inactivation of pathogens in human feces during …

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


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


Armelle Stella JIBIA PALO June 2011 Page iii

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’’


Armelle Stella JIBIA PALO June 2011 Page iv

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


Armelle Stella JIBIA PALO June 2011 Page v

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.


Armelle Stella JIBIA PALO June 2011 Page vi

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


Armelle Stella JIBIA PALO June 2011 Page vii

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


Armelle Stella JIBIA PALO June 2011 Page viii

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


Armelle Stella JIBIA PALO June 2011 Page ix

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


Armelle Stella JIBIA PALO June 2011 Page x

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


Armelle Stella JIBIA PALO June 2011 Page 1

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



the work. Results of the analyses are presented and discussed in third chapter and to end, the

conclusion and some recommendations.



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



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



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.



� 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



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).



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



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.



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.



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.



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



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



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



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



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



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



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



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



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



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



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



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



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.



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



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



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



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



(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.



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).



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.



BIBLIOGRAPHY BERTOLDI, A., VALLINI, G. and PERA, A. 1982. The biology of composting: A review.

1982

CHRISTIENSEN et al, 2002. Strategies for evaluating the sanitary quality of composting.

Journal of

Applied Microbiology.2002

COMMISSION, EUROPEAN. 2009. Working group of DG. Working document on

Biological Treatment of Biowaste. 2009.

DEL PORTO, D. and STEINFIELD, C. 2000. The composting toilet system book. Concord

Massachusetts : USA, Center for Ecological Pollution Prevention (CEPP), 2000.

GALLIZI, Katharina 2003 Co-composting reduces Helminth eggs in fecal sludge 2003

HAUGH, R.T. 1980. Compost engineering:principles and practice. Michigan : Ann Arbor

Science, 1980.

KAIZER, J. 1996. Modeling compostingas a microbial ecosystem: a simulation approach.

1996. pp. 91:25-37.,

KONE, D et al, 2007 Helminth eggs efficiency by faecal sludge dewatering and co-

composting in tropical climates. 2007

LOPEZ ZAVALA et al. 2004. Biological activity in the composting reactor of the biotoilet

systems. 2004.

LOPEZ ZAVALA et al. 2004. 2004. Temperature effect on aerobic biodegradation of feces

using sawdust as a matrix. 2004.

MICHAUD, Lili. 2007 . Tout sur le compost:Le connaître, le faire,l'acheter, l'utiliser.

Québec : Multimondes, 2007.

MORGAN, Peter. 2007. Toilets that make compost. Stockholm : Practical Action

Publishing, 2007.

MUSTIN, Michel. 1987. Le compost:Gestion de le matière organique. Paris : François

DUBUSC, 1987.



PETERS et al.2000 Succession of microbial communities during hot composting, 2000

POLPRASERT, C., WANGSUPHACHART, S. and MUTTAMARA, S. 1980.

Composting nightsoil and water Hyacinth in the tropics. Bangkok, Thailand : s.n., March

1980. p. 4.

RAMIREZ, B 2000 The effects of time and temperature on the fate of pathogens and

indicator bacteria during municipal waste waster sludge-mesophilic anaerobic

digestion, air-drying, and composting, 2000

SIDHU et al, 2009 Human pathogens and their indicators in biosolids: a literature review,

2009

TONNER-KLANK, L., et al. 2006. Microbiological assessments of compost toilets: In situ

measurements and laboratory studies on the survival of fecal micccrobial indicators using

sentinel chambers. 2006.

ZNAÏDI, Ibrahim El Akram, 2002 Etude et évaluation du compostage de différents types de

matières organiques et des effets des jus de composts biologiques sure les maladies des

plantes 2002



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



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.

�� %� ��

��

�� % ��

� ��



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

�� ! "# $ %��&$ ''( / %�& ! �* $ %��&$ ''( +! ( �& / ,� � -



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



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


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


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



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


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


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


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

inactivation of pathogens in human feces during …

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