional farms. In the 40

farms from where vegetable samples were obtained, all organic farms (32) and four

conventional farms reported using aged or composted animal manure as a crop fertilizer.

The researchers found that 92% of all vegetables samples were positive for coliforms,

while only 8% were positive for E. coli. Additionally, the study revealed that E. coli

prevalence on organic produce was approximately three times higher than on

conventional produce. Importantly, whereas E. coli O157:H7 was not detected on any of

the produce (conventional or organic), Salmonella was detected on two organic produce

samples: one head of lettuce, and one green pepper.

Islam et al. (2004, 2005) investigated the survival of Salmonella and E. coli

O157:H7 on vegetables treated with contaminated compost and irrigation water. They

report that S. Typhimurium was detected on carrots and radishes for 203 and 84 days,

respectively, after seeds were sown for those crops. E. coli O157:H7 was detected on

carrots 168 days, and onions 74 days after the introduction of contaminated compost or

irrigation water into the soil. Avery et al. (2004) also reported the presence of E. coli

O157 for six weeks (either through direct plating or enrichment culture) on vegetation

when grown in soils where contaminated bovine and ovine wastes were applied to the

soil surface. In examining the transfer of E. coli O157:H7 to fresh produce from manure,

lettuce seedlings were grown in soil with contaminated bovine manure (Johannessen et

23
al., 2005). Though E. coli O157:H7 was not isolated from any parts of the analyzed

lettuce samples, non-pathogenic E. coli was detected in lettuce during harvesting.

Guo et al. (2001) investigated the survival of salmonellae on and in tomatoes after

inoculating stems and flowers before and after fruiting of the plant. They reported that

37% of the harvested tomatoes were positive for Salmonella. Also, salmonellae were

detected in the scar tissue of the plant stem and the pulp of the fruit. This study suggests

that Salmonella may be internalized in tomatoes, if present on an injured plant. Solomon

et al. (2002) provided compelling evidence as to why untreated wastes should not be

applied to agricultural crops. In their study, lettuce seeds were sown in sandy loam soil

contaminated with manure, and lettuce plants were contaminated with green fluorescent

protein-labeled E. coli O157:H7 at high pathogen concentrations (ca. 107 – 108 CFU/g or

ml). Using confocal microscopy, their results revealed that E. coli O157:H7 colonies

were present in the edible parts of the plant, presumably taken up through the vascular

system of the plant. In 2006, a massive E. coli O157:H7 outbreak occurred in the United

States linked to contaminated spinach, which was due to fecal contamination in the field

where the spinach was grown (FDA News, 2006). This outbreak resulted in two deaths

and over 200 illnesses. In March 2007, it was reported that the spinach implicated in the

outbreak was organically grown, though the producers of the spinach would not indicate

whether raw or improperly treated manure was used during production (The American

Conservative Union Foundation, 2007).

The incorporation of contaminated material necessary for the growth of crops,

such as fertilizers and irrigation waters, can pose a great danger to the safety of the food

24
supply. Waste materials used when growing produce must undergo treatment before

application to land to ensure that no pathogens are being introduced into the food chain.

Summary

It is evident that animal wastes, especially manures, are loaded with many

microorganisms that may be pathogenic to humans. The use of untreated or improperly

treated manures can be a biological hazard to the environment, animals, and food

supplies. Studies and governmental regulations suggest that composting is an effective

means of inactivating pathogens in manures. Treatment of manures on the farm could

help limit transmission of foodborne pathogens to foods, especially fruits and vegetable

which are commonly grown in field and consumed raw. Though it has been shown that

composting results in pathogen inactivation, there are insufficient scientific studies

concerning the mechanisms of pathogen inactivation under field conditions.

The objectives of our study were as follows:

 Surveying the different methods of composting poultry wastes implemented by

poultry farmers.

 Determining the efficacy of composting methods implemented by poultry farmers

by detection of indicator and pathogenic microorganisms over different compost

locations.

 Investigating the inactivation of E. coli O157:H7 at different locations within the

compost heap under field conditions.

25
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transition from mesophilic to thermophilic phase in composting. Bioresource
Technology. 95: 145 – 150.

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Available at: http://fsrio.nal.usda.gov/news_article.php?article_id=4189. Article
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You, Y., S. C. Rankin, H. W. Aceto, C. E. Benson, J. D. Toth and Z. Dou. 2006.

Survival of Salmonella enterica serovar Newport in manure and manure-amended
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Zhao, T., M. P. Doyle, J. Shere and L. Garber. 1995. Prevalence of enterohemorrhagic

Escherichia coli O157:H7 in a survey of dairy herds. Applied and Environmental
Microbiology. 61:1290 – 1293.

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32
 CHAPTER TWO

MICROBIOLOGICAL SURVEY OF COMPOSTING OPERATIONS ON SOUTH

CAROLINA POULTRY FARMS

Abstract

It is well-known that poultry wastes are a reservoir for Salmonella spp. and

Listeria monocytogenes which are microorganisms commonly responsible for foodborne

illnesses. Active composting is a practical method used to treat poultry wastes on the

farm. In this study, nine compost heaps at different stages in the composting process

were analyzed on four poultry farms in upstate South Carolina. Both the materials used

and composting methods differed among the farms surveyed. At Farms G and M, new

materials were combined in heaps which had previously been composted In the

surveyed heaps, 71% of all internal samples contained moisture contents of less than

40%, which is considered as the minimum necessary for active composting. Ninety-one

(91) of 141 compost samples analyzed were positive for coliform populations ranging

from 1.00 to 6.00 log10 CFU/g. Approximately 94% of the surface samples analyzed

were positive for coliforms, compared to less than 50% of the internal samples. Seventy-

six percent of the surface samples were positive for presumptive Salmonella spp. Neither

E. coli O157:H7 nor L. monocytogenes was detected in any of the samples. Among

finished compost samples (n=21), ca. 62%, 33%, and 14% were positive for coliforms,

presumptive Salmonella, and presumptive

33
Listeria, respectively. Results indicate that the conditions in the compost surface were

suitable for pathogen survival. The introduction of new materials into previously treated

composting heaps could reintroduce indicator and pathogenic bacteria into the

composting system, and allowed extended survival. Additionally, farmers should be

educated in the composting process and adhere to composting guidelines, to ensure that

composts produced at their facilities are free of bacteria which may be pathogenic to

humans.

Introduction

In the United States, broiler chickens generate 12 to 23 billion kilograms of waste

each year (Nachman et. al, 2005). Much of this waste is applied back to land for crop

growth, since poultry litter contains valuable nutrients, such as nitrogen, phosphorus and

potassium (CAST, 1996). However, this process may have adverse impacts on the

environment as poultry litter contains microorganisms that could be pathogenic. In

addition to litter treatment, safe disposal of poultry mortalities is also important, as

analysis of broiler carcass rinses indicated that 20% and 15% of sampled carcasses were

positive for Salmonella spp. and Listeria monocytogenes, respectively (USDA, 1995).

While poultry mortalities can be treated through rendering, burial or incineration (Donald

and Blake, 1992), those methods can be expensive, attract scavengers that could harm

livestock, crops or humans, or aid in the transmission of disease, respectively. Poultry

litter and carcasses need to be properly treated; studies report that pathogens are able to

persist in feces, water and soils for extended periods of time (Islam et. al, 2005; You et.

34
al, 2006), and may have extended survival on produce (Islam et. al, 2004; Mukherjee et.

al, 2004).

Composting is a microbe-driven process where organic material is digested into

humus-like material (Richard et. al, 1998). Yard wastes, municipal solid waste, and

agricultural wastes, such as animal mortalities and manures, are often used as substrates

for composting.

While composting can be conducted using a variety of methods (Elwell et. al,

2001), if properly performed, all methods can safely result in the degradation of animal

mortalities. Additionally, pathogens may be inactivated from manure and manure based

materials, resulting in the creation of a biologically stable, nutrient rich product for crop

utilization. The most widely accepted method for the composting of poultry wastes (litter

and carcasses) in roofed bins was proposed by researchers at the University of Maryland

and accepted by the United States Department of Agriculture (USDA) (Murphy and Carr,

1991). Though composting carcasses in roofed bins is the preferred method, open heaps

and windrows may also be used depending on the carbonaceous material used in the

compost (Gonzalez and Sanchez, 2005). Poultry litters are commonly treated through

deepstacking, as it is a simple and cost-effective method of processing the material.

Chaudry et al. (1998) and Kwak et al. (2005) demonstrate that deepstacking poultry litter

can result in the complete inactivation of indicator and pathogenic bacteria.

Methods used in composting poultry wastes vary greatly among poultry farms, as

the techniques employed depends on the facilities and equipment available. The

objective of our study was to survey poultry farms in South Carolina to determine if the

35
methods used for composting on different sites and compost ingredients influenced the

survival of indicator bacteria and pathogens in compost.

Materials and Methods

Farm recruitment: Four poultry farms (A, G, M, and S) in the upstate of South

Carolina were recruited for participation in this study. Table 1 summarizes the

composting information provided by the farm managers overseeing composting at the

selected sites. The survey of compost heaps from Farm A, G, and M was conducted from

November 2004 through August 2005, while Farm S heaps were monitored from

November 2006 through January 2007. The compost heaps at Farms A, G, and M were

sampled nearly monthly, whereas heaps at Farm S were sampled on days 0, 3, 7, 14, 21,

30, and 60.

Description of Surveyed Heaps: During the survey, Farm A had two heaps,

known as initial and premix, that were analyzed. The initial heaps was constructed so

that one part of chicken litter was combined with two parts of pine fines, and the premix

heaps were composed of one part of the initially composted material (i.e, that had

previously undergone a phase of heating) to two parts of pine fines. Three composting

heaps were initially monitored at Farm G. The initial heap was composed of fresh wood

chips, chicken mortalities and chicken litter. The cured heap was composed of initial

composting materials that had been turned and composted for 8 weeks, and the

intermediate heap was a mixture of fresh and cured compost material. Farm M had two

heaps for analysis when the survey began. The first phase bin contained material that had

not completed a heating phase of composting. The second phase bin contained material

36
that had completed one heating phase of compost, and been inverted after the addition of

water so that another heating cycle may be reached. Farm S had two heaps for analysis

during the survey; one contained pine fines and fresh poultry litter, while the other

contained cured compost mixed with fresh poultry litter. Heaps surveyed were monitored

for 185, 132 and 288 days into composting, on Farms A, G, and M, respectively. Farm S

heaps were monitored from day 0 through 60 days of composting.

Temperature measurement in the compost heaps: Compost temperature and

oxygen content data were collected using an OT-21 temperature and oxygen sensor

(Demista Instruments, Arlington Heights, IL) during each sampling date prior to the

opening of the compost heaps. Compost temperatures in duplicate measurements were

taken at Farm M at the surface (0 – 5 cm), 40 cm, and 60 cm depths, while Farms A and

G temperatures were monitored at the surface (0 – 5 cm), 60 cm and 90 cm depths. At

Farm S, temperatures were monitored at the compost surface (0 – 5 cm), and depths 30

cm and 60 cm from the floor of the compost staging area. At Farms A, G, and M,

measurements were recorded from the location in which the compost surface was

breached.

Compost heap sampling: Compost heaps on Farms A and S were turned weekly,

whereas heaps on Farms G and M were turned only when the materials were moved to

another bin. Samples were taken from each available heap at different locations within

the heap over sampling dates to ensure that prior openings of the heaps did not influence

conditions present of the procured samples. During each sampling, the heaps were

opened with a shovel that had been sanitized with 70% ethanol and wiped dry with paper

towels.

37
 Both the surface and internal samples were taken from the locations described

above for temperature measurements. Five subsamples from different positions at each

location were removed with a large sterile sampling spoon, and mixed well in a 2-gallon

sampling bucket sanitized with 70% ethanol and wiped with dry paper towels. The

composite samples (ca. 250 – 350 g), taken in duplicate, were placed into sterile sample

bags. All samples were analyzed within 3 h upon return to the laboratory.

Bacterial enumeration and enrichment: Twenty-five (25) g of sample was

combined with 225 ml of Universal Pre-enrichment Broth (UPB; Becton Dickinson,

Sparks, MD) and homogenized using a stomacher (Brinkman Instruments, Inc.,

Westbury, NY) at medium speed for 1 min. Aliquots of the homogenized samples were

used for the enumeration of total bacterial and E. coli/coliform populations. Sample

homogenates were serial diluted (1:10) using 0.1% peptone water, plated on Tryptic Soy

Agar (TSA; Becton Dickinson) using an Autoplate® 4000 spiral plater (Spiral Biotech

Inc., Bethesda, MD), and incubated at 30oC and 55oC, for total mesophilic and

thermophilic bacterial populations, respectively. E. coli and coliform populations were

determined through plating 1-ml aliquots of the sample dilutions on E. coli/coliform

Petrifilm™ (3M Microbiology Products, St. Paul, MN) and incubated at 37oC for 24 h.

The remaining homogenized sample-UPB mixtures were incubated at 37oC with

shaking for 24 h, and 1-ml aliquots were transferred into 9-ml of Tetrathionate (TT)

Broth (Becton Dickinson), Fraser Broth (Becton Dickinson), and modified tryptic soy

broth (mTSB; Becton Dickinson) with novobiocin (Oxoid Ltd., Basingstoke, Hants, UK),

for Salmonella spp., L. monocytogenes, and E. coli O157:H7, respectively. All three

selective enrichment broths were incubated at 37oC for 24 h. One ml of each selective

38
enrichment culture was transferred to sterile Eppendorf tubes, and briefly treated with 10

µl of Anti-Salmonella, Anti-Listeria, or Anti-E. coli O157 DynalBeads® (Dynal Biotech,

ASA, Oslo, Norway) following the manufacturer’s procedure. The final DynalBead®

suspension was then streaked on either XLT-4 agar (Becton Dickinson), Oxford agar

(Becton Dickinson) with modified Listeria antibiotic supplements (Oxoid Ltd.,

Basingstoke, Hants, UK), or Sorbitol MacConkey agar (SMAC; Becton Dickinson) with

Methylumbelliferyl Glucuronide (MUG) (Oxoid Ltd., Basingstoke, Hants, UK) for

Salmonella spp., L. monocytogenes, or E. coli O157:H7 detection, respectively.

Presumptive positive Salmonella spp. colonies were confirmed using the

Salmonella Latex Test Kit (Oxoid Ltd., Basingstoke, Hants, UK) and serotyping was

performed at a Food and Drug Administration (FDA) laboratory. Presumptive positive

Listeria colonies were confirmed by streaking on Listeria CHROMagar® for growth

typical of L. monocytogenes.

Moisture content and pH determination: Approximately 1 g of each sample

was weighed in a tared aluminum dish. The compost samples were then dried in an oven

(Blue M Electric Company, Blue Island, IL) at 105oC for 24 h for moisture content

determination. Analysis of pH was performed by adding 1 g of sample into 50 ml of

deionized water and stirring the solution for 5 min. A pH meter (Orion Research Inc.,

Boston, MA) was used to determine the pH of the samples.

Statistical Analysis: Bacterial count data were converted to log10 CFU/g for

statistical analysis. An analysis of variance (ANOVA) for a completely randomized

design with repeated measures across dates was conducted to determine if general

differences existed between treatment means using the general linear model (GLM)

39
procedure. Specific comparisons among parameters determined at different locations in

the compost heaps at each sampling date were accomplished with Fisher's least

significant difference (LSD) test. All statistical analysis was performed using the

Statistical Analysis System 9.1 (SAS; SAS Institute, Cary, NC).

Results

Parameters of surveyed compost heaps

Four poultry farms in the upstate area of South Carolina used windrow, static

heap or bin composting systems as a means of treating poultry mortalities and chicken

litter. The farm practices varied among the farms, especially in composting methods and

composting components used (Table 1). Farm managers at Farms A and M were aware

of and tried to adhere to composting guidelines, whereas managers at Farms G and S

were experimenting with composting as a waste treatment method. Compost heaps at

Farm A were considerably larger than those present at the other three farms, but the heaps

were turned weekly, as were heaps at Farm S. New materials were periodically added

into the heaps at both Farms G and M; additionally, there was no set schedule for heap

turning at Farm G, whereas heaps were turned at Farm M after there had been an internal

decline in temperature, indicating the completion of one phase of heating. Farm G was

the only farm that did not monitor compost temperatures, while none of the farms

monitored the moisture content and pH of the compost. The composts produced on

Farms A and M sold their finished composts commercially, whereas the compost from

the other two farms were applied directly to the fields on the respective farms.

40
Temperatures, pHs, moisture contents and oxygen levels of surveyed composting heaps

The temperature profile for the heaps at Farm S (Figure 1) indicates the complete

heating cycle of composting. Maximum temperatures achieved in the initial and compost

mix heaps were 52 and 38oC, respectively. Some active composting heaps at Farms M

and S did not achieve temperatures above 55oC, the level recommended for pathogen

inactivation, on sampling dates. In contrast, temperatures of up to 64 and 55oC were

detected during sampling days at Farm A and G, respectively (Table 2). In the cured

compost heaps at Farms A and G, temperatures were detected ≥55oC at internal locations;

however, temperatures in excess of 26oC were not detected on all sampling days at Farm

M.

Higher temperatures (≥50oC) were maintained in the heaps of Farm A, which

employed windrow composting. In contrast, composting temperatures were generally

lower than 45oC in heaps at Farm M except for two sampling dates after new material

was added into the heaps.

The pH values of active composting heaps at Farms A, M and S were mildly

acidic to mildly alkaline, whereas pH values at Farm G were in the alkaline range during

the survey (Table 2). The pH values for the cured heaps at Farms A, G and M followed

the same trend that was observed for the heaps in active composting at those farms (Table

2). The average pH values from samples taken from all farms were in the following

order: Farm G>Farm M>Farm S>Farm A.

Lower moisture contents were usually detected at the heap surface. However, in

the Farm G heap undergoing active composting, the moisture content was 67.4% in the

surface of the compost on one sampling date. This was due to the incorporation of

41
compost material into the sample that had been exposed to rain. Over all, it was found

that approximately 71% of all internal samples contained less than 40% moisture.

Oxygen contents of the heaps were low among across all farms, however.

Microaerophilic conditions existed within the heaps across Farms A, G and M, as oxygen

contents ranged from 0 – 6% (Table 2). Heaps at Farm S had oxygen contents ranging

from 2 – 17%, through the end of composting (Table 2). At Farm M, the heaps had low

oxygen contents at the onset of composting; however, as composting progressed, the

moisture content of the heaps decreased while the oxygen content increased.

Effect of adding new material into a composting heap

Figures 2a and 2b reveal the coliform populations present in the active

composting heaps at Farms G and M, respectively. Active composting on Farm G

resulted in the elimination of coliforms inside the heaps after 43 days of composting

(Figure 2a). However, the addition of partially composted material between days 43 and

77 after the onset of composting resulted in an increase in coliforms greater than 1.5 log10

CFU/g in the surface of the heap (Figure 2a). Additionally, the introduction of fresh

waste material between days 112 and 147 of composting caused increases of ca. 1.5, 3,

and 1 log10 CFU/g in the surface, 60 cm, and 90 cm depths, respectively, of the heap

undergoing active composting at Farm G (Figure 2a). In Farm M, application of a new

layer of fresh waste material into the active composting bin resulted in increases of

coliform populations of ca. 3 and 1 log10 CFU/g, respectively at the surface and 40 cm

sampling locations between days 54 and 113 after the onset of composting. Coliforms

42
were not detected on the surface of the compost, but in the internal locations due to an

inversion of the composted material one day prior to the final sampling date at Farm M.

Changes of bacterial populations during composting

Thermophilic bacteria: The mesophilic and thermophilic bacterial populations

were in the range of 4.4 -9.6 and 5.1 – 10 log10 CFU/g, respectively, in the surveyed

heaps. Thermophilic bacterial populations in the heaps in active composting at Farm S

were ca. 2.5 log10 CFU/g less than those present in the heap at Farm A. During sampling

dates, temperatures greater than 50oC were commonly detected within the Farm A heap.

This data illustrates that high populations of thermophilic bacteria are correlated with

active composting.

Indicator and presumptive pathogenic bacteria: Detection of coliforms and

presumptive pathogens in surveyed heaps during active and cured composting are

presented in Tables 3a and 3b, respectively. Coliforms were detected in 100% (6 of 6),

83% (5 of 6), 86% (6 of 7), and 100% (14 of 14) of the surface samples from the active

compost heaps of Farms A, G, M, and S, respectively (Table 3a). Inside the compost

heaps on farms A, G, and M, coliforms were detected in ca. 22 – 71% of the internal

samples. In contrast, at least 93% of all internal samples contained coliforms in the heaps

of Farm S. In cured compost heaps on Farms A, G, and M 100% of all surface samples

contained coliforms and up to 83% of all internal samples contained coliforms (Table

3b).

In all heaps of Farms A, G and M, presumptive Salmonella was not detected in

any of the internal samples of the heaps except for one sample in the heap of Farm M

43
(Tables 3a and 3b). E. coli, coliforms and presumptive Salmonella spp. were detected in

100% of the surface samples in the heaps of Farm S, as compared with ca. 71% of the

internal samples (Table 3a). Presumptive Listeria was detected in ca. 31% of all surface

and interior samples of compost heaps in active composting, but were only detected on

the surface of the cured compost heaps. Through further analysis on Chromagar®, no

Listeria isolates collected exhibited growth typical for L. monocytogenes.

Abiotic and biotic analysis of finished compost

In the finished composts, pHs ranged from mildly acidic to mildly alkaline,

depending on the farm and locations of the compost heaps (Table 4). Sixty-two (62)

percent of all samples contained enumerable coliforms. E. coli was only detected in two

samples, both from Farm S. Farm M was the only farm surveyed whose finished

compost did not contain either presumptive Salmonella or Listeria at any sampled

location.

Discussion

Poultry wastes, specifically litter, have many agricultural benefits. Poultry litter,

along with other animal manures, contains nitrogen and phosphorus elements important

to crop growth. As a result, application of poultry litter to land is an accepted “best

management practice” routinely performed in agriculture (Chapman, 1996). Poultry litter

is also used as a feed source for ruminant animals. In spite of the agricultural benefits of

poultry litter, there are drawbacks to its use. Spreading untreated poultry litter could

result in the introduction of pathogenic microorganisms Escherichia, Pseudomonas,

44
Salmonella, Staphylococcus (Srivastata et al., 1972), Campylobacter (Montrose et al.,

1985), and Clostridum (Ogonowski et al., 1984) into soil and water where extended

survival is possible. Additionally, feeding untreated poultry wastes to animals could

result in the transfer of pathogens to other food animals. Due to these reasons, it is

important that poultry wastes are treated before use. Guidelines have been proposed to

suggest conditions that should be targeted to ensure that effective composting occurs

(Richard et. al, 1998). Carbon-to-Nitrogen (C:N) ratios of 20:1 – 40:1, and moisture

contents in the range of 40 – 60% are deemed as parameters that are either “acceptable”

or “optimal” for composting. This survey revealed that the C:N ratios for Farm S heaps

were in the range of 10:1 to 16:1, below acceptability standards for composting.

Studies have shown that co-composting poultry wastes with other substrates allow

for increases in temperatures suitable for pathogen inactivation. Atkinson et al. (1996)

composted a poultry litter – pine sawdust mixture (C:N ratio of 25:1) in compost reactors,

and reported that the temperature increased to 55oC after 20 h of composting and

remained at that level for 16 days. When composting liquid poultry manure with barley

wastes, Guerra-Rodríguez at al. (2003) reported that the compost reached and held

temperatures in excess of 60oC for more than 10 days.

Microbial inactivation in poultry litter during on-farm composting

Limiting pathogen introduction into the environment is one of the most important

benefits in composting poultry litter, as it is widely known that these materials harbor

bacteria associated with foodborne diseases. In poultry litter deep stacked in wooden

bins (1.0 × 1.0 × 1.2 m), E. coli, Salmonella, and Shigella were not detected through 2, 2,

45
and 1 days when present in initial populations of 3.45 – 3.54, 2.18 – 2.32, and 1.40 – 1.70

log10 CFU/g, respectively (Kwak et al., 2005). It is generally accepted that elevated

temperatures in composting is the major mechanism resulting in the elimination of

indicator and pathogenic bacteria during composting. In this study, heaps at Farm A

were large in size, and usually held elevated internal temperatures and moisture contents

above 30%. Consequently, frequencies for detecting presumptive salmonellae and

listeriae were low inside of the heaps (Tables3a and 3b). Our study also demonstrated

that there was a positive correlation between heap moisture and elevated internal heap

temperatures. The heap at Farm S which contained an initial moisture content of 37%

(initial mix) reached an internal temperature in excess of 50oC after one week of

composting. Conversely, the compost mix heap at the farm had an initial moisture

content of ca. 21%, and the maximum temperature reached was 38oC. Chaudry et al.

(1998) reported that deepstacking poultry litter at various moisture contents (15, 25 and

35%) resulted in 4 and 3 log10 CFU/g reductions of total and fecal coliforms,

respectively, after one week. These researchers also report that there was a positive

correlation between heap moisture and achieved temperatures.

Microbial inactivation in poultry litter and chicken carcasses during composting

Poultry litter and carcasses are routinely composted together in a farm

environment. Studies have demonstrated that this practice can eliminate pathogenic

bacteria. Blake et al. (1994) performed a field survey where 12 mini-composters were

monitored for the presence of indicator and pathogenic microbes. Throughout the survey,

temperatures in all heaps were above the 55oC threshold and Salmonella, Campylobacter

46
jejuni, and L. monocytogenes were not detected in any of the samples. Lawson and

Keeling (1999) composted hens according to the prescribed USDA method and reported

heap temperatures in excess of 56oC after 3 days of composting. Additionally,

composting was completed in two months, with ambient temperatures ranging from -1 to

17.5oC, resulting in the decomposition of carcass tissues and complete Salmonella

inactivation. In a study of composting hatchery wastes with poultry litter, Das et al.

(2002) reported that 99.9% of E. coli was eliminated and Salmonella was rendered non-

detectable through composting. Those researchers suggest that elevated temperatures in

excess of 60oC were a major factor causing pathogen inactivation.

In our study, Farms G and M that participated in this survey composted poultry

carcasses and poultry litter together. The presence of indicator bacteria and presumptive

pathogens detected in the active compost heap of Farm M may be explained by the

presence of sublethal temperatures in those heaps as detected on the sampling dates. In

contrast, Farm G heaps usually held elevated temperatures inside the compost heaps.

Farm practices influencing pathogen survival in compost

A few studies surveyed the presence of either indicator bacteria or pathogens in

poultry litter compost. Martin and McCann (1998) investigated the presence of indicator

and pathogenic bacteria in poultry litter on Georgia farms. Out of the 86 samples tested,

64 samples were composted for various amounts of time (from ≤ 1 month through ≥ 4

months). Neither E. coli O157:H7 nor Salmonella spp. were detected in any sample

analyzed. Additionally, only 5 out of 86 samples contained quantifiable coliforms,

ranging in populations from 2.0×101 – 8.0×102 CFU/g. Hartel et al. (2000) compared the

populations of coliforms present in interior and exterior locations of deepstacked poultry

47
litter heaps which were deepstacked from 1 to 104 weeks. Coliforms were not detected

from the interior location of any of the deepstacked heaps, but 1 out of 13 heaps

contained coliforms at the exterior location. Our results were in agreement with the

above study, as we observed that the surface of the compost heaps allowed the extended

survival of indicator and pathogenic microorganisms.

Surveys of poultry litter, mentioned above, suggest that the deepstacking of those

materials effectively eliminates indicator and pathogenic bacteria. Recently, Lasardia et

al. (2006) examined several types of composts available for sale on the Greek market for

indicator and pathogenic bacteria. All composts analyzed, including those not derived

from municipal solid wastes and animal manures, contained fecal coliforms in

populations of at least 5×102 CFU/g. Although Salmonella spp. were not detected in any

analyzed samples, 17 and 96% of the samples analyzed were found to contain

Staphylococcus aureus and Clostridium perfringens, respectively. Though that study did

not exclusively focus on the composting of waste materials, it serves as evidence that

compost may not always completely render composts free of coliforms and pathogens

from compost. In the analysis of finished compost produced at the farms in this survey,

data revealed that coliforms, and often presumptive Salmonella, were detected in the

samples, commonly on the surface. This is a potential hazard, as the presence of

Salmonella may result in regrowth of the pathogen if the conditions are suitable.

Furthermore, application of contaminated compost into agricultural land may allow for

the contamination of soil, and more importantly, crops that may be grown in those fields.

During this survey, it was found that compost practices among farms were highly

variable. On Farm G the temperature of the heaps were not monitored, while no moisture

48
was added or adjusted into the system or the ingredients during composting on Farm S.

Though three of the farms indicated that they did monitor temperature, the monitoring

was performed infrequently, and was only measured at the center of the heaps. This

could be misleading, as the data from our survey shows that the temperatures in the

compost heaps differ depending on location within the heap. Additionally, the farms

which added moisture in the compost heaps had no procedures to determine the amount

of moisture added, and if it was acceptable for proper composting to occur. Two farms,

A and S, maintained set schedules for the turning of the composts. Overall, the oxygen

content of the heaps were low. However, these heaps did not contain appropriate

moisture, as it widely known that moisture decreases air spaces within compost heaps. A

lack of oxygen in compost heaps could result in the uneven decomposition of composting

substrates and reduce amount of heat produced, impacting pathogen abatement.

Importantly, both Farms G and M, which composted poultry carcasses, mixed

fresh waste material into compost heaps that had already undergone a heating cycle.

While this may be a common practice, it is unsafe microbiologically. The addition of

new material into compost which has undergone heating allows for the possibility of

pathogens surviving the compost process, as microbial activity will be reduced due to

inadequate C:N ratios and moisture contents during composting Because these materials

are not subjected to the elevated temperatures which occur during active composting, any

pathogens associated with the fresh material may cause repopulation of the compost with

pathogens. Also, there is a risk for any pathogens reintroduced into the heap to become

heat resistant, as sublethal temperatures may cause the induction of heat-shock proteins.

Staging areas and equipment used in housing and maintaining compost should also be

49
cleared of any untreated materials, as the use of contaminated equipment may result in

repopulation of pathogens into compost. Russ and Yanko (1981) suggest that composts

held in the mesophilic range with moisture contents of ca. 20% and C:N ratio greater than

15:1 may allow for Salmonella repopulation.

Other factors influencing pathogen survival in compost

Several environmental factors may contribute to the reduction of pathogens in

compost. Exposure to ultraviolet light has been suggested as a factor (Nicholson et al.,

2005); however, it is not applicable in this study as composting at each farm was

conducted under a roofed area. Temperature on the compost surface can be “ruled out”

as a factor, as Bush et al. (2007) state that temperatures near the outside edges of stacked

poultry litter changes with ambient temperature. Sundberg et al. (2004) have suggested

that low pH of compost may affect the transition into the thermophilic phase of

composting, thereby influencing pathogen reduction; however, the pH values detected in

this survey were not in the range that would delay entrance into the thermophilic phase.

Although desiccation has also been suggested as a condition which would result in

pathogen inactivation (Redlinger et al. 2001), in this study, presumptive pathogenic

bacteria were often detected in the compost locations in which moisture contents were

lowest. This data suggests that lower water activity (aw) may result in prolonged

pathogen survival. A study by Himathongkham (1999) reveals that salmonellae may

survive for ca. 30 days at aw of 0.5 and 0.38, and nearly 100 days at an aw of 0.07.

Detection of presumptive pathogens (Salmonella and Listeria) also occurred

during the survey in internal samples of the heaps; however, detection frequencies were

50
not comparable to that of the surface samples. An explanation for the lower detection

rates found in internals samples could be the combination of the temperature along with

the production of ammonia during composting. While ammonia release was not

investigated in this study, production of this gas may have had a bactericidal effect on the

pathogens investigated. Elwell et al. (1998) reported that due to low C:N ratios, the

composting of unamended chicken litter results in the release of large amounts of

ammonia. Additionally, Turnbull and Snoeyenbos (1973) indicated that elevated levels

of ammonia have a “salmonellacidal” effect. Importantly, it has been demonstrated that

decreased water activity and gassing with 1% ammonia resulted in reductions of 2.5, 3,

and 4 log10 CFU/g for L. monocytogenes, S. Typhimurium, and E. coli O157:H7,

respectively, in chicken litter.

While it is likely that elevated temperatures and ammonia release could be the

factors causing pathogen destruction inside of the compost heaps, confounding factors

such as low oxygen content and microbial competition may have contributed to pathogen

inactivation. The persistence of presumptive pathogens may be explained by the

expression of heat shock proteins due to exposures to sublethal temperatures during the

mesophilic composting phase (Brinton and Droffner, 1994).

Conclusions

Results from this study revealed that temperatures of compost heaps on poultry

farms are commonly below levels recommended for pathogen destruction. Additionally,

factors which may influence the progression of composting, such as moisture content,

and C:N ratio, usually do not fall within the acceptable ranges for composting.

51
Importantly, indicator and pathogenic bacteria usually persist on the compost surface.

The addition of new materials into treated compost may cause inadequate composting

and permit the reintroduction of pathogens into composts.

The survey of composting on poultry farms revealed that the substrates and

methods implemented in composting vary. When composting poultry wastes,

carbonaceous materials and water should be added to increase the C:N ratio and moisture

content, respectively, to ensure that conditions are acceptable for microbial activity.

Composts should be regularly aerated in order to decrease the likelihood of both uneven

decomposition of organic matter and pathogen destruction. Care should be taken that

facilities used to house and maintain the compost are free of any materials that may re-

introduce pathogens into the compost. Additionally, the addition of fresh, untreated

materials into previously heated compost should be avoided.

Farmers should be educated so that they know under what conditions composting

should be performed in order to produce safe compost. Also, practical measurements

should be developed which make composting easier for farmers to correctly implement

composting guidelines on their farms. Importantly, strategies should be implemented to

reduce the survival of potentially pathogenic bacteria on the compost surface.

Acknowledgements

We would like to thank all participating farms in this survey. This research was

funded by a grant from the USDA-CSREES.

52
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55
 Figure Legend

Figure 2.1: Temperature profiles of the composting heaps on Farm S. Values are the

averages of results of two heaps at each sampling location. Results from the initial mix

heap were obtained at the surface (×) and at two locations within the heaps (60 cm, ◊; 90

cm, □). Results obtained from the compost mix heap were obtained at the surface (○) and

at two locations within the heaps (60 cm, ♦; 90 cm, ■).

Figure 2.2: Impact of adding new material on coliform populations in the initial compost

heap (Farm G). The arrow (↓) indicates that new materials were added into the heaps

between the sampling periods. The darkened circle (●) indicates an inversion of the heap

one day before sampling.

Figure 2.3: Impact of adding of new material on coliform populations in Bin #1 compost

heap (Farm M). The arrow (↓) indicates that new materials were added into the heaps

between the sampling periods.

56
 Table 2.1: Compost heap setup and composting practices on South Carolina poultry farms

Farm Composting Compost dimensions Compost Carbon:Nitrogen Turning Temperature Length of Use of
(# of heaps) method (L×W×H in meters) composition (C:N) ratio frequency monitoring compostingd compost

A (2) Windrow 24.0 × 10.0 × 12.0 Chicken litter and N/Aa Weekly Yes 2 mon Sold to
“pine fines” consumers

G (3) Static Heap 3.6 × 3.5 × 1.6 Chicken litter, N/A Only when No 6 – 8 wk Land
chicken mortalities, moved to new Application
and wood chips bin

M (2) Bin 2.0 × 2.0 × 2.0 Chicken litter, N/A Only when Yes 4 – 6 mon Sold to
chicken mortalities, moved to new consumers
and wood shavings bin

S (2) Static Heap 2.0 × 1.0 × 0.90 Chicken litter and 16:1b; 9.7:1c Weekly Yes 2 – 3 mon Land
“pine fines” Application

a
57

: Initial compost sample was not examined for this parameter.

b
: C:N ratio of the “Initial Mix” compost heap located at Farm S.
c
: C:N ratio of the “Compost Mix” compost heap located at Farm S .
d
: Denotes the length of time farm managers usually compost waste material on-farm.

57
 Table 2.2: Summary of surveyed compost heaps during active composting

Total bacterial counts (Log CFU/g)

Composting Heap Farm Temp. range (oC) Oxy. content (%) pH range Moisture Mesophilic Thermophilic (55oC)b Coliform counts
type (inside heaps) (inside heaps) content (%) (30oC)a (log cfu/g)

Active Initial A 28 – 64 0 – 2c 5.4 – 7.7 10.6 – 44.6 4.5 – 9.6 6.3 – 10.0 NDh – 5.9

Initial G 36 – 55 0 – 8d 8.3 – 9.0 7.2 – 55.1 4.4 – 8.6 5.1 – 8.3 ND – 4.3

Bin #1 M 14 – 51 6 – 18e 6.0 – 8.6 5.7 – 67.4 5.5 – 8.6 6.2 – 8.5 ND – 5.6

Bothi S 13 – 52 1 – 18f 6.7 – 8.6 11.2 – 37.6 6.1 – 7.6 5.9 – 7.4 ND – 6.0

Cured Premix A 36 – 60 1 – 3c 4.8 – 8.4 8.2 – 52.6 5.3 – 9.3 6.0 – 9.5 ND – 6.0

Cured G 42 – 56 1 – 5d 8.0 – 8.6 8.1 – 37.8 5.5 – 8.3 5.5 – 7.5 ND – 3.9
58

Bin #2 M 5 – 26 N/Ag 6.4 – 8.9 10.9 – 52.9 7.1 – 8.6 6.7 – 7.9 ND – 3.5

a
: Temperature value given represents the incubation temperature to enumerate mesophiles.
b
: Temperature value given represents the incubation temperature to enumerate thermophiles.
c
: Measurement began 130 days after the onset of composting.
d
: Measurement began 43 days after the onset of composting.
e
: Measurement began 203 days after the onset of composting.
f
: Measurement began at day 0 of composting.
g
: N/A: Not applicable, measurement was not taken
h
: ND: Not detected, detection limit <1 log CFU/g.
i: Data from the Initial and Compost Mix heaps were combined.

58
 Table 2.3: Prevalence of coliforms, E. coli, and presumptive pathogens in compost undergoing
first heating phase

Farm Heap Location (in Coliforms E. coli Presumptive Presumptive

cm)a Salmonella spp. Listeria spp.

A (n=18)b Initial <5 (n=6)c 6 2 6 1

60 (n=6) 3 0 0 1
90 (n=6) 4 0 0 1
G (n=24) Initial <5 (n=6) 5 1 4 1
60 (n=9) 2 0 0 4
90 (n=9) 2 1 0 1
M (n=21) Bin #1 <5 (n=7) 6 2 3 1
40 (n=7) 5 1 0 1
60 (n=7) 4 0 1 0
59

d
S (n=42) Both heaps <5 (n=14) 14 14 14 9
60 (n=14) 13 10 13 7
30 (n=14) 14 10 14 6

a
: Sampling locations of compost heaps as described in Materials and Methods.
b
: Total number of samples taken from a particular farm over the course of the survey.
c
: Total number of samples taken from each location at a particular farm over the course of the survey.
d
: Data from the Initial and Compost Mix heaps were combined, as both contained fresh poultry litter when composting
began.

59
 Table 2.4: Prevalence of coliforms, E. coli, and presumptive pathogens in compost
undergoing second heating phase

Location Presumptive Presumptive

Farm Heap Coliforms E. coli
(in cm)a Salmonella spp. Listeria spp.
A (n=18)b Premix <5 (n=6)c 6 2 6 1
60 (n=6) 5 0 0 0
90 (n=6) 3 0 0 0
G (n=9) Cured <5 (n=3) 3 0 1 1
60 (n=3) 1 0 0 0
90 (n=3) 0 0 0 0
M (n=9) Bin #2 <5 (n=3) 3 0 0 0
40 (n=3) 2 0 0 0
60 (n=3) 2 0 0 0
60

a
: Sampling locations of compost heaps as described in Materials and Methods.
b
: Total number of samples taken from a particular farm over the course of the survey.
c
: Total number of samples taken from each location at a particular farm over the course of the survey.

60
 Table 2.5: Abiotic and biotic analysis of finished compost samples

Farm Heap Days into Location pHb MC (%)c Temp. Thermo. Meso. Coliforms E. coli Salmonellah Listeriai
composting (cm)a (oC) (log CFU/gd) (log CFU/ge) (log CFU/g)

A Premix 193 <5 6.65±0.08 26.41±0.69 14±0.00 9.31±0.02 9.34±0.16 - - + -

60 5.60±0.03 42.57±0.45 48±0.00 7.56±0.03 6.82±0.67 2.48±0.67 - - -
90 6.15±0.06 42.48±3.09 36±1.41 7.52±0.22 6.07±0.27 2.98±0.46 - - -
Premix 193 <5 7.27±0.01 15.14±0.88 26±0.00 8.53±0.11 8.04±0.08 3.80±0.14 - + -
60 7.18±0.00 22.86±0.91 53±1.41 6.60±0.15 5.39±0.55 - - - -
90 8.40±0.08 52.59±2.52 46±1.41 7.48±0.11 5.34±0.34 - - - -
G Cured 147 <5 7.70±0.30 8.17±1.30 19±0.00 7.36±0.15 7.76±0.03 2.72±0.45 - + -
60 8.31±0.08 27.73±0.64 53±0.71 5.71±0.05 5.54±0.12 - - - -
90 8.02±0.15 25.51±0.72 39.5±0.71 5.52±0.08 5.56±0.04 - - - -
M Bin #1 342 <5 7.53±0.04 18.80±0.40 27±0.00 6.60±0.02 6.71±0.06 - - - -
40 7.90±0.02 34.58±2.52 29±0.00 6.52±0.13 6.52±0.14 2.78±0.25 - - -
60 8.11±0.04 40.49±2.06 32±0.00 6.79±0.10 7.33±0.17 4.00±0.09 - - -
61

Bin #2 342 <5 6.67±0.30 10.93±0.93 -1±0.00 7.47±0.01 7.28±0.08 3.05±0.21 - - -

40 6.60±0.49 52.88±2.97 5±0.00 7.19±0.17 6.69±0.07 - - - -
60 6.67±0.16 48.79±2.33 6+0.00 7.18±0.05 6.65±0.01 - - - -

S Compost Mix 60 <5 8.27±0.06 14.77±2.03 3±0.00 6.23±0.01 6.84±0.09 2.86±0.04 1.40±0.00 + -
60 8.42±0.00 19.30±0.02 13±0.00 6.56±0.06 6.82±0.12 1.69±0.05 - - -
30 8.55±0.01 20.48±0.27 13±1.41 6.49±0.01 6.61±0.06 1.95±0.01 - - -
Initial Mix 60 <5 8.46±0.09 14.76±1.03 3±0.00 6.24±0.14 6.93±0.21 3.70±0.02 2.02±0.05 + +
60 8.38±0.01 20.34±0.90 13.5±0.71 6.35±0.04 6.60±0.03 1.62±0.03 - + +

30 8.57±0.06 22.31±0.05 14.5±0.71 6.38±0.07 6.79±0.02 2.05±0.05 - + +

a
: Locations of the samples of the composting heaps as described in the Material and Methods.
b,c
: Values represent the mean of the data +/- the standard deviation.
d,e
: Values represent the mean of the log10 counts of the thermophilic and mesophilic bacteria, respectively, +/- the
standard deviation.
f,g
: + and -, represents that the parameter has been detected or not detected in the sample, respectively.
h,i: Indicates the presence of presumptive Salmonella and Listeria isolates, respectively.

61
 Figure 2.1.

60

50

40
Temperature (oC)

30

20

10

0
0 10 20 30 40 50 60 70
Time (Day)

62
Figure 2.2.

4.50
A
4.15
Surface 60 cm 90 cm
4.00 A

3.69

3.44
3.50

B
Coliform counts (log10 cfu/g)

3.00
Ba 2.76 2.81

2.57
2.50
B

2.00
2.00 \
1.76

1.50

C
1.00
0.80

0.50

0.00
15 43 77 112 147
Time (days after onset of composting)

a
:Means with the same letter at each sampling date are not significantly
different (P>0.05).

63
Figure 2.3

6.00
A
5.59
5.50 Surface 40 cm 60 cm

5.00
A
4.50
4.50 A

B 4.00
Coliform counts (log10 cfu/g)

4.00
3.65

3.50 A
B
A 3.01 A
3.00 Aa A A 2.78
2.61 2.54
2.49 2.48 2.44
2.50 B B A
2.07
1.96 1.94
2.00
B

1.50
1.24

1.00

0.50

0.00
54 113 160 203 229 264 342
Time (days after onset of composting)

a
:Means with the same letter at each sampling date are not significantly
different (P>0.05).

64
 CHAPTER THREE

FATE OF Escherichia coli O157:H7 DURING ON-FARM DAIRY

MANURE-BASED COMPOSTING

Abstract

Studies were conducted to determine the fate of Escherichia coli O157:H7 in

dairy manure-based compost in a field setting. Two trials were performed involving

duplicate compost heaps constructed on an outdoor, fenced site. The compost heaps, ca.

1.3 m3, were comprised of dairy manure, old hay, feed waste, a sawdust-calf feces

mixture, and fresh hay. Samples of the composting mixture were inoculated with stx-

negative E. coli O157:H7 B6914 at initial cell numbers of 107 and 105 CFU/g for Trial 1

and Trial 2, respectively. Individual sample bags were placed on the surface and at three

locations (top, center, and bottom) within each heap. Though compost heaps achieved

temperatures of 50oC or above at all internal locations for at least 7 days, temperature

stratification was observed. In Trial 1, E. coli O157:H7 was detected by enrichment

through 14 days within the heaps. When inoculated with 105 CFU/g in Trial 2, E. coli

O157:H7 was detected only through days 2, 2, and 5 at the top, center, and bottom

locations, respectively. For both trials, the pathogen survived at the heap's surface for up

to 4 months. The indicator microorganism, E. coli, was inactivated at a rate similar to

that of E. coli O157:H7. Results indicate that composting, with periodic heap turning,

can be a practical approach to inactivating E. coli O157:H7 in cattle wastes on the farm.

65
Our data also suggests that failure to maintain heaps through turning may allow E. coli

O157:H7 survival for months at the compost surface.

Introduction

Cattle are a primary reservoir of Escherichia coli O157:H7 which shed the

pathogen into the environment through their fecal matter. Application of animal waste to

agricultural land as fertilizer or as soil conditioner is generally accepted as a practical

waste management practice in the U.S. (CAST, 1996). However, application of manure

to the land may introduce the pathogen into the soil that supports the growth of food

crops such as vegetables. The application of manure and manure slurries to soil provides

an opportunity for contamination of produce, drinking water, and irrigation water with

the pathogen (Ogden et al., 2001; Islam et al., 2004; Islam et al., 2005; Mukherjee et al.,

2005). Importantly, studies have revealed that E. coli O157:H7 can survive for extended

periods of time in manure (Wang et al., 1996; Himathongkham et al., 1999), manure

heaps (Kudva et al., 1998), and in manure-amended soil (Ogden et al., 2001; Jiang et al.,

2003; Avery et al., 2004; Nicholson et al., 2005).

Some foodborne disease outbreaks have been associated with manure

contamination of fresh produce with E. coli O157:H7 (Cieslak et al., 1993; Chapman et

al., 1997). For example, an outbreak of E. coli O157:H7 infection among members of

four families was associated with potatoes grown in soil fertilized with cattle manure on

the family farm (Chapman et al., 1997). In another instance, a woman acquired E. coli

O157:H7 infection from eating her garden-manured vegetables that were inadequately

washed (Cieslak et al., 1993). Most recently, a large E. coli O157:H7 outbreak

66
associated with bagged baby spinach was linked to cattle feces present in a field of one of

four California ranches implicated in the outbreak (CDC, 2006; FDA, 2006).

Composting is used often on farms to manage large amounts of animal wastes.

When properly performed, composting is a practical method that can effectively kill

pathogens in manure, as well as weed seeds, viruses, and nematodes (Misra et al., 2003).

While some studies suggest that ammonia gas (Nicholson et al., 2005), desiccation

(Redlinger et al., 2001; Nicholson et al., 2005), and microbial antagonism (Ichida et al.,

2001) are factors that contribute to pathogen reduction during composting, the most

influential mechanism is likely temperature elevation caused by metabolically active

microorganisms during composting. Several studies investigating the survival of E. coli

O157:H7 in compost have been performed in laboratory-scale bioreactors (Lung et al.,

2001; Jiang et al., 2002; Hess et al., 2004). Results from these studies further confirmed

that the increased temperatures were the predominant mechanism affecting the

inactivation of E. coli O157:H7 and E. coli, and coliforms.

The objective of this study was to determine if the composting of dairy manure

performed in a field setting under uncontrolled environmental conditions would

effectively eliminate E. coli O157:H7 and indicator bacteria, E. coli and coliforms,

throughout compost heaps.

Materials and Methods

Preparation of compost heaps: Two composting trials were conducted both in

2005; one during the summer and the other in the fall. For each trial, a sawdust-dairy

manure mixture, fresh hay, waste cattle feed and old hay were mixed thoroughly with a

67
front-end loader at a ratio of 28:16:8:1, respectively, on a weight basis. All materials

used in the composting trials were obtained from a single dairy farm and were

accumulated no more than 2 weeks before the start of composting. The dairy manure

used was collected daily from a herd of ca. 125 lactating cows and stored with minimal

movement to prevent aeration, which could have potentially caused heating of the

materials. None of the materials collected was subjected to any treatment prior to

composting. The materials were thoroughly mixed, and split into two separate heaps

with the aid of a front-end loader. Each heap was conical in shape and approximately 1.2

m in height by 2 m in width. During composting, the two heaps were on a 25-m by 16-m

concrete slab surrounded by a gated fence and were not covered or protected from

environmental conditions.

Preparation of bacterial cultures: An avirulent green fluorescent protein (GFP)

- labeled E. coli O157:H7, strain B6914, provided by Dr. Pina Fratamico at the United

States Department of Agriculture, Agricultural Research Service – Eastern Regional

Research Center, was used for the study. A frozen stock culture of E. coli O157:H7 was

thawed and streaked on tryptic soy agar (TSA; Becton Dickinson, Sparks, Md.)

containing 100 μg ampicillin/ml (Sigma Chemical Co., St. Louis, Mo.) (TSA-A), and

incubated at 37oC for 24 h. After two transfers in tryptic soy broth (TSB; Becton

Dickinson) containing 100 μg ampicillin/ml (TSB-A), one ml of the TSB-A culture was

inoculated into 500 ml of TSB-A, and incubated with agitation (125 rpm) for 18 to 24 h.

The bacterial culture was sedimented by centrifugation at 5,000 × g for 15 min at 4oC,

and twice washed with sterile 0.85% saline. The optical density of the bacterial

68
suspension was adjusted to ca. 0.5 at 600 nm to obtain cell numbers of ca. 5 x 108

CFU/ml. Cells were enumerated by plating serial dilutions of the inoculum on TSA-A.

Sample preparation and placement: For both composting trials, a small heap

(ca. 30,000 g) of compost was set aside and underwent extensive mixing with a shovel.

Ten kg of this compost mixture was weighed, and inoculated with 10 ml of a fresh

culture of the GFP-labeled E. coli O157:H7 using a mister with a sterile spray nozzle,

followed by rinsing with 6 ml of sterile deionized water. The compost was continually

mixed by hand, wearing sterile gloves for approximately 10 minutes after inoculation to

distribute the inoculum. Compost ingredients of Trial 1 were inoculated with ca. 107 CFU

E. coli O157:H7/g, whereas those of Trial 2 were inoculated with ca. 105 CFU/g.

Approximately 30-g portions of the inoculated compost samples were inserted

into Tyvek® self-seal pouches (8.89 cm x 13.33 cm, DuPont, Wilmington, Del.) with

sterile spoons. Tyvek® is a chemically inert material made of spunbounded olefin and

mylar that provides sterile packaging protection and is breathable to allow the exchange

of oxygen and water. It does not rot or mildew. These pouches maintained environmental

conditions that were similar to the environment inside the heaps and enabled control of

their position inside the heaps. Compost samples were placed inside the composting

heaps at three locations of 30, 50, and 70 cm depths measured from the base of the

concrete slab (Fig. 1). Duplicate sample bags were placed at each location inside each

heap. All sample bags were color-coded to enable identification of the samples, as

settling of the compost could cause some movement of the samples. Surface samples (ca.

35 g) were positioned by placing the inoculated compost inside a sterile weighing dish

69
(14 cm x 14 cm x 5 cm), and sample dishes were secured to the top of the heaps with thin

strings.

Temperature and oxygen measurements: An OT-21 oxygen and temperature

sensor (Demista Instruments, Arlington Heights, Il.) was used to record the temperature

and oxygen concentrations at three locations inside each heap. Both temperature and

oxygen concentrations were measured daily for up to 14 days for Trial 1 and 21 days for

Trial 2. Thereafter, temperature and oxygen concentrations were monitored at 30, 60,

and 120 days for both trials.

Sample collection and heap maintenance: Samples were obtained at 0, 3, 7, 14,

21, 30, 60, and 120 days of composting for Trial 1 and at 0, 1, 2, 3, 5, 7, 14, 21, 30, 60,

and 120 days for Trial 2. At each sampling, the temperature of the compost was

determined at each specified location before any internal samples were removed from the

compost heaps. Once the collection of data was completed, the compost heaps were

opened with a shovel and the samples were collected aseptically. All tools and containers

used for sampling were sanitized with Enviroquant sanitizer (Vestal Laboratories, St.

Louis, Mo.), and wiped dry with sterile paper towels.

The compost heaps were turned and mixed well mechanically using a front-end

loader, and the remaining sample bags were placed back to the previous locations as soon

as the heap was turned and reconstructed manually to approximately the same height as

achieved at day 0. For Trial 2, on sampling days 1, 2, and 5, the heaps were not turned

but the sample bags attached to a long string were pulled out of the heaps manually. All

samples were analyzed in the laboratory within 4 h of sampling.

70
 Microbiological analysis of compost samples: Ten g of sample was added to 90

ml of Universal pre-enrichment broth (UPB; Becton Dickinson) in a stomacher bag and

macerated using a Stomacher® 400 Circulator (Seward Ltd., West Sussex, UK) for 1

min. The mixture was then serially diluted (1:10) with sterile 0.85% NaCl solution, and

plated on TSA in duplicate using an Autoplate® 4000 spiral plater (Spiral Biotech Inc.,

Bethesda, Md.). The plates were incubated at 30oC and 55oC for 24 h, for enumerating

mesophilic and thermophilic bacteria, respectively. The remaining portions of the

samples in UPB were incubated at 37oC for 24 h for pathogen detection.

TSA-A was used for enumerating the GFP-labeled E. coli O157:H7. The plates

were incubated at 37oC for 24 h, and then examined under a UV light using a Gel Doc

2000 imager (Bio-Rad Laboratories, Inc. Hercules, Ca.) for typical green fluorescent

colonies. Selected green fluorescent colonies were further confirmed using the E. coli

O157 latex test kit (Oxoid Ltd., Basingstoke, Hants, UK). In addition to direct plating,

the compost samples were enriched in UPB for 24 h, and then selectively enriched in 9

ml of TSB-A at 37°C for 24 h. The selective enrichment broth (500 µl) was mixed with

10 µl of anti-E. coli O157 Dynabeads® (Dynal Biotech, ASA, Oslo, Norway) according

to the manufacturer’s instructions. The anti-E. coli O157 Dynabeads® were washed twice

with the buffer, and the final suspension of Dynabeads® was plated on TSA-A through

quadrant streaking. Following incubation at 37oC for 24 h, the colonies were observed

under UV light for green fluorescence, and confirmed using the E. coli O157 latex test kit

as described above.

Both E. coli and coliforms were enumerated using 3M E. coli/coliform Petrifilm™

(3M Microbiology Products, St. Paul, Mn.), with 1-ml dilutions plated in duplicate and

71
incubated at 37oC for 24 h. Blue colonies surrounded with gas bubbles on Petrifilm™

were counted as E. coli, whereas all colonies producing gas were considered as coliforms.

When E. coli/coliform counts were not detectable on Petrifilm™, the compost samples

were analyzed using the 3-tube most probable number (MPN) method as specified in the

Food and Drug Administration Bacteriological Analysis Manual (FDA-BAM) (FDA,

2002). MPN tubes exhibiting the presence of gas were streaked on Levine Eosin

Methylene Blue (L-EMB; Becton Dickinson) agar to determine if the samples contained

E. coli.

Moisture content, pH, and C:N ratio determinations. Approximately 1 g of

each sample was weighed, and analyzed for both moisture content and pH determinations

as described previously (Jiang et al., 2002). About 100 g of initial compost mixture in

duplicate were sent to the Agricultural Service Lab at Clemson University for C:N ratio

testing.

Statistical analysis: Bacterial count data were converted to log10 CFU/g for

statistical analysis. Specific comparisons of plate counts among different locations in the

compost heaps at any date, and in some cases over all dates, were accomplished with

Fisher's least significant difference (LSD) test. Using the GLM procedure, an analysis of

covariance was performed comparing the populations of commensal E. coli to those of E.

coli O157:H7 at each sampling location over the duration of the study to determine if

correlations between those two counts exist. For composting temperature data, an

analysis of variance (ANOVA) for a completely randomized design with repeated

measures across dates was conducted to determine if general differences existed among

72
different locations. All calculations were performed using either the GLM or the MIXED

procedure of the Statistical Analysis System (SAS 2001, Cary, Nc.).

Results

Outdoor compost heap construction: Two trials were conducted in upstate

South Carolina in 2005, with Trial 1 initiated in the summer and Trial 2 in the fall. In

each trial, two compost heaps were constructed, and duplicate sample bags were placed

in each heap at each location for selected sampling intervals. A summary of composting

ingredients and results of analyses are shown in Table 1. Both the carbon to nitrogen

ratio and the moisture contents of the compost heaps were in the acceptable ranges for

composting.

Temperature profiles: Self-heating inside the compost heaps occurred rapidly

soon after all compost ingredients were mixed. Figures 2a and 2b reveal the temperature

stratification present in the compost heaps of two trials. Temperatures at all locations

inside heaps were significantly (p<0.05) different for Trial 1, whereas the temperatures at

only the top and center locations were significantly (p= 0.2526) different from the bottom

location in Trial 2. Overall, the composting temperatures from the highest to lowest at

those locations were in the following order: top>center>bottom>surface. While

temperature stratification was present throughout the heaps in active composting,

temperatures were elevated above 50oC during thermophilic composting at all locations

in the heaps for at least 7 days in Trial 1, and 14 days in Trial 2. The temperature at the

top location for all heaps was above 50oC for at least 30 days, and above 55oC between

14 and 21 days. The maximum temperature achieved occurred at the top location of the

73
heaps in both trials, with 65oC at day 6 and 62oC at day 11, in Trials 1 and 2,

respectively. At day 21, in Trial 1, temperatures gradually declined, whereas

temperatures fell sharply in Trial 2. This was due to the lower ambient temperature in

Trial 2 which concluded in the winter.

pH and moisture content determinations: The changes of pH values and

moisture content in the compost through 120 days of composting for Trial 1 are shown in

Table 2. The results for Trial 2 were very similar to those for Trial 1 (data not shown).

In both trials, the compost mixture was mildly alkaline at day 0. Through day 7,

however, the pH in the compost decreased slightly to neutral within the heaps.

Thereafter, internal compost samples returned to mildly alkaline levels, reaching a

maximum of 10.01 at day 21 in Trial 1 and 9.43 in Trial 2, both at the bottom location.

Overall, pH values of samples inside compost heaps were not significantly (p>0.05)

different during most sampling days for both trials. For the surface samples in both trials,

the pH decreased gradually from mildly alkaline to near neutral by the end of the

composting trials.

Throughout most of the sampling intervals in both trials, the moisture contents of

the internal samples changed very little. In Trial 1, moisture contents were not

significantly (p>0.05) different inside the heaps, except for days 14, 21 and 60 of

composting. However, surface samples at most sampling days contained significantly

(p<0.05) less moisture than the internal samples.

Soon after turning the compost heaps, the oxygen concentrations inside the heaps

decreased rapidly, to 1 to 5 % depending on location (data not shown).

74
 Survival of E. coli/coliform populations during composting: For Trial 1, both

coliforms and E. coli cell numbers at the top and center locations of the compost heaps

decreased ca. 4.9 and 4.6 log10 CFU/g within 3 days, respectively, and to non-detectable

levels by direct plating at 7 days and beyond (Table 3). However, both E. coli and

coliforms were positive after enrichment culture for up to 14 days of composting. Both

E. coli and coliforms were inactivated slowly at the bottom of the compost heaps, and

detectable by direct plating through 3 and 7 days, respectively. Both E. coli and

coliforms were inactivated more rapidly in Trial 2 than in Trial 1 (Table 4). The

populations of indicator bacteria decreased to undetectable levels by direct plating at 2

days of composting at top and center locations, and were eliminated after 5 days at the

bottom location (Table 4). By day 7, all internal samples were negative for both E. coli

and coliforms even after enrichment culture. In surface samples of Trial 1, both E. coli

and coliforms were detected by direct plating for at least 14 days and 120 days of

composting, respectively. E. coli were positive by enrichment culture in surface samples

throughout 120 days of composting. In contrast, both indicator bacteria were inactivated

more slowly in surface samples of Trial 2. Overall, the E. coli/coliform cell numbers

were significantly (p<0.05) greater at surface and bottom locations than those at top or

center locations up to 3 days and 5 days of composting for Trials 1 and 2, respectively.

Survival of E. coli O157:H7 during composting: Two different inoculation

levels of E. coli O157:H7 were used. The lower inoculation level, ca. 105 CFU E. coli

O157:H7/g, would likely be found in feces, whereas higher populations of ca. 107 CFU E.

coli O157:H7/g allowed for the determination of pathogen inactivation rates through 5

log CFU/g during composting.

75
 There was a ca. 6 log10 CFU/g reduction of E. coli O157:H7 at 3 days after

composting began for samples obtained from both the top and center locations, as

compared with a 4 log10 CFU/g reduction at the bottom location (Figure 3a). Samples

from inside the heaps were E. coli O157-positive by enrichment culture through 14 days

at all three locations. In contrast, E. coli O157:H7 was enumerated directly from

compost on the surface of heaps for up to 2 weeks, and detected by enrichment culture

for at least 4 months, when the study was terminated. Overall, the surface samples had

significantly (p<0.05) greater E. coli O157:H7 cell numbers from day 3 until the

completion of the study, whereas internal samples were not significantly (p<0.05)

different over the same sampling interval.

Rapid inactivation of E. coli O157:H7 in compost heaps with a lower inoculum is

shown in Fig. 3b. The pathogen was detected using enrichment culture through 2 days of

composting at the top and center of heaps and through 5 days at the bottom. However, E.

coli O157 was detected for at least 30 days by direct plating and for at least 4 months by

enrichment culture in samples from the heap surface. Statistical analysis of pathogen

survival data from Trial 2 revealed that there were no significant (p>0.05) differences in

E. coli O157:H7 cell numbers among the different sampling locations on day 1 of

composting. After 2 days, there were no significant (p>0.05) differences between the

surface and bottom samples, or between the top and center samples. After day 3 of

composting, E. coli O157:H7 cell numbers in the surface samples were significantly

(p<0.05) greater than those from all internal samples.

In both trials, the correlations were positive between cell numbers of both E. coli

O157:H7 and E. coli at each sampling location, indicating that inactivation of E. coli and

76
E. coli O157:H7 populations are correlated. The p-values of the correlation for Trial 1

were 0.5818, 0.7860, 0.7254, and 0.9518 for the surface, top, center and bottom

locations, respectively. In Trial 2, p-values were 0.2565, 0.5880, 0.6410, and 0.6810 for

the aforementioned locations, respectively.

Discussion

Only a few reported studies have addressed the inactivation of E. coli O157:H7 by

composting in a field setting (Larney et al., 2003; Hutchinson et al., 2005; Pourcher et al.,

2005). In those studies, entire compost heaps were inoculated with the pathogen and only

composite samples were collected and analyzed for the presence of the target pathogen.

Considering the highly heterogeneous nature of compost ingredients and the stratification

of temperature that occurs throughout the heaps, the dynamics of E. coli O157:H7

inactivation at different locations of the heaps would be anticipated to be highly variable.

To address this variability, we strategically placed small contained portions of thoroughly

mixed, inoculated compost mixture at different locations of the compost heap. This

method enabled us to easily control the position of compost samples and subsequently

determined how the stratification of temperature, pH, and moisture content of compost at

different locations in heaps affected E. coli O157:H7 inactivation.

USDA prescribes to organic growers that composting operations maintain a

temperature in the range of 55-70oC for a minimum of 3 days for static aerated pile

systems and 15 days with 5 turns for windrow systems (NOSB, 2002). The optimal ratio

of the carbon to nitrogen (C:N) for active composting is 25:1 to 30:1; however, 20:1 to

40:1 is considered an acceptable range for active composting (Richard et al., 1998). The

77
C:N ratio in both of our trials was within the acceptable range, and the temperature within

each heap at the top location was above 55oC for 14 to 21 days. However, the

temperature profiles for both composting trials revealed there was temperature

stratification throughout the heaps. The warmest location within the heap was slightly

above the geometric center, and the coolest site was at the bottom near the concrete pad.

Results of previous studies in bioreactors or compost heaps have revealed temperature

stratification within the compost (Jiang et al., 2002; Hutchinson et al., 2005). Therefore,

the temperature measurement for the compost heaps should be clearly defined in terms of

location.

In addition to temperature, oxygen content, pH value, and moisture content were

determined at each sampling location at each sampling interval, while C:N ratios of the

Day 0 composting materials were determined. Results revealed that within the compost

heaps a microaerophilic condition prevailed. Therefore, additional aeration should be

applied to extend active composting, since only turning a heap, as was done in this study

was not sufficient to maintain highly aerobic conditions. Our survey of several

composting operations on poultry farms revealed generally low oxygen levels (0-6%)

within composting heaps as well (data not shown). During active composting, microbial

activity slightly reduced the pH (Table 2), which is consistent with previous studies

(Sundberg et al., 2004). The moisture content of the internal samples changed very little

in both trials during active composting (thermophilic phase), suggesting that moisture

content had minimal influence on pathogen inactivation. However, the moisture content

of the initial compost mixture may have affected the rapid onset of self-heating at the

beginning of composting, which in turn also affected pathogen inactivation. For these two

78
trials, although the initial compost mixture and C:N ratio were very similar, Trial 1 heaps,

which had a higher initial moisture content due to heavy rain prior to mixing compost

ingredients, began heating more slowly than heaps in Trial 2. Moisture content

fluctuations occurred in compost surface samples in both trials; however, this was not

unexpected because all of the heaps were exposed to the elements of the environment.

Periods of precipitation and dry conditions occurred throughout both trials, and were the

cause of the variation in moisture content of surface samples. It is important that initial

compost parameters fall with the acceptable to optimal ranges for composting to ensure

that conditions for microbial activity are suitable; thereby allowing an the increase in

temperature necessary for pathogen inactivation.

Several studies, using laboratory-scale bioreactors under well-controlled

environmental conditions, have revealed that composting can effectively inactivate E.

coli O157 in manure in times ranging from 72 h to less than 14 days (Lung et al., 2001;

Jiang et al., 2002; Hess et al., 2004). Only a few studies have addressed the inactivation

of E. coli O157:H7 in composted manure heaps under field conditions; however, it was

not indicated whether or not those studies were performed using an optimal C:N ratio and

moisture content of compost materials. Nicholson et al. (2005) determined composting

dairy manure in unturned solid manure heaps reached temperatures greater than 55oC,

and E. coli O157 inoculated at ca. 2.7 – 5.2 log10 CFU/g could not be detected after one

week In our study, the E. coli O157:H7 survived for up to 14 days when the initial cell

numbers were ca.107 CFU/g and composting was performed during the summer, but

became undetectable within 5 days when the inoculation cell numbers were ca. 105

CFU/g and composting was carried out during the fall. The E. coli O157:H7 inactivation

79
rates were also affected by the elevated temperatures and how rapidly the self-heating

occurred within the heaps. At locations in the compost heap where temperatures were

higher, i.e., the top and center, the reduction of E. coli O157:H7 and E. coli/coliform cell

numbers was significantly (p<0.05) more rapid than that at the cooler locations such as

the bottom and on surface (Fig. 3 & Table 3 & 4). Hutchinson et al. (2005) reported

extended survival of E. coli O157 in heaped mixtures of dairy cattle manure and beef

cattle manure for 32 and 93 days, respectively, despite heap temperatures in excess of

50oC within 5 days of the onset of composting. The authors suggest that the pathogen

may have survived by the induction of heat shock proteins during the longer mesophilic

phase. However, it is also possible that detection of the pathogen was due to the

sampling procedure used, as the samples for the study were composites of compost

material throughout the heaps. An uneven inactivation of E. coli O157:H7 at different

locations of the heaps would be expected. Variation in the composting length for E. coli

O157:H7 inactivation from different studies can be explained by many variables that

occur, including the types of raw materials, C:N ratios of compost mix, size of heaps,

ambient temperature, frequency of turning, initial cell numbers of target pathogens, strain

variation, seasons of the year and geographical locations.

Both E. coli and coliforms have been widely used as indicators of fecal

contamination, although coliforms have minimal significance as fecal indicators (Doyle

and Erickson, 2006). In this study, commensal E. coli cell numbers in the top locations

of compost heaps were reduced by ca. 4 and 7 log10 CFU/g in 3 days at >55oC for Trials

1 and 2, respectively, which are rates similar to those observed for E. coli O157:H7 in the

same locations. Statistical analysis of bacterial survival at each sampling location

80
revealed that inactivation of E. coli and E. coli O157:H7 populations correlated. While

complete pathogen and indicator organism inactivation occurred in internal samples

within 2 weeks of composting for both composting trials, coliforms, commensal E. coli,

and E. coli O157:H7 in surface samples survived for up to 4 months. This is in direct

contrast with results of Hutchinson et al. (2005), who reported that the pathogens they

inoculated could not be detected after 8 days in surface samples of the heaps they

monitored, and Nicholson et al. (2005), who reported pathogen survival at the surface

was comparable to survival in the main body of the heap. Although desiccation and UV

exposure could contribute to pathogen reduction in surface samples, the lack of elevated

temperatures was likely the primary factor influencing pathogen inactivation rates in this

study. Greater survival of E. coli O157:H7, E. coli and coliforms in compost surface of

Trial 2 most likely resulted because of the lower ambient temperatures to which the

compost heaps were exposed. While it is unknown how the strain used in our study

differs from other strains of E. coli O157:H7 in terms of desiccation resistance, there is

evidence that the strain of E. coli O157:H7 can be a factor in its persistence in bovine

feces at low moisture contents (Wang et al., 1996). The extended survival of E. coli

O157:H7 on the compost surface is problematic because surviving pathogens can spread

to the surrounding environment such as soil, water, and agricultural crops that are being

harvested for human consumption. Results from a four-month study using the compost

mixture described in this experiment, without any aeration of the heap, showed that the

maximum internal heap temperature was 56oC. Additionally, after the decline in

temperature, regrowth of coliforms in the heaps occurred (data not shown). Hence, it is

important to maintain active composting by frequent turning of heaps to ensure that all

81
composted materials are subjected to elevated temperatures generated during active

composting.

Conclusions

Our results indicate that the compost composition and heap size utilized were

adequate to achieve active composting. The stratification of temperature at different

locations of the heaps affected the rates of E. coli O157:H7 inactivation; however, ca. 7

log CFU E. coli O157:H7/g inside compost heaps could be inactivated within 2 weeks if

active composting occurred during this period of time. Importantly, our results revealed

that both indicator bacteria and E. coli O157:H7 can survive up to 4 months on the

surface of compost heaps and this could serve as a source of pathogen contamination of

the surrounding environment. Finally, the methods used in this study can be used as a

model to evaluate the fate of other pathogenic microorganisms such as Salmonella spp.,

Listeria monocytogenes, viruses, and parasites in compost.

Acknowledgements

We thank Steve Waggoner, Ricky Tingle, and Ronnie Ducworth at LaMaster

Dairy Farm, Clemson University, for assistance in setting up and turning the compost

heaps, and Dr. James Rieck for assistance with statistical analysis. This research was

supported by a USDA-CSREES National Integrated Food Safety Initiative grant (project

2004-51110-02162).

82
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 Figure Legend

Figure 3.1: Temperature profiles of the composting heap for Trial 1. Values are the

averages of results of two heaps at each sampling location. Results were obtained at the

surface (◊) and at three locations within the heaps (Top, □; Center, ∆; and Bottom, ×)

which were 70, 50, and 30 cm, respectively, from the surface of a concrete pad on which

the compost heaps were built. Arrows indicate days of precipitation events.

Figure 3.2: Temperature profiles of the composting heap for Trial 2. Values are the

averages of results of two heaps at each sampling location. Results were obtained at the

surface (◊) and at three locations within the heaps (Top, □; Center, ∆; and Bottom, ×)

which were 70, 50, and 30 cm, respectively, from the surface of a concrete pad on which

the compost heaps were built. Arrows indicate days of precipitation events.

Figure 3.3: Fate of E. coli O157:H7 at different locations within the heaps during

composting of Trial 1 on the Surface (◊) and at three locations within the heaps (Top, □;

Center, ∆; and Bottom, ×).

Figure 3.4: Fate of E. coli O157:H7 at different locations within the heaps during

composting of Trial 2 on the Surface (◊) and at three locations within the heaps (Top, □;

Center, ∆; and Bottom, ×).

86
 Table 3.1 Composition, and biotic and abiotic analyses of experimental compost heaps at the beginning of each trial.

Parameter Description or results

Compost mixture cow manure, calf barn sawdust bedding, feed waste, fresh hayab

Compost dimensions triangular shape, ca. 2 m in base width and ca. 1.1 - 1.2 m in heightab

C:N ratio 22:1ab

pH 7.9a; 8.0b
Moisture content 63.6%a; 56.5%b
Mesophilic bacterial counts 1.3x108 CFU/ga; 2.3x108 CFU/gb
Thermophilic bacterial counts 1.1x108 CFU/ga; 1.4x109 CFU/gb
E. coli O157:H7 absent in 25 gab

Salmonella spp. absent in 25 gab

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a,b
Indicates parameters or results for Trial 1 or Trial 2 of composting, respectively.

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 Table 3.2 pH values and moisture content of Trial 1 compost samples.

pH values or moisture content (%) on composting daya:

Parameter Location in heap 0 3 7 14 21 30 60 120
pH
Surface 7.90±0.06 A 8.58±0.21 B 9.17±0.13 B 8.34±0.27 A 8.45±0.09 A 7.79±0.09 A 7.39±0.04 A 7.14±0.04 A
Top 7.90±0.06 A 7.37±.014 A 7.90±0.38 A 8.53±0.35 AB 9.88±0.45 B 9.94±0.31 B 8.62±0.46 B 8.53±0.69 B
Center 7.90±0.06 A 7.20±0.39 A 8.42±0.17 AB 8.92±0.22 BC 9.98±.012 B 9.80±0.20 B 8.85±0.24 B 8.50±0.46 B
Bottom 7.90±0.06 A 7.63±0.29 A 8.32±0.40 AB 9.25±.015 C 10.01±0.35 B 9.71±0.62 B 9.41±0.16 C 9.07±.039 B
Moisture
content
(%)
Surface 63.6±1.1A 68.9±1.2 B 64.2±6.4 A 4.25±0.52 A 5.30±0.08 A 8.50±0.01 A 3.27±0.45 A 5.77±0.78 A
Top 63.6±1.1A 63.1±0.6 A 60.7±2.2 A 61.1±0.7 C 61.5±2.2 C 56.1±11.7 B 64.3±2.1 C 58.2±2.1 B
Center 63.6±1.1A 63.7±1.5 A 60.7±1.7 A 59.2±2.6 BC 58.3±2.6 BC 57.9±5.9 B 58.9±3.4 C 51.4±15.2 B
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Bottom 63.6±1.1A 63.3±0.8 A 62.8±0.8 A 57.3±3.4 B 48.7±7.7 B 50.1±11.9 B 21.7±4.0 B 48.4±17.1 B

a
Values with different capitalized letters are statistically different (p<0.05) on the sampling day.

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 Table 3.3 Fate of E. coli and coliforms at different locations of heaps during composting (Trial 1).

Avg. log CFU/g at days of compostinga:

Indicator bacteria Location in heap 0 3 7 14 21 30 60 120

Coliforms

Surface 7.20±0.11 Aa 5.37±0.08 B 5.57±0.12 C 4.00±0.01 B 5.40±0.03 3.68±0.19 2.60±0.08 2.10±0.08

Top 7.20±0.11 A 2.52±0.16 A 1.40±0.00A 1.40±0.00 A NDb ND ND ND

Center 7.20±0.11 A 2.26±0.40 A 1.40±0.00 A 1.40±0.00A ND ND ND ND

Bottom 7.20±0.11 A 4.86±0.04 B 2.52±0.16 B 1.40±0.00A ND ND ND ND

E. coli

Surface 6.40±0.26 A 5.12±0.07 B 5.29±0.01 B 4.00±0.01 B 1.40±0.00 1.40±0.00 1.40±0.00 1.40±0.00

Top 6.40±0.26 A 1.96±0.00 A 1.40±0.00 A 1.40±0.00 A ND ND ND ND

Center 6.40±0.26 A 1.79±0.27 A 1.40±0.00 A 1.40±0.00 A ND ND ND ND

Bottom 6.40±0.26 A 4.72±0.08 B 1.40±0.16 A 1.40±0.00A ND ND ND ND

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a
Values with different capitalized letters are statistically different (p<0.05) on the sampling day.
b
ND: not detected.

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 Table 3.4 Fate of E. coli and coliforms at different locations of heaps during composting (Trial 2).

Avg. log CFU/g at days of compostinga:

Indicator Location in
bacteria heap 0 1 2 3 5 7 14 21 30 60 120

Coliforms

Surface 7.12±0.04 Aa 7.10±0.02 C 7.05±0.01 C 7.04±0.01 B 6.75±0.04 B 6.04±0.04 5.52±0.43 5.93±0.06 4.97±0.07 4.61±0.03 3.35±0.41
b
Top 7.12±0.04 A 5.69±0.04 A 1.40±0.00 A ND ND ND ND ND ND ND N/Ac

Center 7.12±0.04 A 5.72±0.07 A 1.40±0.00 A ND ND ND ND ND ND ND N/A

Bottom 7.12±0.04 A 6.00±0.11 B 3.67±2.62 AB 1.40±0.00 A 1.40±0.00 A ND ND ND ND ND N/A

E. coli

Surface 7.05±0.03 A 7.02±0.03 C 7.00±0.01 C 6.94±0.03 B 6.65±0.02 B 5.70±0.06 4.85±0.43 5.59±0.11 4.54±0.02 4.06±0.08 2.71±0.23

Top 7.05±0.03 A 5.15±0.06 A 1.40±0.00 A ND ND ND ND ND ND ND N/A

Center 7.05±0.03 A 5.60±0.08 A 1.40±0.00 A ND ND ND ND ND ND ND N/A

Bottom 7.05±0.03 A 5.91±0.11 B 3.62±2.56 AB 1.40±0.00 A 1.40±0.00 A ND ND ND ND ND N/A

90

a
Values with different capitalized letters are statistically different (p<0.05) on the sampling day.
b
ND: not detected.
c
N/A: not applicable, sample not taken.

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Figure 3.1

80

70

60

50
Temperature (oC)

40

30

20

10

0
0 15 30 45 60 75 90 105 120
Time (Day)

Figure 3.2

70

60

50
Temperature (oC)

40

30

20

10

0
0 15 30 45 60 75 90 105 120
Time (Day)

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Figure 3.3

7
E. coli O157:H7 counts (log CFU/g)

2
Detected by
1 enrichment

0
0 15 30 45 60 75 90 105 120
Time (Day)

Figure 3.4

5
E. coli O157:H7 counts (log CFU/g)

2
Detected by
enrichment
1

0
0 15 30 45 60 75 90 105 120
Time (Day)

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 CHAPTER FOUR

CONCLUSION

In this study, a survey of South Carolina poultry farms and a challenge study

determining the survival of E. coli O157:H7 were conducted. In both studies, extended

survival of indicator and pathogenic microorganisms were detected in the compost

surface for extended periods of time. However, within the body of the heaps, detection of

pathogens was lower. In our survey of poultry farms, our results indicated that

composting illustrated that composting was not performed under recommended

conditions at the surveyed farms. Results from our challenge study revealed that E. coli

O157:H7 may be inactivated within two weeks from the onset of composting within the

body of the compost heap. In conclusion, our results demonstrated that composting

under appropriate conditions results in the inactivation of pathogenic bacteria.

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