Publicly
Armelle Stella JIBIA PALO
Works directed by: Dr. Ryusei
Mr. Seyram SOSSOU
Ms. Lydie YIOUGO
Panel Members:
President: Dr Franck LALANNE
Members and correctors: Dr. Ynoussa MA
Mr. Seyram SOSSOU
Ms. Lydie YIOUGO
THESIS SUBMITTED IN FULFILLMENT FOR THE
MASTER OF WATER AND ENVIRONMENTAL ENGINE
OPTION
INACTIVATION OF PATHOGENS IN HUMAN FECES DURING COMP
ublicly defended on 10 th June 2011 by
Armelle Stella JIBIA PALO
Ryusei ITO
Seyram SOSSOU Research Engineer
Lydie YIOUGO PhD Student
UTER GVEA
Dr Franck LALANNE
Dr. Ynoussa MAÏGA
Mr. Seyram SOSSOU
Ms. Lydie YIOUGO
Promotion
THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF
MASTER OF WATER AND ENVIRONMENTAL ENGINE ERINGPTION: WATER AND SANITATI ON
INACTIVATION OF PATHOGENS IN HUMAN FECES DURING COMP OSTING PROCESS USING SAWDUST
AS MATRIX
Promotion [2010/2011]
PARTIAL DEGREE OF
ERING ON
INACTIVATION OF PATHOGENS IN HUMAN FECES ESS USING SAWDUST
‘‘Inactivation of pathogens in human feces during composting process using sawdust as matrix’’
Armelle Stella JIBIA PALO June 2011 Page i
“Every accomplishment starts with the decision to try”
Anonymous
‘‘Inactivation of pathogens in human feces during composting process using sawdust as matrix’’
Armelle Stella JIBIA PALO June 2011 Page ii
DEDICATION
To my late father JIBIA DANIELJIBIA DANIELJIBIA DANIELJIBIA DANIEL whose
memory is always there: You’re still alive in
my heart and you will be proud of me.
To my beloved and sweet mother JIBIA JIBIA JIBIA JIBIA nnnnéeéeéeée
TCHATCHOUANG Anne SotchéTCHATCHOUANG Anne SotchéTCHATCHOUANG Anne SotchéTCHATCHOUANG Anne Sotché: More than
a mother, a great woman who inspires me.
I dedicate this Master thesis
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ACKNOWLEGMENTS
I don’t know how to express my gratitude to GOD for His ineffable graces and His
benedictions so I will just say, THANK YOU LORD.
This work has been a great challenge to deal with and it would not have been possible without
some help.
So, I would like to thank:
The Deutscher Akademischer Austauch Dienst DAAD for their financial support
during these two years of master degree.
Dr. Ryusei ITO for the theme proposal and for his disponibility and support.
My supervisors Mr. Seyram SOSSOU and Ms. Lydie YIOUGO for their frame,
guidances and their entire disponibility. Through these I thank all the teaching corps of
2iE.
Dr. Mariam SOU/DAKOURE and Mr Boukary SAWADOGO for their help.
The 2iE ‘s Laboratory LEDES ‘’Laboratoire Eau Dépollution Ecosystème et Santé’
and it’s whole staff for their welcome and the support especially Mr. Moustapha
OUEDRAOGO and Mr. Pierre KABORE for their assistance and Mr. Kader
CONGO and Ms. Emeline BITIE for their kind collaboration.
I want to deeply thank the workers of the 2iE’s construction site without whom the project
would have not taken place for their entire and kind participation.
I would like also to express my gratitude to those who have walked with me during those
years:
Especially my mother Mrs. JIBIA Anne Sotché for her indefectible support
My brothers and sisters Nelly, Eric, Alain, Brice, Gaëlle, Danielle for all they do.
The big families JIBIA , SOTCHE and OUAHA for their presence
The families NONO Joseph, BIKE Moïse and WETHE Joseph for their support
All my friends and especially my all days accomplices Ruth NGANLO , Tatiana
TANKEU , Olivier TAPSOBA, Gael NDANGA for their presence and support
My brothers and sisters of’’ Cellule de Prière Evangélique du 2iE’’ and the ’’Bon
Berger Gospel choir’’ for their spiritual and fraternal support.
My classmates and future colleagues: Thank you for those sweet years
And finally, You, who are more than a friend, for your prayers.
Special thanks to those who have helped me and who were not mentioned there
’’To All of you, may Almighty GOD bless you beyond your expectations’’
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ABSTRACT
The inactivation of pathogens in compost made from human feces and mixed with sawdust
was evaluated during 15 days. Sawdust was weighed and put in the composting reactor and
each day a known amount of feces was added and mixed. Total Coliforms, Fecal Coliforms,
Fecal Streptococci and Helminth Eggs were analyzed every three (03) days in compost
samples which having been previously subjected to temperature variation and pH increasing.
Results show that coliforms and fecal streptococci behaved differently with variation of
temperature and pH increasing. In addition, both temperature and pH had a positive influence
on some parasites inactivation since no Helminth eggs were found after fifteen (15) days in
the composting reactor.
Results on temperature and pH monitoring also suggest that pathogens inactivation is
optimum 50°C and pH increase up to 12 contribute to total inactivation of Helminth eggs.
Keywords: Compost, pathogens inactivation, human feces, sawdust, pH, temperature, total
coliforms, fecal coliforms, fecal streptococci, Helminth eggs
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RESUME
L'inactivation des microbes pathogènes dans du compost à base de fèces humains et de la
sciure de bois a été évaluée pendant 15 jours. La sciure de bois a été pesée et mise dans un
réacteur de compostage et chaque jour, une quantité connue de fèces a été ajoutée et mélangée
à la sciure. Les coliformes totaux, les coliformes fécaux, les streptocoques fécaux et les œufs
d'helminthe ont été mesurés dans des échantillons de compost prélevés tous les trois (03)
jours, et ayant été au préalable soumis à une variation de température et une augmentation du
pH.
Les résultats montrent une influence remarquable de la température et du pH sur la charge en
coliformes et les streptocoques fécaux dans le compost. Ces paramètres ont également une
influence positive sur l'inactivation des œufs d'helminthe, ces derniers étant indétectables dans
les échantillons de compost après 15 jours dans le réacteur de compostage.
Les résultats indiquent aussi que la température optimale pour l'inactivation des
microorganismes étudiés est 50 °C et que l'élévation du pH à des valeurs supérieures à 12
favorise également l'inactivation totale des œufs d'helminthe.
Mots-clés: Compost, inactivation des microbes pathogènes, fèces, sciure de bois, pH, température,
coliformes totaux, coliforms fécaux, streptocoques fécaux, Œufs d’Helminthes.
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ABBREVIATIONS AND ACRONYMS
2iE: International Institute for Water and Environmental Engineering
TC: Total Coliforms
FS: Fecal Streptococci
FC: Fecal Coliforms
IM: Inorganic matter
OM: Organic matter
LEDES : Laboratoire Eau Dépollution Ecosystèmes et Santé
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LIST OF CONTENTS
DEDICATION ........................................................................................................................... ii
ACKNOWLEGMENTS ............................................................................................................ iii
ABSTRACT .............................................................................................................................. iv
RESUME .................................................................................................................................... v
ABBREVIATIONS AND ACRONYMS ................................................................................. vi
LIST OF CONTENTS ............................................................................................................. vii
LIST OF TABLES .................................................................................................................... ix
LIST OF FIGURES .................................................................................................................... x
GENERAL INTRODUCTION .................................................................................................. 1
CHAPTER I: LITERATURE REVIEW .................................................................................... 2
1. About composting ........................................................................................................... 2
1.1. Definitions ................................................................................................................ 2
1.2. Composting process: Ins and outs ............................................................................ 2
1.2.1. Composting methods ............................................................................................ 3
1.2.2. Parameters in Composting process ...................................................................... 3
2. Composting toilet ............................................................................................................ 4
2.1. Types of composting toilet ....................................................................................... 4
2.2. Particular case of Bio-toilet ..................................................................................... 5
3. Pathogens in compost ...................................................................................................... 5
3.1. Influence of temperature .......................................................................................... 8
3.2. Influence of pH ........................................................................................................ 8
CHAPTER II: MATERIALS AND METHODS ..................................................................... 10
1. Methodological approach .............................................................................................. 10
2. Experimental site ........................................................................................................... 10
3. Production of compost .................................................................................................. 10
3.1. Construction of pilot scale composting toilet ........................................................ 10
3.2. Matrix: The sawdust .............................................................................................. 13
3.3. Composting process with feces feeding ................................................................. 13
4. Lab-Scale Studies .......................................................................................................... 14
4.1. Compost tests ............................................................................................................. 14
4.2. Physical and chemical Analyses ................................................................................ 15
4.3. Bacteriological Analysis ............................................................................................ 15
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4.4. Parasitological Analyses ............................................................................................ 16
CHAPTER III: RESULTS AND DISCUSSION ..................................................................... 17
1. Results ........................................................................................................................... 17
1.1. Physicals and chemical analyses ............................................................................ 17
1.1.1. Evolution of temperature .................................................................................... 17
1.1.2. Evolution of pH .................................................................................................. 17
1.1.3. Evolution of Electric conductivity ..................................................................... 18
1.1.4. Evolution of moisture content ............................................................................ 19
1.1.5. Evolution of Organic and Inorganic matter ........................................................ 19
1.2. Bacteriological results ............................................................................................ 20
1.3. Parasitological results ............................................................................................ 25
2. Discussion ..................................................................................................................... 27
2.1 Chemical and physical characterization ................................................................. 27
2.2 . Pathogens Inactivation ......................................................................................... 28
CONCLUSION AND RECOMMENDATIONS ..................................................................... 30
BIBLIOGRAPHY .................................................................................................................... 31
APPENDICES .......................................................................................................................... 33
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LIST OF TABLES
Table 1 Pathogens and fecal indicators content in waste water and sludges ............................ 7
Table 2 : Daily weight of feces put in the reactor .................................................................... 14
Table 3 : Initial concentration of microorganisms in CFU compost ........................................ 20
Table 4 : increased pH during sampling ................................................................................... 23
Table 5 : Prevalence of Helminth eggs in compost (n=10) ...................................................... 25
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LIST OF FIGURES
Figure 1: Behaviour of microorganisms opposite to temperature (Mustin, 1987) ..................... 8
Figure 2 Influence of pH of medium on growth’s rate of microorganisms (E.Coli) (Demeyer
et al., 1981) ................................................................................................................................. 9
Figure 3 : Sensitivity of microorganisms due to pH (Mustin, 1987) ......................................... 9
Figure 4 : Model 1 of composting toilet ................................................................................... 11
Figure 5 : Model 2 of composting toilet ................................................................................... 12
Figure 6 : Composting reactor .................................................................................................. 13
Figure 7 : Evolution of temperature during composting .......................................................... 17
Figure 8 : Evolution of pH during composting ........................................................................ 18
Figure 9 : Evolution of Electric conductivity (EC) during composting ................................... 18
Figure 10 : Evolution of moisture content during composting ................................................ 19
Figure 11 : Evolution of Organic matter (OM) during composting ......................................... 19
Figure 12 : Evolution of Inorganic matter during composting ................................................. 20
Figure 13 : Influence on temperature on Total coliforms ........................................................ 21
Figure 14 : Influence of temperature on Fecal coliforms (FC) ................................................ 22
Figure 15 : Influence of temperature on fecal streptococci (FS) .............................................. 22
Figure 16 : Influence of pH on total coliforms (TC) ................................................................ 24
Figure 17 : Influence of pH on fecal coliforms (FC) ............................................................... 24
Figure 18 : Influence of pH on fecal streptococci (FS) ............................................................ 25
Figure 19: Influence of temperature on Ankylostoma duodenanale A.d eggs during
Composting .............................................................................................................................. 26
Figure 20 : Influence of pH on Ankylostoma duodenanale (A.d) during composting ............. 27
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GENERAL INTRODUCTION
For agriculture, people use many types of fertilizers such as chemicals one and also compost
made by raw biodegradable materials. However, the price of chemical fertilizer is getting
more and more expensive. Utilization of human feces seems to be a great alternative and
another way to make compost for plants because most part of the nutrients as nitrogen,
phosphorus, potassium, copper, iron, etc… necessary to the growth of the plants are present in
the feces. Thus, the effective utilization of nutrients is becoming important but the direct
utilization on plants will make bad effect due to easy biodegradation part of organic matter
and also to pathogens, parasite eggs etc.
The composting toilet with sawdust as a bulky matrix also called bio-toilet is a type of toilet
which produces the compost used as fertilizer. This compost should not be noxious for plants
so inactivation of pathogens should be one of its main purposes. Although many studies have
been conducted on the composting toilets, most of them mainly focus on agricultural value of
compost as a fertilizer rather than on microorganisms’ activity in the system (Del Porto and
Steinfield, 2000 in Lopez Zavala et al., 2004).
The monitoring of pathogens is a difficult, lengthy procedure unsuited to routine
application (Burge, 1983 in Periera-Neto et al., 1986). Commonly, inactivation rate of
pathogens in composting process is affected by several factors such as temperature, pH, and
moisture content. The use of indicator organisms whose removal characteristics are similar to
those pathogens is a shorter, more convenient technique which is the reason for their use in
this study (Periera-Neto et al., 1986). Knowledge of how we can get a sufficient level of
pathogens’ risk minimization is essential to get better working conditions of the composting
toilet.
The aim of this study is to analyze the effect of pH and temperature on inactivation rate of
indicator bacteria and helminths eggs during stabilization of compost in a bio-toilet reactor.
This must lead to:
� Characterize chemical and microbial aspects of compost,
� Study pathogens inactivation over composting process,
� Propose optimal conditions for compost production in a bioreactor.
The work presented in this thesis is subdivided in three chapters. After the
introduction of the work with general information and problem statement, the first chapter,
the literature review, presents the former studies on the inactivation of pathogens and the
composting process. The second chapter is talking about materials and methods used during
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the work. Results of the analyses are presented and discussed in third chapter and to end, the
conclusion and some recommendations.
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CHAPTER I: LITERATURE REVIEW This chapter presents composting process and methods, types of composting toilet and also
the former studies on pathogens inactivation.
1. About composting 1.1. Definitions
Mustin (1987) defines composting like a controlled biological process of conversion and
valorization of the organic substrates in a stabilized and hygienic product, similar to compost,
rich in humic compounds.
Haugh (1980) defines composting like the biological decomposition and the stabilization of
the organic substrates under conditions which allow the development of the thermophilous
temperatures, result of a calorific production of biological origin with obtaining an end
product sufficiently.
Other authors cited by (Polprasert et al., 1980) involves that composting is an aerobic reaction
of microorganisms in metabolizing the waste materials into a stabilized product called
“compost”.
Finally, for (European Commission, 2009), composting means the autothermic and
thermophilic biological decomposition of separately collected biowaste in the presence of
oxygen and under controlled conditions by the action of micro- and macro-organisms in order
to produce compost.
According to those definitions composting can be understood as a biological and an
aerobic decomposition of organic materials by micro-organisms under controlled conditions
into a stabilized substance called compost comparable to humus which is used as a fertilizer
on soils without any impacts on the environment. During composting, microorganisms such
as bacteria and fungi break down complex organic compounds into simpler substances and
produce carbon dioxide, water, minerals, and stabilized organic matter (compost).
1.2. Composting process: Ins and outs
Making compost is one of the best ways of protecting the environment. It also allows the
reduction of pollution but the most important benefit is the safeguarding and the improving of
soil productivity (Michaud, 2007). Indeed, composting makes possible to recycle the various
putrescible and biodegradable matters such as food scraps, agricultural materials, industrial
processing wastes, sludges or urines. Most often, there is a primary raw material to be
composted and other materials are added. Organic materials have rarely all of the
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characteristics needed for efficient composting, so other materials (amendments or bulking
agents) must be blended to achieve the desired characteristics. Amendments can be added to
adjust moisture content, C/N ratio, or texture.
1.2.1. Composting methods Whatever the nature of the method used, some conditions appear essential for the production
of good compost:
� Organic materials blended to provide the nutrients that support microbial activity and
growth, including a balanced supply of carbon and nitrogen (C:N ratio)
� Sufficient oxygen to support aerobic organisms
� Moisture levels that uphold biological activity without hindering aeration
� Temperatures needed by microorganisms that grow best in a warm
medium.
Depending on the way of composting, there are various methods of composting:
� Domestic or family composting: It is practiced on a small scale in the families
� Composting called intermediate practiced on a larger scale than family;
� Vermicomposting for those who do not have the possibility to make compost outside;
� Industrial composting generally practiced with commercial goal.
1.2.2. Parameters in Composting process (Mustin, 1987) said that main factors affecting composting process are related to life
conditions of microorganisms like:
� Lacunary oxygen rate definite as oxygen rate in air of vacuums, it operates a
primordial role in aerobic composting of solid wastes. The composting process
consumes large amounts of oxygen. If there is not enough oxygen, the process slows,
and odors may result.
� Moisture content: microorganisms need water to support their metabolic processes
and to help them move. A moisture content range between 40 to 60 percent is
recommended for most materials. Below 40 percent, microbial activity slows. It ceases
below 15 percent. When moisture levels exceed 65 percent, air in the pore spaces of the
raw materials is replaced by water, leading to anaerobic conditions, odors, and slower
decomposition.
� Temperature: microorganisms release heat while they work, so temperature is a good
indicator of the composting process. Composting process use two scales of
temperature: mesophilic and thermophilic. Although the ideal temperature for initial
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stage of composting is between 20 and 45 °C, later on, for thermophilic organisms
having taken control of subsequent steps, a temperature between 50 and 70 °C is ideal.
Higher temperatures characterize aerobic composting process and are indicators of an
important microbial activity. Pathogens are generally destroyed at 55°C and more,
when critical point of elimination of casual seeds is 62°C.
� pH: The pH level is an indicator of the acidity or alkalinity of the composting
material, measured on a scale from 0 (very acidic) to 14 (very alkaline), with 7 being
neutral. Most of bacteria involved in composting have their optimum growth between
pH 6 and 8, while fungi are more tolerant.
� C/N ratio: During aerobic fermentation active stage, microorganisms consume
between 15 to 30 times more carbon than nitrogen in the substratum. At initial stage of
composting C/N ratio value, which is about 30 decreases constantly during
composting process to stabilize between 15 and 8 in matured compost.
2. Composting toilet Composting toilet is a “win-win system’’ with several advantages:
� Firstly, the toilet is dry: It’s a great alternative to other common systems where water
using is obliged.
� Secondly, it products compost which is used as a fertilizer for plants.
� It provides sanitation systems
� Avoid expensive pipe networks to transport feces and treat them in a mixing
wastewater system.
2.1. Types of composting toilet Morgan (2007) presented three types of composting toilet:
� The single pit compost toilet: In this concept, the pit is shallow, about 1.0 to 1.5 m
deep, and the toilet site is temporary. Excreta, soil, ash, leaves are added to the pit.
The toilet consisting of a ring beam, slab and structure; moves from one site to the
next at 6 to 12 months intervals. The old site is covered with soil and left to compost
and a tree is planted there preferably during the rainy season.
� The double alternating pit compost toilet: In this concept, there are two
permanently sited shallow pits, about 1.5m deep and dug close to each other for
alternate using. For a medium sized family (about 5 persons) the pit takes about 12
months to fill up and this same period allows sufficient time for the mix of excreta,
soil, ash and leaves to form compost which can be excavated. Every one year pit is
excavated while the other becomes full.
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� The Urine-diverting toilet: In this concept, there is one permanent site but there is no
pit. Urines and faeces are separated and faeces fall into a 20 liters bucket held into a
brick vault. Wood ash and soil are added after every deposit. The contents of the
bucket are removed regularly and placed in another site (secondary compost site) to
make compost. The urine is collected in a plastic container.
2.2. Particular case of Bio-toilet The bio-toilet is an important subsystem of the Onsite Wastewater Differentiable
Treatment System (OWDTS) for treating the toilet wastes such as feces, urine, and toilet paper
(Lopez Zavala et al., 2002a in Lopez Zavala et al., 2004). Bio-toilet is the name of a dry toilet
or composting toilet that uses sawdust as a bulky matrix for bioconversion of human excreta into
compost which can be used either as organic fertilizer rich in Nitrogen, Potassium, and Sodium,
or as a soil conditioner (Kitsui and Terazawa, 1999; Del Porto and Steinfeld, 2000 in Lopez
Zavala et al., 2004).
On the other hand, the bio-toilet system differs from conventional composting systems since:
(i) Human excreta are treated.
(ii) The composting reactor of the bio-toilet system is provided with heating and mixing
systems that ensure a continuous thermophilic-aerobic biodegradation process and almost
uniform temperature distribution.
(iii) The moisture content in the composting reactor is kept in the range of 50 to 60% by heating,
mixing, and ventilation.
(iv) The system is managed with the aim of accelerating decomposition, optimizing efficiency, and
minimizing any potential environmental or nuisance problems like odor.
(v) Traditional composting systems have batch configurations where drying is an important process
for the proper management and operation; whereas the bio-toilet is a continuous feeding system
where both drying and biodegradation rate of organic matter are important because urine and
feces are daily added into the composting reactor
Affordability of the bio-toilet system depends on several factors; among them, the capacity of
microorganisms for reducing and stabilizing the organic matter contained mainly in feces.
Additionally, composting process in such toilets is conducted under mesophilic temperatures
(Del Porto et al., 2000).
3. Pathogens in compost To ensure affordability of composting toilets, we have to make sure that compost do not
present any healthy risk and will not be harmful for plants. For compost to be freely used, the end-
product should have a very low concentration in pathogens and it’s also important to guarantee
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against regrowth of pathogens (Kawata et al, 1977; Millner et al.1977, 1980; Burge et al.1979;
Cooper and Golueke 1979; Strauch and Berg 1979; Commision of European communities 1981;
Dean and Lund 1981; World health Organization) as cited by (Bertoldi, et al., 1982). Among
pathogens of concern, some of them are used as indicators to model effective microbial activity. The
best indicators of the potential presence of pathogens are the facultative enteric organisms such as
fecal coliforms.
The inactivation of pathogens in the biosolids depends on several factors such as
temperature (Martin et al., 1990), moisture content (Ward et al., 1981; Russ and Yanko, 1981) and
competition from indigenous microflora (Hussong et al., 1985; Sidhu et al., 2001; Pietronave et al.,
2004). Other factors such as predation, pH, sunlight, oxygen, soil type and texture also influence
pathogens inactivation. The degree to which these factors influence survival of pathogens can vary
from one pathogen to another. However, few results from these studies are comparable due to the
lack of standardized methods and because very few authors have mentioned the detection limits of
the methods used. Compiled data from some of the studies are presented in Table 1 .
The Danish legislation stipulates that untreated fecal waste should not be used in private gardens.
To qualify for that use, it must undergo hygienic treatment to inactivate pathogens, for example
exposure to a temperature of 70 °C for at least 1 h or similar treatment with the same result (Danish
Ministry of the Environment, 2003). Others suggest that 55 °C for 2 weeks would inactivate all
pathogens (Feachem et al., 1983). However, such temperatures are not normally reached during
simple storage of feces in single household compost toilets (Carlander and Westrell, 1999) as cited
by (Tonner-klank, et al., 2006).
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Table 1 Pathogens and fecal indicators content in waste water and sludges
Range Mean References
Total coliforms 1.9xl08 to l.lxl010 5.6 xl09 Soares et al., 1992
Faecal coliforms
9.2xl07 to 1.7xl09 8.9 xl08
9.3xl06 to 1.7xl09 8.5 xl08 Gibbs et al., 1994
7xl01 to l.lxl05 3.4x10 4 Payment et al., 2001
3.6 xl07 Dahab and Surampalli, 2002
3.4xl06 Lasobars et al., 1999
E. coli
3xl02 to 6.2xl04 1.5xl04 Payment et al., 2001
4.4xl05to l.lxl06 Pourcher et al., 2005
Faecal streptococcus
3.7xl05 to 6.6xl07 1.5xl07 Soares et al., 1992
3.5xl05 to 1.0xl08 5 xl07 Gibbs et al., 1994
3x102 to lx104 5.1xl03 Payment et al., 2001
2.1 xl07 Dahab and Surampalli, 2002
1.5xl05 Lasobars et al., 1999
1.58 xl04 Moce-Llivina et al., 2003
Enterococci 7.2xl05 to 2.6xl06 Pourcher et al., 2005
Salmonella
1.1xl03 to 5.9xl03 2.9x103 Gibbs et al., 1994
1.2 to 1.3 Pourcher et al., 2005
6.2x103 Dahab and Surampalli, 2002
3.1xl04 to 8.1xl04 5.6 xl04 Gibbs et al., 1994
Source: Jatinder and al (2008) modified
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3.1. Influence of temperature Temperature is one of the most important factors that affecting microbial growth and
biological reactions.
For (Kaizer, 1996) as cited by (Lopez Zavala et al., 2004), temperature can exert an effect on
biological reactions in two ways: by influencing the rates of enzymatically catalyzed reactions
and by affecting the rate of diffusion of substrate to the cells.
Lopez Zavala et al (2004) and Mustin (1987) said that microorganisms are classified into
three groups depending on temperature range presented in Figure 1 below. Second group as
mesophilic organisms is main concerned in biological processes and grow well over
temperature range of 10-35°C they can even grow up to 45°C. The optimum temperature for
the mesophilic bacteria is around 35°C; they perish at 40-45°C. In most composting toilet
systems, mesophilic bacteria are dominant. However, in the composting reactor of the bio-toilet the
temperature distribution can vary widely from 20°C to 70°C and thermophilic temperatures
between 50° C and 60° C are dominant in 44.5% of the reactor volume:
Figure 1: Behavior of microorganisms opposite to temperature (Mustin, 1987)
Temperatures higher than 70-80° C inhibit growth of main microorganisms present, and then
slowing down decomposition of organic matter.
3.2. Influence of pH According to Mustin (1987), if microorganisms are not able to regulate their own
temperature, they can on the other hand regulate their intern pH. The curve expressing effect
of medium’s pH on growth’s rate looks like a parabola as presented on Figure 2. It indicates
that optimal growth’s rate pH is between 6 and 8.
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Figure 2 Influence of pH of medium on growth’s rate of microorganisms (E.Coli)
(Demeyer et al., 1981)
Mustin (1987) also inscribe the sensitivity of microorganisms due to pH as shown in Figure 3.
Most of bacteria have optimal pH growths near to the neutral pH.
Figure 3 : Sensitivity of microorganisms due to pH (Mustin, 1987)
If pH of composting environments depends on the starting substratum, it can also be modified
by the metabolism of growing microorganisms. There are anaerobic microorganisms which
stretched to neutralize their own medium life and make it more favorable.
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CHAPTER II: MATERIALS AND METHODS
This work was conducted in two phases: the production of the compost and the lab-scale
studies to evaluate the effect of physical parameters, temperature and pH on the inactivation
of indicator bacteria and Helminth eggs in compost. This chapter present methodological
approach adopted and also tools which have been used to implement activities on pilot and at
laboratory scale.
1. Methodological approach To obtain the results of our study, we have adopt a methodological approach which gone
through three steps
� the preliminary works has consisted to: (i) the construction of the pilot the
temporary toilet made of wood and also acquisition of an experimental
composting reactor made of stainless steel and (ii) bibliographical research of
articles, revues in order to make the literature review of the subject
� Study’s work concerning essentially the experimental work and the analysis in
laboratory:
� Data treatment and final report: this step notes the end of the work and it
consists to(i) Analyze different informations on the subject (ii) Carry and
understand the study’s results (iii) Finalize the report
2. Experimental site The composting toilet pilots were located in International Institute for Water and
Environmental Engineering 2iE Foundation based in Ouagadougou. All the experimentations
were carried out in Water, Depollution, Ecosystems and Health laboratory (LEDES in French)
from the water and sanitation department of the 2iE.
3. Production of compost
For the production of compost, there are two processes: one is composting process with feces
feeding (10 days) and one for bacteriological and parasitological analyses (15 days)
3.1. Construction of pilot scale composting toilet
An experimental platform was planned, made of wood to be the pilot of the composting toilet.
Two (02) different types of urine diverted toilet tagged model 1 and model 2 in the document
were set up.
� Model 1 was planned on English toilet concept i.e. with a chair just as shown on
Figure 4. It was used by the personal of LEDES.
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The model was put near flushing toilet site of the laboratory separated by a fake wall
to allow better privacy.
� Model 2 was planned on Turkish toilet concept with a direct hole for defecation as
shown on (Figure 5 picture a). However, an anal washing area for people using water
to wash their body after defecation and a hand washing system were also provided
(Figure 5 pictures b and d). This model was used by people working in building
construction inside 2iE campus. Notice that the choice has been made on them
because they are representative sample of nutritional habits of rural and suburban
people. The model was put into a bloc 1m x 2m with walls made of wood and an
aeration area in order to avoid odors and mosquitoes.
Figure 4 : Model 1 of composting toilet
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Figure 5 : Model 2 of composting toilet
(a) (b)
(c) (d)
Hole for defecation
Hole for urine
Tank for washing body water
Washing area
Hand washing system Instructions for use
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Armelle Stella JIBIA PALO June 2011 Page 13
For every model, instructions for use were detailed and put for users (Figure 5 picture c).
Feces were collected in a plastic bag introduced in a bucket and urines were collected in a 20
liters’ tank.
Figure 6 presents a composting reactor with a mixing system made of stainless steel for feces
and sawdust mixing.
Figure 6 : Composting reactor
3.2. Matrix: The sawdust
Before putting feces in the reactor, an amount of sawdust has already been put in. This
amount of sawdust is put only on time during the experiment and the quantity put in the
reactor was about 2,7Kg. Sawdust is used because of its great value of C: N ratio which is
between 100 and 500 (Center for Environmental Farming systems, 2005).
3.3. Composting process with feces feeding
After toilets’ installation and the effective utilization by people, the feces collection was
performed every day, in the two (02) toilets. As mentioned above, feces were collected in
plastic bags in order to facilitate the manipulation. After weighting, the daily amount of feces
collected are put in the reactor and mixed with the existing sawdust.
The feces were collected during ten (10) days and the amount of feces put in the reactor is
presented in table 2.The total amount represent about 14Kg of feces.
Mixing system
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Table 2 : Daily weight of feces put in the reactor
After about two weeks of collection and mixing of feces, the compost is produced and ready
for analyses in laboratory.
4. Lab-Scale Studies
For the lab-scale studies, the second process of composting starts and remained 15 days.
4.1. Compost tests
Experiment last 15 days and every three (03) days of this period, samples are taken in the
reactor for the various analyses at the laboratory leading a total of 6 samples with the first day
one. For each sample, pH, temperature and moisture content were measured to determine the
initial conditions of mixed feces/sawdust.
After these measurements, the sample was subdivided in 8 subsamples of 50g each. Four (04)
subsamples were incubated during 24h in a drying oven at the temperatures 30°C, 37°C,
44°C, 50°C and the other four subsamples were having their pH increased by addition of
calcium hydroxide Ca(0H)2 in the proportions 0,25g; 0,5g; 0,75g and 1g.
Those temperatures were selected because 37°C and 44°C are usual bacteria and parasites
growth temperature; 30°C and 50°C are the extreme temperatures for bacterial culture. The
DAYS Daily weight of feces
( in Kg of fresh matter)
Day 1 1,320
Day 2 0,559
Day 3 1,600
Day 4 1,145
Day 5 0,852
Day 6 1,750
Day 7 1,302
Day 8 1,730
Day 9 2,503
Day 10 0,855
Total 13,616
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proportions of calcium hydroxide were selected in order to make the pH of the subsample
more alkaline.
After incubation and variation of the pH, the analyses begin.
4.2. Physical and chemical Analyses Physical and chemical parameters analyzed in compost samples are pH, electric conductivity,
temperature, moisture content, organic and inorganic matter.
� Temperature Temperature was determined using a probe thermometer (Digital Thermometer 343) in
compost. Temperature was taken at the starting of the experiments and then every (03) days
during 15 days.
� pH and electric conductivity pH was determined on the taken sample by the following procedure:
� 5g of compost in 10 ml of distilled water.
� Agitate the mixture in a beaker which contains a magnetized bar using the
mechanical agitator during 30 minutes
� Let the mixture rest for 30 other minutes and then carry out the reading. For the
reading a multi parameter probe (WTW 330 i) is used which one will have taken
care to calibrate with water distilled at the beginning.
The pH-meter indicates the pH of the solution and the conductivity-meter the electric
conductivity of the solution in µs/cm
� Moisture content (MC), Organic matter (OM) and Inorganic matter These parameters were determined through the weight loss at 105°C and 550 °C by the
procedures established in the Standard methods (1995) detailed in Appendix 1.
4.3. Bacteriological Analysis All the bacteriological analysis performed in this part of the study included total coliforms,
fecal coliforms and fecal streptococci. Bacterial cultures were performed using the spread-
plating procedure. The results of the examination of Petri dishes and the dilutions are reported
by counting the organisms in the sample and the most real number is taken.
� Preparation of culture media
The culture media used for bacteriological analysis of and were respectively ‘’ Chromocult’’
for total Coliforms and fecal Coliforms and ‘’ Slanetz and Bartley’’ for Fecal Streptococci.
� Sampling and preparation of solutions
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10g of compost’s subsample is taken and added to 90 ml of a solution called Ringer 1for the
preparation of mothers’ solutions.
For dilutions; the dilution factor used is 10-1
� Bacterial culture 0,1 ml of each solution (mother and diluted) is taken and put it in Petri dishes, spread it out
with small rakes and incubate at 37°C for 24h and 48h respectively for total coliforms and
fecal Streptococci; at 44°C for 24h for fecal coliforms.
The numbers of Total Coliforms, Fecal Coliforms, Presumed E.Coli and Fecal Streptococci
are determinate by counting method which consists to number all the microorganisms present
in the petri plates and to choose the most probable one.
4.4. Parasitological Analyses The parasitological analyses performed only included helminths eggs. They were performed
using US EPA protocol (1999) modified by Schwartzbrod (2003) detailed in Appendix 2.
1 It is an optimal solution allowing the maintenance of the microorganisms while avoiding any proliferation
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CHAPTER III: RESULTS AND DISCUSSION
The results presented in this part are the results of the lab-scale studies included the
physical/chemical parameters and the inactivation rate of pathogens in the compost.
1. Results 1.1. Physicals and chemical analyses
1.1.1. Evolution of temperature Figure 7 presents the evolution of temperature during the composting process.
Figure 7 : Evolution of temperature during composting
The result shows that the peak is observed at the first day of the experiment at 35,8°C and it
decreases until the reach of 29°C the fifteenth day which get closer to the ambient
temperature. The process of composting is mesophilic. This long duration of mesophilic stage
can be explained by the nature of matrix which is not rich in carbon.
1.1.2. Evolution of pH Figure8 present the evolution of pH during the composting process. This pH is only alkaline
and decreased from 9,32 to less than 8. According to (Mustin, 1987), it favors the mesophilic
flora and an intensive production of carbonic acid gas.
25
27
29
31
33
35
37
0 3 6 9 12 15
Tem
pera
ture
°C
Composting Days
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Figure 8 : Evolution of pH during composting
1.1.3. Evolution of Electric conductivity Electric conductivity showed a variation during the process (Figure 9).
Figure 9 : Evolution of Electric conductivity (EC) during composting
A peak is observed at 1109µS/cm the 9th day and the lowest value is observed the 3th day.
7,5
7,7
7,9
8,1
8,3
8,5
8,7
8,9
9,1
9,3
9,5
0 3 6 9 12 15
pH
Composting Days
750
800
850
900
950
1000
1050
1100
1150
0 3 6 9 12 15
EC
(µS
/cm
)
Composting days
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1.1.4. Evolution of moisture content During the composting process the moisture content is always decreasing as Figure10 shows it.
Figure 10 : Evolution of moisture content during composting
1.1.5. Evolution of Organic and Inorganic matter Organic matter decreases with time from the first day to the sixth, becomes a little bit stable
from the sixth to the ninth day. (Figure11)
Figure 11 : Evolution of Organic matter (OM) during composting
70
72
74
76
78
80
82
0 3 6 9 12 15
Moi
stur
e co
nten
t (%
)
Composting days
60
62
64
66
68
70
72
74
76
78
80
0 3 6 9 12 15
OM
(in
% o
f dry
mat
ter)
Composting days
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Inorganic matter as for it increases with time from the first to the sixth day. It becomes a little
bit stable from the sixth to the ninth day and decreases after (Figure 12)
Figure 12 : Evolution of Inorganic matter during composting
A summarization of all the chemical and physical parameters during composting has been
done and presented in Appendix 3.
1.2. Bacteriological results Before seeking the influence of temperature and pH, the initial concentration of the
microorganisms of Total Coliforms (TC), Fecal Coliforms (FC), Presumed E.Coli and Fecal
Streptococci (FS) has been determined and summarized in table 3.
Table 3 : Initial concentration of microorganisms in CFU compost
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
Total Coliforms 5,20E+05 2,60E+05 7,00E+02 1,40E+05 9,80E+04 7,30E+04
Fecal Coliforms 1,10E+05 4,50E+06 0,00E+00 1,19E+05 5,50E+04 1,70E+04
Presumed E.Coli 2,00E+03 0,00E+00 0,00E+00 0,00E+00 0,00E+00 6,00E+03
Fecal Streptococci 2,00E+05 8,90E+04 0,00E+00 1,52E+04 2,00E+03 1,40E+04
0
5
10
15
20
25
30
35
40
0 3 6 9 12 15
IM in
% o
f dry
mat
ter
Composting days
Inorganic matter (IM) in compost
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1.3.1. Influence of temperature
As mentioned above, the collected samples were incubated at 30°C, 37°C, 44°C and 50°C and
the numbers of microorganisms were evaluated and the results are detailed in Appendix 4.
a) Total Coliforms In relation with the initial concentration of Total Coliforms, there is a decrease observed for
all the temperatures, TC decreased the day 0 of the experiment, inhibit or totally absent the
third, sixth and ninth days but for the twelfth day and the fifteenth day, a regrowth is
observed.
b) Fecal Coliforms
Fecal coliforms behave practically the same as total coliforms.
c) Fecal Streptococci
Fecal Streptococci decreased during all the process. They are inhibited the 9th and 12th day at
44°C and the 6th and 9th day at 50°C. A regrowth is immediately observed the 15th day.
The greater decrease for all the pathogens is observed at 50°C, so it’s seems to be an
appropriate temperature for inactivation of pathogens.
Graphics showing the influence of temperature are presented on Figures 13, 14, 15
Figure 13 : Influence on temperature on Total coliforms
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
0 3 6 9 12 15
CF
U/g
Composting Days
30°C 37°C 44°C 50°C Initial concentration of TC
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Armelle Stella JIBIA PALO June 2011 Page 22
Figure 14 : Influence of temperature on Fecal coliforms (FC)
Figure 15 : Influence of temperature on fecal streptococci (FS)
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
0 3 6 9 12 15
CFU/g
Composting Days
30°C 37°C 44°C 50°C Initial concentration of FC
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
0 3 6 9 12 15
CF
U/g
Composting Days
30°C 37°C 44°C 50°C Initial concenntration of FS
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1.3.2. Influence of pH
As mentioned above, an amount of calcium Hydroxide was added to the collected samples in
order to increase the pH respectively in proportions of 0,25g, 0,5g, 0,75g, 1g. The increased
pH are summarized in table 4.
Table 4 : increased pH during sampling
Initial pH
Final pH
0,25g of Ca(OH)2
0,5g of Ca(OH)2
0,75g of Ca(OH)2
1g of Ca(OH)2
Day 0 9,32 9,34 10,33 12,05 12,53
Day 3 9,14 9,32 10,3 11,78 11,98
Day 6 9,01 9,98 10,4 12,21 12,52
Day 9 8,62 10,9 12,24 12,45 12,61
Day 12 8,2 9,85 10,49 11,72 12,22
Day 15 8,01 8,95 9,12 11,61 12,09
A very great increase of pH is noted.
The numbers of microorganisms were evaluated as shown in Appendix 5.
a) Total Coliforms
In relation with the initial concentration of TC, there is a decrease observed with pH better
than the one with temperature. Indeed, TC decreased the day 0 of the experiment, inhibit or
totally absent the third, sixth and ninth days but for the twelfth day and the fifteenth day, a
regrowth is observed.
b) Fecal Coliforms
A decrease of concentration of FC is also observed better than the one with temperature.
c) Fecal Streptococci
Fecal Streptococci decrease during all the process. They are inhibited the 6th and 9th day at
50°C. A regrowth is immediately observed the 12th and 15th days. We also observed that more
the pH increase better it has a great influence of inactivation of pathogens. Addition of
calcium hydroxide and more generally increasing of pH to an alkaline one seems to be an
appropriate way to inactivate pathogens.
Graphics showing the influence of pH are presented on Figures 16, 17, 18.
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Armelle Stella JIBIA PALO June 2011 Page 24
Figure 16 : Influence of pH on total coliforms (TC)
Figure 17 : Influence of pH on fecal coliforms (FC)
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
0 3 6 9 12 15
CF
U/g
Composting Days0,25 g →pH [8,95 to 9,34] 0,5 g→pH [9,12 to 12,24]
0,75 g→pH [11,61 to 12,45] 1 g→pH [11,98 to 12,61]
Initial concentration of TC
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
1,00E+07
0 3 6 9 12 15
CF
U/g
Composting Days
0,25 g →pH [8,95 to 9,34] 0,5 g→pH [9,12 to 12,24]0,75 g→pH [11,61 to 12,45] 1 g→pH [11,98 to 12,61]Initial concentration of FC
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Armelle Stella JIBIA PALO June 2011 Page 25
Figure 18 : Influence of pH on fecal streptococci (FS)
1.3. Parasitological results Parasitological analyses have revealed a high prevalence of Helminth eggs in feces as shown
in table 5:
Table 5 : Prevalence of Helminth eggs in compost (n=10)
Parasites Ankylostoma duodenanale
Ascaris lumbricoïdes
Trichiuri trichiura
Schistosoma mansoni
Numbers in 1g of dry feces
544 66 17 17
The predominance of Ankylostoma duodenanale eggs is explained by the nutritional habits of
the studied people.
The variation of temperature was maintained ≥ 50°C and pH was maintained at an alkaline
one, thus indicating the positive effect of both parameters on the inactivation of Ankylostoma
duodenanale eggs.
1,00E+00
1,00E+01
1,00E+02
1,00E+03
1,00E+04
1,00E+05
1,00E+06
0 3 6 9 12 15
CF
U/g
Composting days
0,25 g →pH [8,95 to 9,34] 0,5 g→pH [9,12 to 12,24]
0,75 g→pH [11,61 to 12,45] 1 g→pH [11,98 to 12,61]
Initial concentration of FS
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1.3.1. Influence of temperature
Figure 19: Influence of temperature on Ankylostoma duodenanale A.d eggs during Composting
In relation with the initial number of A.duodenale, the reduction of the number of
Ankylostoma duodenanale eggs greatly decrease with time and tend towards zero for all the
temperatures except 30°C.
0
50
100
150
200
250
300
350
0 3 6 9 12 15
Num
bers
of e
ggs
Composting Days
30°C 37°C
44°C 50°C
Initial A.d
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1.3.2. Influence of pH
Figure 20 : Influence of pH on Ankylostoma duodenanale (A.d) during composting
In relation to the initial number of A.duodenale, the reduction of the number of Ankylostoma
duodenanale eggs greatly decrease with time and tend towards zero for all the temperatures
except 0,25g (pH between 8,95 and 9,34).
2. Discussion It was mentioned above that the inactivation of pathogens is an important factor to assess an
effective utilization of compost made of feces. Knowledge of the effect of temperature and pH
on that inactivation allows proper design criteria of the composting toilet and reduces the
waiting time after the toilet utilization.
2.1 Chemical and physical characterization A maximum temperature of 35,8°C was measured in the composting reactor. Thus,
temperatures always decrease until the reach of ambient temperature. This long duration of
mesophilic stage can be explained by the nature of matrix used. However, temperature in the
compost was higher than the environmental temperature, indicating that an active microbial
population was present. Normally pathogens are expected to be killed after 2 weeks at 55°C
0
50
100
150
200
250
300
350
0 3 6 9 12 15
Num
bers
of e
ggs
Composting Days
0,25 g →pH [8,95 to 9,34] 0,50 g→pH [9,12 to 12,24]
0,75 g→pH [11,61 to 12,45] 1 g→pH [11,98 to 12,61]
Initial A.d
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(Feachem et al, 1983).Our results show that after 2 weeks at 50 °C pathogens are inactivated
(a great log reduction is observed).
During all the process, pH decreases and this can be explained by the acido -genesis process
with an intense production of carbon dioxide. In most standards that define pH limits,
compost should have a pH value within the range of 6.0-8.5 to ensure compatibility with most
plants(Hogg et al., 2002) as cited by (Lasaridi et al., 2005), a condition not met by the four of
our compost samples.
The range of electric conductivity is within 803µS/cm and 1109µS/cm which is in agreement
with the Greek Standards’ which upper limit is at 4dS/cm, a level considered tolerable by
plants of medium sensitivity.
For the experiment, Organic matter content decreased, and as expected IM increased over the
composting process. The active biodegradation of the organic matter is the resident microbial
community during the different stages of composting (Peters et al., 2000).
2.2 . Pathogens Inactivation As mentioned above, temperature is one of the most important factors of inactivation of
pathogens. The results showed that temperature has an effect on inactivation of pathogens.
During the composting process, which is mesophilic in our case, the optimal temperature
should be around 35°C as suggest by (Feachem et al., 1983 in Lopez Zavala et al, 2004) but
we retained that 50°C was the better temperature for pathogens inactivation.
The increase of pH, basic pH, has a great effect on pathogens inactivation especially on
Helminth eggs which has a die-off observed. The pH was basic during all the process and the
addition of calcium hydroxide just increased it to another basic high value. Alkaline pH ≥ 12
was retained as a good parameter for inactivation of pathogens.
At the end of the composting, coliforms were inactivated but enterococci were present
because of their resistance. In fact, international surveys of biosolids quality report that
enterococci, coliphages, and Clostridia show greater resistance to inactivation compared to
fecal coliforms during composting (Christensen et al., 2002). Sometime in biosolids,
Enterococci may better represent health risks associated with more resistant pathogens (Sidhu
et al., 2009).
The changes in chemical composition that were observed during the composting process
suggest that microbial ecology was changing. This would imply that a very heterogeneous
microbial population existed in the composting.
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Total coliforms, fecal coliforms, and fecal streptococci densities were reduced by more than
99,9 % by using liquid lime stabilization at a pH ≥12,0 in full scale studies in Lebanon, Ohio
(US EPA, 1979 as cited by Ramirez,2000).
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CONCLUSION AND RECOMMENDATIONS
The primary objective of this thesis was to evaluate the effectiveness of temperature variation
and pH increasing on inactivation rate of indicator bacteria and helminths eggs in stabilized
compost. To reach this objective, compost was made during 10 days and lab-scale studies
performed on the stabilized compost during 15 days.
The following conclusions were supported by the results of this research:
� After incubation to different temperatures and addition of calcium hydroxide,
reductions of densities of Total coliforms, fecal coliforms, fecal streptococci and
Helminth eggs were observed during the days of experiments.
� Fecal coliforms and total coliforms have practically the same behavior and their
reduction is appreciable.
� Fecal streptococci also reduce but are more resistant than the coliforms.
� Log reduction of indicators bacteria is observed for the reach of 50°C of temperature
and also an alkaline pH of about11.
� Predominance of Ankylostoma duodenanale eggs was observed in prevalence of
Helminth eggs and their total removal was reach after the 15 days of the experiment
for temperature ≥30°C and a basic pH between 11 and 12.
� The changes in chemical composition that were observed during the composting
process suggest that microbial ecology was changing and different substrates were
being used. This would imply that a very heterogeneous microbial population existed
in the composting
According to all those conclusions we can established that the waiting time after the
utilization of composting toilet can be reduced if some conditions are taken into account
The influence of temperature variation and the increase of pH on the inactivation of pathogens
is clearly point up.
Researches on pathogens inactivation constitute a great study for affordability of composting
toilet. Our work not being exhaustive, as recommendations, further researches are welcome.
These further researches should focus on moisture content variation and also effect of time,
effect of type and size of the composting matrix on the compost.
It is recommend also to enlarge the target of the study to another scale or another part of the
population in order to ensure conditions of composting toilet use.
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Armelle Stella JIBIA PALO June 2011 Page 33
APPENDICES Appendix 1: Standard method for measurement of Moisture content (MC), inorganic matter
(IM) and organic matter (OM) ................................................................................................. 34
Appendix 2 : Method for parasitological analysis ................................................................... 35
Appendix 3 : Summarization of physical and chemical parameters ........................................ 36
Appendix 4 : Concentration of Bacteria in compost in CFU /g after temperature variation ... 37
Appendix 5 : Concentration of Bacteria in compost in CFU /g after pH increasing ............... 38
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Armelle Stella JIBIA PALO June 2011 Page 34
Appendix 1: Standard method for measurement of Moisture content (MC), inorganic matter
(IM) and organic matter (OM)
For moisture content, take 10 g of compost in a tare and put it in the stove at 105°C for 24
hours. Come the other day and weight the dry compost. MC is determined by the formula:
Where PT the weight of the tare
PH weight of the tare charged before drying and
PDthe weight of the tare charged after drying.
For IM, dry compost obtained before is put in another stove at 550 °C for three (03) hours
IM is the difference between the weight of dry compost and weight of mineral matter
The result is expressed in percentage of dry matter.
Where PD the weight of dry matter
PM weight of mineral matter
Organic matter is the difference between the dry weight at 1105°C and the one at 550 °C.
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Armelle Stella JIBIA PALO June 2011 Page 35
Appendix 2 : Method for parasitological analysis
� Homogenize the compost sample for 30 seconds using a blender with 22,000 rpm (the blended sample should be equivalent to 10 g TS ;liquid composts may be blended undiluted ,while dewatered or dried composts should be resuspended in tap water to give a volume of approx.400 ml
� Screen the compost through a 160 µm screen with 21 of water per sample
� Collect the filtrate in the same 2 l contained. Let it settle overnight or for 3 hours (for
water, start at this stage). Suck up as much of the supernatant as possible
And place the sediment in a 450 ml centrifugal flask
Rinse the 2-litre contained 2 to 3 times
� Centrifuge at 400 g for 3 min. (1450 rpm)
Pour the supernatant and resuspend the deposit of 150 ml with ZnSO4 Of 1.3 density
Homogenize with a spatula
� Centrifuge at 400 g for 3 min. (1050 rpm)
Pour the ZnSO4 supernatant in a 2 litres flasks and dilute it with at least 1 l of water. Let it
settle for 3 hours. Suck up as much of the supernatant as possible and resuspend the
deposit by shaking, empty it in 2 tubes of 50 ml and clean 2 to 3times with deionized
water place the rinsing liquid in the 50 ml tubes
Centrifuges at 480 g for 3 min.(1600 rpm)
� Regroup the deposits in a tube of 50 ml and centrifuge at 480 g for 3 minutes
� Resuspend the deposits in a 15 ml acid /alcohol (H2SO4+C2H5OH)
Or 5 ml acetic acid solution
And add 10 ml ethyl ether or 5 ml ethyl acetate
Shake and open occasionally to let out the gas
� Centrifuge at 660 g for 3 min.
Suck up as much supernatant as possible to leave less than 1 ml of liquid
� Read at microscope
� For helminths eggs Quantification, use the formula
Where V = volume of initial sample compost K = constant related to the performance of the method (k = 1.42)
Reduce the weight of dry compost diluted
���� ! "# $ %���&$ ''( / %�& ! �* $ %���&$ ''( +! ( �& / ,� � -
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Armelle Stella JIBIA PALO June 2011 Page 36
Appendix 3 : Summarization of physical and chemical parameters
Range Average Standard Deviation
Temperature (°C) 29 35,8 32 2,4
Moisture content(%) 72 80 75 3
pH 8,01 9,32 8,72 1
Electric Conductivity (µS/cm) 803 1109 919 108
Inorganic matter(% of Dry matter)
22,2 33,4 27,5 4,8
Organic matter(% of Dry matter)
66,6 77,8 72,5 4,8
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Appendix 4 : Concentration of Bacteria in compost in CFU /g after temperature variation
Influence of temperature for Total Coliforms
Temperature Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
30°C 4,70E+03 6,20E+04 0,00E+00 0,00E+00 6,00E+03 1,10E+03
37°C 4,50E+03 1,00E+00 0,00E+00 0,00E+00 0,00E+00 3,40E+04
44°C 8,00E+02 1,00E+00 0,00E+00 0,00E+00 0,00E+00 2,40E+03
50°C 9,00E+02 1,00E+00 0,00E+00 0,00E+00 9,00E+03 0,00E+00
Influence of temperature for Fecal Coliforms
Temperature Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
30°C 4,10E+03 3,00E+04 0,00E+00 0,00E+00 1,00E+03 1,80E+03
37°C 3,20E+03 0,00E+00 0,00E+00 0,00E+00 0,00E+00 8,00E+04
44°C 6,00E+02 1,00E+00 0,00E+00 0,00E+00 0,00E+00 1,00E+04
50°C 0,00E+00 0,00E+00 0,00E+00 0,00E+00 3,00E+03 0,00E+00
Influence of temperature on Fecal Streptococci
Temperature Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
30°C 3,90E+04 1,8E+03 3,00E+02 2,00E+02 8,30E+04 1,50E+04
37°C 5,50E+03 5,00E+02 6,00E+02 1,00E+02 3,50E+04 1,02E+05
44°C 4,10E+03 1,00E+02 1,00E+02 0,00E+00 0,00E+00 2,30E+03
50°C 1,00E+02 4,00E+02 0,00E+00 0,00E+00 2,00E+03 5,00E+02
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Appendix 5 : Concentration of Bacteria in compost in CFU /g after pH increasing
Influence of pH on Total Coliforms
Amount of Ca(OH)2
Final pH range
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
0,25 g 8,95 to 9,34 5,00E+02 0,00E+00 0,00E+00 0,00E+00 8,20E+04 0,00E+00
0,5 g 9,12 to 12,24 1,00E+02 2,00E+03 0,00E+00 2,00E+03 6,30E+04 1,40E+03
0,75 g 11,61 to 12,45 1,70E+03 1,00E+03 0,00E+00 1,00E+03 1,80E+04 3,90E+03
1 g 11,98 to 12,61 1,10E+03 8,00E+03 0,00E+00 1,00E+03 1,00E+03 6,40E+04
Influence of pH on Fecal Coliforms
Amount of Ca(OH)2
Final pH range
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
0,25 g 8,95 to 9,34 1,00E+02 0,00E+00
0,00E+00 1,00E+00 2,10E+04 0,00E+00
0,5 g 9,12 to 12,24 1,00E+02 2,51E+05
0,00E+00 3,00E+03 1,00E+04 5,00E+02
0,75 g 11,61 to 12,45 5,00E+02
0,00E+00 0,00E+00 0,00E+00 6,00E+03 1,00E+03
1 g 11,98 to 12,61 1,00E+03 1,40E+04
0,00E+00 0,00E+00 4,00E+03 1,90E+04
Influence of pH on Fecal Streptococci
Amount of Ca(OH)2
Final pH range
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
0,25 g 8,95 to 9,34 1,02E+04 1,8E+03 5,00E+02 4,00E+02 1,04E+05 3,90E+03
0,5 g 9,12 to 12,24 7,00E+02 2,5E+03 1,40E+03 1,00E+02 1,00E+03 1,40E+03
0,75 g 11,61 to 12,45 8,00E+03 15,7E+03 7,00E+03 0,00E+00 0,00E+00 2,40E+03
1 g 11,98 to 12,61 0,00E+00 8,00E+02 0,00E+00 0,00E+00 1,00E+03 4,00E+02