WATER SUPPLY IN TANZANIA AND PERFORMANCE OF LOCAL …13925/... · 2008-06-02 · Water supply in...

TRITA-LWR PhD Thesis 1042 ISSN 1650-8602 ISRN KTH/LWR/PHD 1042-SE ISBN 978-91-7415-016-2

WATER SUPPLY IN TANZANIA

AND PERFORMANCE OF LOCAL

PLANT MATERIALS IN

PURIFICATION OF TURBID

WATER

Nancy Jotham Mwaisumo Marobhe

June 2008

Nancy Jotham Mwaisumo Marobhe TRITA LWR PhD Thesis 1042

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© Nancy Jotham Mwaisumo Marobhe TRITA LWR PhD Thesis 1042 Doctoral Thesis Department of Land and Water Resources Engineering Royal Institute of Technology (KTH) SE-100 44 STOCKHOLM, Sweden

Water supply in Tanzania and performance of local plant materials in purification of turbid water

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DEDICATION

To Isaac, Emmanuel, Mutaju, Tusajigwe and Obed

Gen. 18:14. Is anything too hard for the Lord? Hymn 11. I need you the every hour. Mwa. 18:14. Kuna neno gani lililo gumu la kumshinda Bwana? Tenzi 11. Nina haja nawe kila saa.


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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Sida/SAREC for financial support, which has made this research possible and has contributed significantly to building human and non-human resource capacity at Ardhi University (ARU). I greatly appreciate the coordination and support of Prof. Dick Urban Vestbro that contributed greatly to the success of PhD studies at ARU and KTH. I wish to extend profound gratitude to my main supervisor, Associate Professor Gunno Ren-man, for his scientific guidance and constructive inputs to my research. Without his tireless sup-port and encouragement, I could not have accomplished this research. I also appreciate the kindness and willingness of his entire family to assist, especially in times of difficulty. My special thanks go to my co-supervisors Associate Professor Gunnar Jacks and Professor Jackson Msafiri for their scientific guidance, valuable discussions and support. I am deeply indebted to Dr. Gunaratna Rajarao for her close supervision in purification and characterisation of the seed proteins and Professor Gunnel Dalhammar for her great interest in my study subject and for providing facilities and materials used for protein and bacterial studies. Their scientific inputs into my research activities have greatly contributed to the success of my studies. My gratitude also goes to EE academic staff members, Dr. Kiunsi R, Dr. Mgana S, Prof. Kas-senga G.R and Prof. Kaseva M.E to mention a few, for fruitful discussions and comments during the preparation of some manuscripts. My sincere thanks go fellow PhD candidates at the De-partment of LWR and Applied Environmental Microbiology at KTH for their wonderful hospi-tality and friendship. Special thanks to Sofia, Kay and Per for tireless assistance in the lab. The encouragement and support of my fellow PhD students from ARU (Merdard, Charles, Felician, Prosper, Tatu, Stanlaus and Boniface) are highly appreciated. Thank you Aira and Britt for the wonderful assistance in administrative as well as social matters. My special thanks go to the leadership in Singida Rural District, the local leadership and villagers in Ilongero Division for their cooperation and assistance during my fieldwork studies in the district. I am also indebted to the District water engineer, Eng. Luanda, J.F, Mr. Shekiondo, H.M, and other staff members at the water department in Singida district for their support during my fieldwork and assistance in analysis of water samples. I would be delighted to see villagers man-age to transform muddy water into clear and bacteriologically safe drinking water and also man-age to improve their income and hence afford to operate and maintain potable water sources. My heartfelt thanks go to my late parents, Mzee Jotham Obed Mwaisumo and mama Andekile Anna Ilomo, without whose wisdom and love I could not have been as I am today. The moral and spiritual support and great love of my beloved sisters, brothers, aunts, cousins and nephews, my sisters and brothers in law, my mother in law, Nyabhele Rukia Mutaju and other relatives are highly appreciated. My very special thanks go to my husband and best friend, Isaac, for his boundless enthusiasm for my studies. His great love, encouragement and spiritual support and paving a way towards my academic achievement is highly appreciated. I sincerely appreciate the effort, time and skills he used in taking care of family in my absence. My special thanks go to my children, Emmanuel-Nyarugere, Mutaju-Ipyana, Tusajigwe-Gloria and Obed-Baraka, for their understanding, patience and moral support during the whole period of my studies. My aunt Laida Kasuga, I thank you so much for your love, caring and faithfulness to our family, may God bless you and reward you accordingly. Finally, above all, I thank the Lord for all he has done, is doing and will do in and through me. Nancy Jotham Mwaisumo Marobhe Stockholm, June 2008


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LIST OF PAPERS APPENDED

I. Marobhe, J.N. 2008. Critical review of water supply services in urban and rural areas of Tanzania. Water Policy, 10(1), 57-71. doi:2166/wp.2007.029.

II. Marobhe, N.J., Renman, G., Gunnar, J. 2007. The study of water supply and traditional water purification knowledge in selected rural villages in Tanzania. In: Indigenous knowl-edge systems and sustainable development: Relevance for Africa. Boon E. and Hens, L (eds). Kamla-Raj Enterprises, Delhi, Indian. Tribes and Tribals, Special Volume No. 1, 111-120.

III. Marobhe, N.J., Renman, G., Jackson, M. 2007. Investigation on the performance of local plant materials for coagulation of turbid river water. Institution of Engineers Tanzania, 8 (3), 50-62.

IV. IV.Marobhe, N.J., Dalhammar, G., Gunaratna, K.R. 2007. Simple and rapid method for purification and characterization of active coagulants from the seeds of Vigna unguiculata and Parkinsonia aculeata. Environmental Technology, 28, 671-681.

V. V.Marobhe, N.J., Dalhammar, G., Gunaratna, K.R., 2008. Effect of coagulant protein from Vigna and Parkinsonia on bacteria isolated from Ruvu River in Tanzania (Submitted to the World Journal of Microbiology and Biotechnology on 15 March 2008).

VI. Marobhe, N.J., Renman, G., Msafiri, J., Mgana S. 2008. Purification of water from small man-made reservoirs by coagulation using purified proteins from Vigna and Parkinsonia seeds (Submitted to Separation and Purification Technology on 5 March 2008).

Papers are reproduced with permission of the journal concerned


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ABBREVIATIONS

CF Citrus fruit juice CM Carboxymethyle DAWASA Dar es Salaam Water Supply and Sewerage Authority DBPs Disinfection-by Products FC Fecal coliforms FTU Formazin turbidity units GOT Government of Tanzania GS Grain size IEX Ion exchange LBB Buffered lauryl broth mg/L milligram per litre µS/cm micro siemens per cm, a measure of electrical conductivity (EC) MO Moringa oleifera MOCP Moringa oleifera coagulant protein MS Mass spectrometry NCIB National Center for Biotechnology Information NOM Natural organic matter NTU Nephelometric unit OD Optical density PACE Parkinsonia aculeata crude seed extract PAP Parkinsonia aculeata protein PPT Parts per thousand RPM Revolution per minute RWSSPs Rural Water Supply and Sanitation Projects SD Standard deviation SDS- PAGE Sodium dodecyl sulphate-Polyacylamide gel electrophoresis THMs Trihalomethanes URT United Republic of Tanzania WHO World Health Organisation WEO Ward Executive Officer VEO Village Executive Officer VUCE Vigna unguiculata crude seed extract VUP Vigna unguiculata protein UV Ultra violet


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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ........................................................................................................................... V LIST OF PAPERS APPENDED ................................................................................................................ VII ABBREVIATIONS.......................................................................................................................................IX TABLE OF CONTENTS.............................................................................................................................XI ABSTRACT..................................................................................................................................................... 1 1 INTRODUCTION....................................................................................................................................... 1

1.1 Background ...................................................................................................................................................... 1 1.2 Motivation........................................................................................................................................................ 2 1.3 Objectives ........................................................................................................................................................ 3 1.4 Thesis outline ................................................................................................................................................... 3

2 OVERVIEW OF WATER SUPPLY IN DEVELOPING COUNTRIES .................................................... 3 2.1 Urban and rural water supply ........................................................................................................................... 3

3 REVIEW OF CONVENTIONAL SURFACE WATER TREATMENT.................................................... 4 3.1 Coagulation and flocculation ............................................................................................................................ 4

3.1.1 Factors affecting coagulation-flocculation...............................................................................................................................5 3.1.2 Health risks associated with chemical coagulation and flocculation........................................................................................6

3.2 Water disinfection and associated health risks.................................................................................................. 6 4 REVIEW OF TRADITIONAL PLANT MATERIALS USED IN WATER TREATMENT ..................... 6

4.1 Traditional natural coagulants .......................................................................................................................... 6 4.1.1 Performance of natural coagulants ........................................................................................................................................7

4.4 Natural plant materials for water treatment in Tanzania .................................................................................. 8 4.4.1 Parkinsonia aculeata ...........................................................................................................................................................8 4.4.2 Vigna unguiculata...............................................................................................................................................................9 4.4.3 Voandzeia subterranea........................................................................................................................................................9 4.4.4 Citrus aurantifolia...............................................................................................................................................................9

5 MATERIALS AND METHODS ................................................................................................................. 9 5.1 Urban and rural water supply in Tanzania ........................................................................................................ 9 5.2 Coagulation experiments under varying physical parameters (Paper III)....................................................... 10

5.2.1 Source of seeds and their chemical composition ....................................................................................................................10 5.2.2 Preparation of seed powder and crude seed extracts .............................................................................................................10 5.2.3 Source of water samples......................................................................................................................................................11 5.2.4 Experimental and analytical procedures .............................................................................................................................11

5.3 Purification and characterisation of active coagulant proteins (Paper IV) ...................................................... 11 5.3.2 Characterisation and analytical procedures .........................................................................................................................11 5.3.2.1 SDS-PAGE Electrophoresis.........................................................................................................................................11

5.4 Antimicrobial effect (Paper V) ...................................................................................................................... 12 5.4.1 Bacterial isolation and identification...................................................................................................................................12 5.4.2 Flocculation and growth inhibition .....................................................................................................................................12 5.5.1 Sampling of charco dam water and coagulation experiments ...............................................................................................12 5.5.2 Analytical methods ............................................................................................................................................................13

6 RESULTS AND DISCUSSION..................................................................................................................13 6.1 Review of urban and rural water supply services in Tanzania (Paper I).......................................................... 13

6.1.1 Urban water supply: The case of Dar es Salaam city .........................................................................................................13 6.1.2 Rural water supply: The case of Singida rural district ........................................................................................................15


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6.2.1 Water supply sources..........................................................................................................................................................16 6.2.2 Traditional water coagulation: Procedures and performances...............................................................................................17

6.3 Effect of varying physical parameters on coagulation of river water with crude seed extracts (Paper III)...... 18 6.3.1 Optimum dosage and seed powder grain size effect ..............................................................................................................18 6.3.2 Effect of raw water pH.....................................................................................................................................................19 6.3.3 Mixing speed and duration ................................................................................................................................................20

6.4 Purification and characterisation of coagulant proteins (Paper IV) ................................................................ 21 6.4.1 Protein purification ............................................................................................................................................................21 6.4.2 Characterisation ................................................................................................................................................................22

6.5 Antimicrobial activity on tropical river water bacteria (Paper V) .................................................................... 27 6.6 Physico-chemical quality (Paper VI) ............................................................................................................... 29

7 CONCLUSIONS AND RECOMMENDATIONS.................................................................................... 30 8 AREAS FOR FURTHER RESEARCH......................................................................................................31 9 REFERENCES.......................................................................................................................................... 32


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ABSTRACT

Water supply services in urban and rural areas of Tanzania were reviewed and specific studies were carried out on water supply and on purification of turbid water sources using locally avail-able plant materials in rural villages of Singida Rural District. The review showed that large pro-portions of urban and rural populations in Tanzania face acute water supply problems mainly due to poor planning, implementation and management of water supply projects, including an inabil-ity to address social, technical, operation and maintenance and financial issues. Laboratory-scale experiments studied the effectiveness of crude seed extracts (CSEs) and purified proteins of Vigna unguiculata (VUP), Parkinsonia aculeata (PAP) and Voandzeia subterranea (VS) seeds, which are used traditionally for clarification of turbid water. The VUP and PAP were purified from CSEs using simple and straightforward two-step ion exchange chromatography. The coagulant proteins are thermoresistant and have a wide pH range for coagulation activity. Coagulation of turbid waters with CSEs, VUP and PAP produced low sludge volumes and removed turbidity along with other inorganic contaminants in line with Tanzania drinking water quality standards. The PAP also showed antimicrobial effect against river water bacteria. Citrus fruit juice (CF) en-hanced the coagulation of turbid water by CSEs and inhibited bacterial growth, rendering it useful for disinfection of water prior to drinking in rural areas. It was concluded that natural coagulants should not be regarded as a panacea for rural water supply problems, but rather a tool in the development of sustainable water supply services in Tanzania. Keywords: Antibacterial effect; Coagulant proteins; Vigna unguiculata; Parkinsonia acu-leata; Ion exchange; Tanzania water supply

1 INTRODUCTION

1.1 Background

Despite widespread recognition of the importance of improved water and sanita-tion and heavy investment by international donors and governments in developing countries in extending water supply systems, more than half the population of rural areas still lack access to clean drinking water (Rondinelli, 1991). The rapid pace of urbani-sation in developing countries is also in-creasing the demand for water in peri-urban communities and cities. Contaminated drinking water and inadequate supplies of water for personal hygiene and poor sanita-tion are responsible for about 4 billion cases of diarrhoea each year that cause 2.2 million deaths, mostly among children under the age of five (WHO, 2000a; WHO, 2003). The deaths from water-borne and water-washed diseases in developing countries are likely to increase due to widespread immuno-compromisation in people with HIV/AIDS, which increases the risk of contracting diarrhoeal disease (Kahinda et al., 2007). In many developing countries, potable water is collected from communal sources which

are either unimproved, such as unprotected wells, unprotected springs and rivers, or improved such as protected wells, boreholes and public standpipes (WHO & UNICEF, 2000a). In most cases the improved com-munal sources are malfunctioning or com-pletely broken down due to poor operation and maintenance, financial constraints, poor project planning and inefficient management systems (Howard & Bartram, 2005; Mugabi et al., 2007a). Due to chronic problems associated with improved water supply such as the cost (both financial and time required to collect water), the quality and the reliabil-ity of the supply, large populations in devel-oping countries, especially in rural areas, are forced to use traditional sources that are polluted. Given the time and costs of developing or rehabilitating improved communal water supply sources, it can be cost-effective in certain circumstances to rely on existing traditional sources (Clasen & Bastable, 2003). This is possible if traditional water sources can be treated using water purifica-tion methods, which are inexpensive and suitable under local conditions. One method


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that has been practised by people in some parts of the developing world is the use of locally available natural coagulants to re-moval turbidity and bacteria in surface water (Schulz & Okun, 1984; Madsen et al., 1987; Ghebremichael et al., 2005). The natural coagulation technology has the potential of replacing or supplementing costly water treatment chemicals in poor countries. In addition, the natural coagulants avoid the human and environmental problems that are associated with the use of water treatment chemicals (Crapper et al., 1973; Christopher et al., 1995; Kaggwa et al., 2001). Using natural coagulants in developing countries could alleviate their economic situation and allow further extension of water supply services to rural areas (Ndabigengesere & Narasiah, 1998). The current research was therefore initiated in order to assess the water supply services in urban and rural areas of Tanzania and to explore the motives and mechanisms behind traditional treatment of domestic water supply. Together, these aims formed the basis for a detailed study on the perform-ance of locally available natural coagulants for production of potable water for rural and peri-urban communities in Tanzania and other developing countries.

1.2 Motivation

A large proportion of the population in urban and rural areas of Tanzania lack access to a reliable water supply, regardless of quality. The studies reported in this thesis

were motivated by the serious water supply problems prevailing in many rural areas and peri-urban areas where people are obliged to use alternative sources that are polluted, with rampant water-borne diseases through-out the country (Fig. 1). Women in rural villages traditionally treat water from these sources using locally available plant seed powder to remove suspended matter. How-ever, it was discovered that although such plant seeds seem to be effective water clari-fiers, good coagulation results were not always obtained due to lack of understand-ing on how the seed powder coagulates turbid waters. Some plant species that pro-duce seeds for water treatment, especially Parkinsonia aculeata, have multiple uses and could be cultivated intensively and help to improve the quality of life of the rural popu-lation. It was anticipated that appraisal of locally available coagulants in scientific studies would not only benefit the rural population but also the urban water utilities, which often produce insufficiently treated water due to inadequate resources at water treatment plants. Thus, it was deemed im-portant to conduct investigations on the performance of traditional coagulants with the aim of producing more potable water. Although Moringa oleifera seed, one of the most effective natural coagulants, has been extensively studied, scientific investigations into the coagulation potential of other lo-cally available natural coagulants in other parts of the developing world are lacking.

Fig. 1. The water supply problems in rural areas in the drier regions of Tanzania. (a) left - Animals carry water long distances; (b) center and (c) right- people fetch water from tradi-tional wells and small dams. Photo: Nancy Marobhe 2006.


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1.3 Objectives

The objectives of this study were to review the water supply services in selected urban and rural areas of Tanzania, to investigate the existence of traditional water coagulation methods and to carry out laboratory studies on the performance of locally available natural coagulants including Vigna unguicu-lata, Parkinsonia aculeata and Voandzeia subter-ranea seeds under varying physical operating parameters. The effects of local coagulants on physico-chemical quality parameters of treated water and their antimicrobial effects on bacteria isolated from river water were also studied. An additional objective was to isolate and characterise the active coagulat-ing agent in the seeds. The specific objectives include: i) To assess the water supply services in Tanzania and investigate traditional methods and procedures for purification of turbid water in rural areas of Tanzania ii) To analyse the effect of varying physical parameters on performance of crude ex-tracts of the locally available seeds (Vigna unguiculata, Parkinsonia aculeata and Voandzeia subterranea seeds) during coagulation of river water samples. iii) To isolate and characterize an active coagulant component in the powdered seeds. iv) To investigate the effects of crude ex-tracts and purified coagulant proteins of Vigna unguiculata and Parkinsonia aculeata seeds on physical and chemical quality pa-rameters of treated water. v) To investigate the effect of purified co-agulant proteins on bacteria isolated from Ruvu River.

1.4 Thesis outline

The water supply situation in developing countries is presented in section 2. A review of conventional water treatment methods with focus on theory and practice of coagu-lation-flocculation, sedimentation and disin-fection is presented in section 3. Application of natural materials in water treatment is covered in section 4. The summary of mate-rials and methods used in the study is cov-

ered in section 5. The results and discussions from the review of water supply situation in Tanzania, traditional water treatment proc-esses, performance of crude seed extracts under varying physical factors, purification and characterisation of coagulant seed pro-teins and the effect of coagulant proteins on physico-chemical quality of treated water including the antimicrobial effects is pre-sented in section 6. Finally, conclusions from the study including proposed areas for further study are given in sections 7 and 8 respectively.

2 OVERVIEW OF WATER

SUPPLY IN DEVELOPING

COUNTRIES

2.1 Urban and rural water supply

Many millions of people, in particular throughout the developing world, use unre-liable water supplies of poor quality, which are costly and are distant from their homes (WHO & UNICEF, 2000a). In particular, water supplied to rural communities world-wide is generally of poorer quality than that in urban areas (Chongsuvivatwong et al., 1994; Sugita, 2006). It is generally accepted that the poor tend to be more vulnerable to disease and have least access to basic ser-vices and water supply is no exception (Pokhrel & Viraraghavan, 2004). The evi-dence suggests that interventions targeted at poor populations could provide significant health benefits and contribute to poverty alleviation (DFID, 2001; WHO, 2002; Gun-dry et al., 2004). Shortage of clean drinking water continues to be a significant problem in many parts of sub-Saharan Africa. The quality and cover-age of services from most of the urban water utilities south of the Sahara remain poor. The situation is becoming worse with high urban population growth rates of 2-6% per year (Njiru & Sansom, 2003). For a long time, measures taken by governments to address gaps in service coverage have con-centrated on building new infrastructure, with little attention being given to improving the efficiency and productivity of water utilities (Wolff & Gleick, 2002). The situa-


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tion is aggravated by consumption of im-ported chemicals such as alum and chlorine in water treatment processes, since these are unaffordable for most poor countries. In Africa, many obstacles, which include bu-reaucratic barriers between ministries, mea-gre funds allocated to rural areas and too few technically qualified persons, have retarded progress towards safe and adequate water supplies and improved sanitation (Muyibi, 1992; Isley, 1995). Factors such as poor reliability of water supply, affordability and distance between a water source and the home have collectively led households to depend on unsafe sources, to reduce the volume of water used for hygiene purposes and to reduce spending on other essential items (Lloyd & Bartram 1991; Cairncross & Kinnear, 1992; Howard, 2002). The attempts to provide potable water by application of inappropriate technologies in urban and rural areas of developing coun-tries have led to severe difficulties and even failure of the projects. Mintz et al. (2001); Banerjee et al. (2007), pointed out that as the provision of safe water infrastructure to the world’s poor will take decades and require significant investment, the health of these people could be protected in the short to medium term by scaling up access to alterna-tive water quality interventions that are effective and inexpensive. The appraisal of natural coagulation technology in the con-text of the present study is among such interventions, which could help a large proportion of the population in rural areas obtain clean drinking water.

3 REVIEW OF

CONVENTIONAL SURFACE

WATER TREATMENT

Water treatment usually comprises water clarification and disinfection processes (Suarez et al., 2002). In conventional water treatment a series of processes including coagulation, flocculation, sedimentation, filtration and disinfection are often involved (Edzwald et al., 1989; AWWA, 1990). A combination of several processes is usually needed to improve the quality of raw water depending on the type of water quality

problems present, the desired quality of the treated water, the costs of different treat-ments and the size of the water system (Kalibbala, 2007). However, it should be understood that the aim in this section is not to review all of the processes that take place in conventional water treatment, but only those processes that are of great significance in water treat-ment using traditional natural coagulants.

3.1 Coagulation and flocculation

Treatment of water to remove turbidity is essential for large- and small-scale (e.g. household) production of drinking water. The removal of turbidity in water treatment is essential because naturally suspended particles are transport vehicles for undesir-able organic and inorganic contaminants, taste-, odour- and colour-imparting com-pounds and pathogenic organisms (Raghu-wanshi et al., 2002). The turbidity of water often results from the presence of colloidal particles that have a net negative surface charge. Thus, electrostatic forces prevent them from agglomerating, making it impos-sible to remove them by sedimentation without the aid of coagulants, which carry counter-ions. Hydrolysing metal salts based on aluminium or iron, also known as pri-mary coagulants, are very widely used in conventional water treatment processes (Diaz et al., 1999). The high cationic charge of these two metal salts makes them effec-tive for destabilising colloids. They act by neutralising the negative charges of the stable colloidal particles (coagulation) and are followed by the addition of organic coagulant aids that enhance particle collision and agglomeration of neutral particles to form dense flocs (flocculation) that can settle easily. Destabilisation of colloidal particles in water is accomplished via ad-sorption and charge neutralisation, adsorp-tion and interparticle bridging, enmeshment in a precipitate and double layer compres-sion (Amirtharajah & O´Melia, 1990; Greg-ory & Duan, 2001). In addition to inorganic coagulants (alumin-ium and iron salts), water-soluble organic polymers, also known as polyelectrolytes, are


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used in water treatment to remove turbidity. Polyelectrolytes can be either natural or synthetic and they can be used as both primary coagulants and coagulant aids (Sanky, 1978; Hashimoto et al., 1991). Polye-lectrolyte primary coagulants are cationic with high charge density and low molecular weight, while synthetic polyelectrolyte co-agulant aids have relatively high molecular weights and facilitate flocculation through inter-particle bridging (Gregory & Duan, 2001). Although polyelectrolytes are more expensive than aluminium and iron salts in terms of material cost, overall operating costs can be lower because of reduced need for pH adjustment, lower sludge volumes, no increase in total dissolved solids in treated water and shorter settling time (Mahvi & Razavi, 2005). However, polyelec-trolytes are not readily available and also costly for most parts of the developing world. The natural polyelectrolytes such as water-soluble proteins released from crushed seed kernels are potential alternatives to synthetic polyelectrolytes. The merits of natural polyelectrolytes (which are extracted mainly from certain plants) over synthetic include safety to human health, biodegrad-ability and a wide effective range of floccula-tion for various colloidal suspensions (Kawamura, 1991; Özacar & Şengil, 2003).

3.1.1 Factors affecting coagulation-flocculation Coagulation and flocculation processes are dependent on numerous inter-related fac-tors, which sometimes makes optimisation of the processes cumbersome. Such factors include the characteristics of the water source, raw water pH, alkalinity and tem-perature, the type of coagulant and coagu-lant aids and their order of addition, dose rates of coagulants, the degree and time of mixing provided for chemical dispersion and floc formation (Rossi & Ward, 1993; Kalib-bala, 2007). For water with low alkalinity coagulant can consume virtually all of the available alkalinity, hence lowering the pH to a level that hinders effective treatment, while high alkalinity waters may require additional chemicals to lower the pH to values favour-able for coagulation.

The performance of the hydrolysing metal salts is significantly influenced by the pH of solution and they have a good coagulation effect within a certain pH range of the water. The coagulation process in water treatment can be modified to facilitate the removal of dissolved organic matter (en-hanced coagulation), which has been re-ported to occur optimally at pH 5-6 (Greg-ory & Duan, 2001) and at maximum rate at pH 4 (Gregor et al., 1997). Low temperature affects the coagulation and flocculation process by altering the coagulant solubility, increasing the water viscosity and retarding the kinetics of hydrolysis reactions and particle flocculation. Poly-aluminium coagu-lants are more effective in cold water than alum, as they are pre-hydrolysed. To achieve effective coagulation, proper mixing is also necessary to allow active coagulant species to be transferred onto turbid water particles. Proper mixing after addition of coagulants into raw water facili-tates optimum removal of fine particles in the supernatant. This is because very fine particles become transformed to aggregates under good mixing condition (Kan et al., 2002). It is commonly observed that particles are destabilised by small amounts of hydrolysing metal salts and that optimum destabilisation corresponds with the neutralisation of parti-cle charge. Larger amounts of coagulants cause charge reversal so that the particles become positively charged and thus restabi-lisation occurs, which results in elevated turbidity levels. Thus, careful control of coagulant dosage is needed to give optimum destabilisation (Gregory & Duan, 2001) and this is determined to a great extent by the consistency of raw water quality. As the present study investigated the performance of natural coagulants that had not been investigated previously, identification of some factors that influence the coagulation process was necessary.


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3.1.2 Health risks associated with chemical coagulation and flocculation Although the water treatment chemicals are effective and used worldwide, scientific evi-dence shows that exposure to chemicals during coagulation with metal salts could be associated with adverse health effects (Dris-coll &. Letterman, 1995). Aluminum, which is the most major component of aluminium sulphate (alum), polyaluminium chloride (PAC) and polyaluminium silico sulphate (PASS), could induce Alzheimer’s disease and other similar related problems that are associ-ated with residual aluminium in treated water (Miller et al., 1984; AWWA, 1990). Moreover, monomers of some synthetic organic poly-mers such as acrylamide have neurotoxicity and strong carcinogenic properties (Crapper et al., 1973; Hashimoto et al., 1991).

3.2 Water disinfection and associated health risks

Disinfection of the clarified water prevents the growth of microorganisms both in the plant and in the distribution system, thus protecting the public from water-borne diseases. Like chemical coagulants, disinfec-tants (chlorine in particular) combine with natural organic matter (NOM) that may be present in water to form trihalomethanes (THMs), which are carcinogenic and/or mutagenic by–products (Tokmak et al., 2004). These THM cannot be removed by conventional treatment methods and thus water to be chlorinated should either be free from natural organics, or if NOM is present an alternative disinfectant should be used. Alternative disinfectants such as chlorine dioxide, chloramines and ozone are also associated with the formation of disinfection by- products (DBPs) that are toxic com-pounds and impart objectionable taste and odour (Kiely, 1997; Sadiq & Rodriguez, 2004). Irradiation with ultraviolet (UV) light is a promising alternative method of disin-fection but it is expensive and leaves no residue and hence another disinfectant is required to disable bacteria and viruses. In addition, UV light can react with nitrate in water to produce nitrite, the precursor for methaemoglobinaemia in infants (Mole et al.,

1999). Although the risks associated with DBPs are low, associations with such dis-eases cannot not be ruled out unambigu-ously (Van Benchosten & Edzwald, 1990; Boisvert et al., 1997; Bove et al., 2002; Woo et al., 2002). The search for disinfectants that are cheap, maintain acceptable microbiologi-cal quality and avoid chemical risks is one of the biggest challenges facing the water treatment industry.

4 REVIEW OF TRADITIONAL

PLANT MATERIALS USED IN

WATER TREATMENT

4.1 Traditional natural coagulants

The use of natural materials for treatment of drinking water in some parts of the world has been recorded throughout human his-tory. However, the natural materials have not been recognised or duly supported due to lack of knowledge on their exact nature and the mechanism by which they function. As a consequence, the natural materials have been unable to compete effectively with the commonly used water chemicals (Ndabigengesere & Narasiah, 1998). In recent years there has been a resurgence of interest in using naturally occurring alterna-tives to currently used coagulants for water treatment in developing countries (Jahn, 1981, 1988), mainly due to cost implications that are associated with inorganic chemicals, synthetic organic polymers and disinfectants (Schultz & Okun, 1984, Ndabigengesere & Narasiah, 1995). There is also an interest in reusing some of the by-products from natu-ral coagulants in other enterprises (Kawa-mura, 1991). Traditionally, treatment of turbid surface water sources is carried out at household level using local materials of plant or animal origin (Okun, 1984; Jahn, 1988, 2001) For example, rural people in Sudan and Malawi, who depend on muddy water from rivers or intermittent streams, natural rain ponds and artificial rain-water catchments for domestic water supply, treat water fetched from such sources using Moringa seeds and other plant and soil materials (Jahn & Dirar, 1979; Eilert et al., 1981). In India, crushed seeds of the


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nirmali tree (Strychnos potatorum) have been used for centuries to clarify muddy water (Tripathi et al., 1976). In Peru, villagers clarify turbid water using cactus leaves and ‘tuna’ (e.g. Opuntia ficus indica Mill.). Tradi-tional water treatment using crushed or chopped Maerua pseudopetalosa (kordala) roots is practised in some parts of Sudan. In Northern Chad and villages around Maiduguri in Northern Nigeria, people use wood ash as a natural water coagulant (Jahn & Dirar, 1979). Knowledge on natural co-agulants is widespread in many parts of the developing world and therefore there is good potential for such knowledge to be used efficiently provided concerted efforts can be devoted to maximising their per-formance through research.

4.1.1 Performance of natural coagulants Studies have been conducted to evaluate the coagulation efficiency of many plant materi-als including extract of Moringa seeds (Ndabigengesere et al., 1995; Ndabigengesere & Narasiah, 1998; Babu & Chaudhuri, 2005; Ghebremichael et al., 2005; Bhuptawat et al., 2007); okra and nirmali seeds (Al-Samawi & Shokralla, 1996); tamarind seeds (Bhole, 1995); extracts of Prosopis juliflora and Cactus latifaria (Diaz et al., 1999); and vegetable tannins (Özacar & Şengil, 2003). The coagu-lation potential of chitosan, one of the effective natural coagulants extracted from the organic skeletal substance in the shells of crustacean has been studied by Huang & Chen (1996); Huang et al. (2000); Divakaran & Pillai (2004). The crude extract of M. oleifera seed is the most extensively studied natural coagulant. Jahn (1984) conducted a preliminary study aimed at improving the coagulation efficiency of Moringa seed based on traditional methods used by women in Sudan. This study revealed that scientific advice and support are necessary to improve the traditional water clarification methods. Laboratory studies using natural and artifi-cial water samples have shown that Moringa seeds are highly effective in coagulating and removing suspended solids from water containing medium to high initial turbidity (Sutherland et al., 1989).

It has also been observed that the reduction in turbidity is associated with significant improvements in bacteriological quality (Grabow et al., 1985; Madsen et al., 1987; Ghebremichael et al., 2005). There are only a few reports on the effect of natural coagu-lants on bacteriological quality of the treated water, as most studies on natural coagulants concentrate on efficacy of turbidity removal. Tripathi et al. (1976) showed that the crude extract of Nirmali seed reduced 50-52% of bacteria after 30 min of settling time of turbid water coagulated by 0.5 mg/L of seed extract. The crude extract and purified proteins of M. oleifera seed have been re-ported to possess antimicrobial properties (Tripathi et al., 1976; Madsen et al., 1987; Suarez et al., 2003; Ghebremichael et al., 2005). Furthermore, Eilert et al. (1981) identified an active antibiotic component 4(α-L-Rhamnosyloxy) benzyl isothiocyanate in M. oleifera and M. stenopetala seeds that acts on several bacteria and fungi. Studies on the antimicrobial effects of Mor-inga seeds have shown that there are specific mechanisms of action of antimicrobial agents on microorganisms (Conte et al., 2007) in addition to the removal of floccu-lated microorganisms by settling along with the colloidal particles in properly coagulated and flocculated water (Casey, 1997). The effect of natural coagulants on turbidity removal and the antimicrobial properties against microorganisms may render them applicable for simultaneous coagulation and disinfection of water for rural and peri-urban people in developing countries. 4.2 Natural biocides

Recent studies have shown that acidification of food and drinking water with acidic consumables, notably Citrus (lime or lemon juice), protects people against cholera (D´Aquino & Teves, 1994; Rodrigues et al., 1997). However, few formal evaluations of the disinfection effects of lime and lemon juice have been conducted and information on their disinfection potential for naturally contaminated water sources is scarce. Due to the chronic water-borne diseases in most parts of the developing countries, efforts are being made to identify less costly, more


8

appropriate and non-toxic biocides to be used in communities without treated or protected water supplies (Anderson, 1991; D´Aquino & Teves, 1994). In this regard lime and lemon trees, that are native in most parts of the tropical and subtropical world, could be among the potential sources of natural biocides of choice, although system-atic studies to evaluate their performance is of paramount importance. 4.3 Purification of coagulating seed proteins

The main problem with crude seed extracts is the release of organic load into the treated water, which encourages microbial decom-position of organic compounds that cause colour, taste and odour problems. These problems are amplified when the treated water is stored for long periods (Ndabigengesere & Narasiah, 1998; Ghe-bremichael et al., 2005). To overcome these problems, researchers working with Moringa have been trying to develop methods for extracting the active coagulating component from Moringa seeds and separate it from inactive proteinous and non-proteinous materials. Ndabigengesere et al. (1995) puri-fied active coagulant protein from Moringa seed by precipitation with ammonium sul-phate, ultrafiltration, dialysis, ion-exchange chromatography and lyophilisation. How-ever, the method is complex and too costly for use in poor countries. Ghebremichael et al. (2005) have developed a simple method based on ion exchange (IEX) chromatogra-phy for purification of M. oleifera coagulant protein that is very appropriate for use in poor countries. The leguminous seed ex-tracts used in the present study were a het-erogeneous mixture of organic and inorganic compounds. Therefore, it was necessary to isolate the active coagulating agent using cheap and straightforward methods that can be easily adopted in poor communities.

4.4 Natural plant materials for water treatment in Tanzania

Most of the plant seeds that are used by rural women in semi-arid parts of Tanzania for treatment of drinking water belong to the genus Fabaceae (legume or pea family).

Some important characteristics of plant species that produce the coagulant seeds are described below.

4.4.1 Parkinsonia aculeata P. aculeata (Jerusalem thorn), also known by vernacular name mkeketa, is a shrubby thorny tree (3-10 m tall) that belongs to the family Fabaceae. It is fast growing, drought tolerant, resistant to biotic stresses and able to grow in different soil types. P. aculeata seeds have a thick, hard coat that allows them to remain viable for many years. This species is an indigenous or planted tree and has spread throughout the tropics for differ-ent uses including fodder, firewood, con-struction materials, fencing and shade (For-oughbakhch et al., 2001) and folk medicine (Andrade-Cetto & Heinrich, 2005; Leite et al., 2007). The large, fragrant, golden-yellow flowers easily attract bees and may give people the opportunity for beekeeping activities. Like many legumes, P. aculeata seeds are very rich in crude protein and digestible fibre, making them a potential source of low-cost protein. However, the seeds are among the most under-utilised leguminous seeds due to lack of adequate knowledge regarding their nutritional com-position. The fruit pulp (pod) can also be eaten and has up to 60% sugar content (Foroughbakhch et al., 2001; Prakash et al., 2001). The pod of the seed contains 1-4 seeds but occasionally up to 11 (Fig. 2). Mature trees typically produce about 5,000 seeds per year, but can produce in excess of 13,000. The rural populations in the central dry areas of Tanzania are mainly crop farm-ers, although keeping of cattle is also prac-tised. Besides clean water, they also need to restore degraded land, improve the fertility of agricultural land and need other basic items such as firewood, building poles, fodder and income-generating activities. The multipurpose, fast growing and nitrogen-fixing P. aculeata trees appear to be the most promising for improvement of the quality of life for people in semi-arid areas.


9

4.4.2 Vigna unguiculata Vi. unguiculata or cowpea is a heat loving, drought resistant crop and plays a role in low input production systems, particularly on small-scale farms (Quass, 1995; Graham & Vance, 2003). Cowpeas are rich in pro-tein and hence regarded as a seed vegetable that provides an inexpensive alternative source of protein in Africa and other parts of the developing world (Popelka et al., 2004). They can be grown as a multi purpose crop for instance the green pods can be used as a vegetable, the remaining part as live-stock fodder and the dry cowpeas as the seed food protein. Cowpeas produce 8-20 seeds per pod and weigh between 5-30 g per100 seeds.

4.4.3 Voandzeia subterranea Vo. subterranea (Bambara groundnut) is an indigenous African legume grown primarily by subsistence farmers (Enwere & Hung, 1996). It is tolerant to drought and resistant to pests and disease, and yields in soils too poor to support the growth of other leg-umes such as groundnuts (Brough & Azam-Ali, 1992). The plant is grown in many parts of Tanzania as food and is commonly used for traditional water treatment in villages located in dry areas.

4.4.4 Citrus aurantifolia C. aurantifolia (lime) belongs to the family Rutaceae. Lime trees are cultivated through-out the tropics and in warm subtropical areas as a commercial crop. In addition to juice production, essential oil is one of the main by-products of citrus processing, usually obtained a by distillation process.

Lemon oil is one of the most important food-flavouring agents and both lemon and lime oil is also used in the production of perfumes and cosmetics (Vekiari et al., 2002; Gamarra et al., 2006). Traditionally, lime is an everyday food ingredient in Tanzania and other African countries. It is also used to prepare beverages and possesses a variety of medicinal applications (Al-Khayri & Al-Bahrany, 2001). It has been reported that the acidic juices have a protective effect on enteric pathogens present in food. Studies confirming the effect of limejuice on coagu-lation and disinfection of drinking water for use in many rural and peri-urban areas may improve the health situation in rural and urban areas of Tanzania that lack potable source and also increase the economic value of the lime.

5 MATERIALS AND

METHODS

5.1 Urban and rural water supply in Tanzania

The review of water supply services in Tanzania was conducted using Singida rural district and Dar es Salaam city as case study areas (Paper 1). Singida rural district is lo-cated in a semi-arid area of central Tanzania while, Dar es Salaam city is located along the coast of the Indian Ocean on the eastern side of Tanzania (Fig. 3). Literature studies, field observations and discussions with stakeholders were used to gather informa-tion about the historical development and social, technical, management, financial and traditional aspects of water supply in the case study areas.

Specific studies on water supply problems were conducted in four villages including, Msange, Mipilo, Mangida and Sifunga, which are located in Singida rural district (Paper II). Structured questionnaires (open-ended and closed questions), focus group discus-sions with key informants and participatory methods were employed to study the exist-ing socio-economic activities, water supply situation and traditional water coagulation methods. Questionnaire interviews were conducted with between 49 and 85 houses,

Fig. 2. Part of a P. aculeata tree showing the mature seed pods.


10

based mainly on systematic random sam-pling. In each village, the first nearest house was selected for sampling, followed by the systematic sampling of houses along the surveydirection. In villages where some houses were distant from each other, for reasons of convenience the nearest houses were selected for interview.

5.2 Coagulation experiments under varying physical parameters (Paper III)

5.2.1 Source of seeds and their chemical composition Dry seeds of Vigna unguiculata (VU), Parkin-sonia aculeata (PA) and Voandzeia subterranea (VS) were obtained from households in Singida rural villages or from local markets and stored at room temperature. The chemi-cal composition of the seed powder of VU, PA and VS was analysed at the Government Chemical Laboratory, Dar es Salaam.

5.2.2 Preparation of seed powder and crude seed extracts The seed kernels were ground into fine powder in a kitchen blender or by pestle and mortar. Seed powders with different grain sizes were obtained by laboratory sieving through mesh sizes that ranged from 0.200 to 0.850 mm using Laborsrebmoschine model EML 200-67. Crude seed extracts (CSEs) were prepared from the seed powder as 1-5% solution (W/V) in distilled water. The suspensions were stirred for 15 min at room temperature (25 ºC ±2) and then filtered through nylon cloth and/or Whatman No. 3 filter to obtain the CSEs. More refined CSEs were ob-tained by filtering the prepared CSEs using either fine fibre material with pore size of about 10 µm, or fibreglass filter with pore size of 0.45 µm, or centrifugation (3000 RPM, 2 min).

L. Victoria

L. Tanganyika

INDIAN OCEANSingida

Singida Rural District

Dar es SalaamL. N

yasa

TANZANIA

35

35

40

40

5 5

00

0

20

AFRICA

Fig. 3. Index map of Tanzania showing the location of the case study areas.


11

5.2.3 Source of water samples Water samples were collected in 20-L plastic buckets from the Ruvu River at Mlandizi, Tanzania, during the dry season of 2006. Samples with different turbidities were prepared by mixing the sampled clear and muddy river water to produce samples with low (50 FTU), medium (205 FTU) and high (500 FTU) turbidity. The muddy water was stirred and allowed to settle for 5 min before mixing with clear water. No fixed volumes of clear and muddy water were established to produce a particular turbidity level.

5.2.4 Experimental and analytical procedures The effect of varying physical parameters on coagulation of turbid water with the CSEs was evaluated using a jar tester (Model Phipps and Bird-PB-700TM). Lime (Citrus aurantifolia), prepared as 1% solution (W/V) from crystals was used to regulate the pH of water samples. Water samples with different turbidities and seed powder with different grain sizes were obtained as described in sections 5.2.3 and 5.2.2 respectively. The effects of slow and rapid mixing intensity and duration on coagulation were deter-mined and the mean velocity gradients were calculated using equation (1). The computa-tion of the mean velocity gradients using relevant experimental values used in this study is shown in Paper III.

VPG n

µ= (1)

Where: G = velocity gradient (s-1) P

n = power dissipated (W)

V = volume of water (m3) µ

= dynamic viscosity of water (N s m-2)

Preliminary coagulation experiments were conducted at rapid and slow mixing speeds of 175 RPM and 40 RPM for durations of 3 min and 40 min respectively. Coagulant dosages were added into beakers during rapid mixing. After settling for 30 min, samples were drawn at 5 cm from the sur-face and residual turbidity were measured using a spectrophotometer model 21 D at wavelength 450 nm.

5.3 Purification and characterisation of active coagulant proteins (Paper IV)

5.3.1 Purification Active coagulant proteins in the CSEs of VU and PA were purified by batch IEX chromatography and gel filtration. The IEX chromatography was carried out using 1 mL of CM sepharose fast-flow matrix cation exchanger with bead size 45-165 µm (Bio-Rad, Sweden). The matrix was equilibrated with 10 mM ammonium acetate buffer (pH 7.2) and CSEs were added at a dosage of 25 mg/L of the matrix. The bound proteins were eluted with 0.3 and 0.6 M NaCl in the same buffer. Eluted proteins were desalted through PD-10 columns pre-packed with sephadex G-25 (Amersham Biosciences AB) using Milli Q water.

5.3.2 Characterisation and analytical proce-dures

5.3.2.1 SDS-PAGE Electrophoresis The molecular masses and purity of the purified proteins were analysed by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) on 10% acrylamide gel using a Mini-PROTEN 2 apparatus (Bio-Rad) and tris-tricine SDS-PAGE (Hultmark et al., 1983). Protein dye binding method (Bradford, 1976) was used to quantify protein concentrations in CSEs samples using bovine serum albumin as a standard. 5.3.2.2 Coagulation activity using small sample volume Raw water samples: A stock of synthetic turbid water samples was prepared by suspending 10 g of kaolin clay in 1 L of tap water. The suspension was stirred for 30 min and left to stand for 24 hr to hydrate particles. The desired turbidity was prepared by mixing a fraction of de-canted clay suspension with tap water. The pH of water samples was kept constant at 7.3 ±1 using 0.1 M HCl. Preparation of Citrus juice and alum: Citrus fruit juice (CF) was squeezed from ripe fruits and filtered through a fine fibre material. The filtrate was allowed to stand for 2 hr and the supernatant was decanted and stored. The pH of the filtrate was 2.55.


12

Aqueous aluminium sulphate (alum) was also prepared as a 5% (W/V) solution. Coagulation activity: One millilitre of clay suspension was em-ployed for measurement of coagulation activity of CSEs, purified proteins, CF and alum. Different dosages of coagulants were added to the water sample to produce a final volume of 1 mL in a semi-micro plastic cuvette (10x4x45 mm, Sarsted Aktiengesell-schaft & Co. Germany) and homogenised instantly. The samples were allowed to settle for 90 min or more and thereafter the ab-sorbancies were measured at 500 nm using UV-visible spectrophotometer (Cary 50 Bio). Coagulation activity was measured as the difference between the absorbance at zero time and absorbance after 90 min of settling time. 5.3.2.3 Effect of thermal treatment, pH and settling time Thermal stability of CSEs and purified proteins was determined at 40, 80 and 95 °C for 0.5 to 5 h in a heating block Different dosages of the cooled protein samples were added into the kaolin water samples to check their coagulation activity. The pH range for coagulation activity was 4.5 to 9.5, while floc-settling studies were carried out from 0.5 to 5 hr.

5.4 Antimicrobial effect (Paper V)

5.4.1 Bacterial isolation and identification Bacterial strains were isolated from water samples collected from the Ruvu River. The strains were isolated on nutrient and McConkey agar (Oxoid) plates at an incuba-tion temperature of 20, 30 and 37°C under aerobic and anaerobic conditions. Pure cultures were obtained by repeated streaking on nutrient agar and incubation at 30°C for 24-48 hr. Bacterial isolates were identified by cultural and morphological characteristics (Krieg & Holt, 1984) and biochemical tests according to Cappuccino & Sherman (1987). Moreover, representative Gram-positive and Gram- negative bacterial strains were identi-fied by API tests and 16S rRNA gene analy-sis (as detailed in paper V). The sequences

were run against the bacteria database using a blast search (NCBI).

5.4.2 Flocculation and growth inhibition Isolates representative of Gram-positive and Gram-negative bacterial spp. were cultured in 0.2 M phosphate buffered Lauryl broth (LBB) at 30°C with shaking overnight. The cultures were diluted 10x with diluted LBB to get the desired microbial density (OD620). In cell flocculation studies, dosages of CSEs and purified proteins were added to the culture suspensions and then these were incubated at 30°C for 4 hr. Cell aggregations were observed under the microscope (Olym-pus BX51 with AnalySIS) and images were captured using a Sony PC 120 camera. For growth curve studies, overnight bacterial cultures diluted in LLB were inoculated into sterile 96-well microtitre plates (Greiner bio-one). Ten microlitres of diluted proteins and CF were added to wells containing 90 µL bacterial cultures. The plates were incubated at 30ºC in a FLUOstar* OPTIMA (LabVi-sion, Sweden) spectrophotometer with shaking at 5 min intervals and changes in the OD620 values were recorded. 5.4.2.1 Effect of purified proteins, crude seed extracts and citrus juice on natural bacteria

The antimicrobial effect of the purified proteins, CSEs and CF was studied on natural river water bacteria using jar tests. Water samples were collected in sterile 1-L plastic bottles during the dry season. After determination of the initial pH, different doses of purified proteins, CSEs and CF were added, followed by rapid and slow mixing of samples. The dose of CF ranged from 0 to 0.9%. Faecal coliforms (FC) in the clarified water were determined using Stan-dard Method 9221E Faecal Coliform Mem-brane Filter Procedure (Standard Methods, 1998) after 30 min-24 hr of settling time. 5.5 Physico-chemical quality studies of coagulated charco dam water (Paper VI)

5.5.1 Sampling of charco dam water and coagulation experiments Water samples were collected in 10-L buckets from Nunguru charco dam, located at Mipilo


13

village in Singida rural district, during the dry season of 2007. Samples were collected from a pipe that discharges into a cattle trough. Coagulation-flocculation experiments were performed using jar tests at rapid and slow mixing speeds of 150 and 40 RPM for dura-tions of 5 and 30 min respectively. The qual-ity parameters of water coagulated with dif-ferent doses of CSEs, 0.6 M NaCl eluted proteins (un-desalted proteins) and alum were determined following the settling phase of 30 min.

5.5.2 Analytical methods Turbidity, pH, TDS, sludge volume, conduc-tivity, alkalinity, concentration of a cation (Fe2+) and anions (NO3

- and PO43-), and

COD in the treated water were analysed according to Standard Methods (1998), as described in Paper VI. For determination of the settled sludge volume, the water samples collected from the Ruvu River (205 NTU) were first coagulated with different dosages of 1% (W/V) CSE of VU and PA in jar test experiments and allowed to settle for 30 min. The clarified water was decanted and 50 mL of the sludge were transferred into settling columns. The volumes occupied by the suspension were recorded for 60 min at intervals of 5 min.

6 RESULTS AND DISCUSSION

6.1 Review of urban and rural water supply services in Tanzania (Paper I)

An overview:

A large proportion of the population in urban and rural areas of Tanzania faces acute water supply problems as regards both quantity and quality aspects. The water supply coverage in rural and urban areas is only 42 and 80% respectively (Table 1). These figures give the impression that a large proportion of the urban population has access to adequate water supply. However, when other factors such as intermittent supply, waiting time in queues at water points, distance to water sources, costs of buying water from street vendors and trucks, and inconvenience caused to those who have taps in their houses in assisting

neighbours are taken into account, they are indicators of inadequate water supply (Mwaiselage, 2003; Katko, 1991). Up to 70% of government expenditure on the water sector comes from external sources and the rural water supply receives a higher funding level than the urban water sector (Table 2). However, these figures show a high discrepancy between the fund-ing levels for rural water supply and the actual water supply coverage, the situation, which indicates poor planning and manage-ment of water supply projects (URT, 2002). The chronic water supply problems are responsible for endemic cholera in most regions of Tanzania (WHO, 2001), with small outbreaks being reported every year and much larger outbreaks occurring every 4-5 years (Fig. 4). Similarly, in Latin America and the Caribbean, about 40% of the popu-lation consume water of substandard quality (Pan American Health Organization, 1994), which creates health risks.

6.1.1 Urban water supply: The case of Dar es Salaam city Dar es Salaam city has a population of about 2.5 million, which is about 30% of the urban population in Tanzania. The growth rate is estimated at 8% per annum. About 70% of the population live in informal settlements with marginal infrastructure development, water being no exception (Chaggu et al., 2004; Kyessi, 2005). The water system in Dar es Salaam city is managed and operated by Dar es Salaam Water Supply and Sewer-age Authority (DAWASA). Similar urban water supply and sewerage authorities (UWSSAs) exist in other cities. However, since 1997 DAWASA has been privatised but its operational achievements are yet to be recorded (Kyessi, 2005; URT, 2006). There is mounting opposition to privatisa-tion of water in developing countries from civil society and consumers that calls for caution in the privatisation process (K’ Akumu, 2006) in order to ensure adequate and sustainable water supply for urban populations. The existing water supply infrastructure for Dar es Salaam city is old, worn-out and


14

inadequate to meet the ever-increasing demand for water (URT, 2002). The total output from the water treatment plants is 270,000 m3 /day, of which only 175,500 m3/day (65%) reach the consumer. More-over, an average of 60 % of the pumped water is lost through worn-out distribution system and only 40% eventually reaches the consumers. Part of the remaining demand is obtained privately, either from boreholes or shallow wells, traditional wells and rivers or streams (Chaggu et al., 2002; Mato, 2002). Sepällä & Katko (2003); the World Bank (2004) also reported that more than one- third of water production in Africa is lost through physical and commercial losses, and revenues are insufficient to cover operating costs and expand service coverage. Similarly,

among the major issues that face the UWS-SAs apart from the poor water supply ser vices are poor billing and revenue collection systems which is magnified by increasing number of payment defaulters (URT, 2002; Mwaiselage, 2003). The current average domestic water tariff for UWSSAs is about US$ 0.25/m3 and about 72% of the UWS-SAs have an operating costs to operating revenue ratio of 1.18 to 2.04, which contrib-utes to the financial incapability of DAWASA and other UWSSAs to maintain their facilities. With the growing urban populations in African countries, reducing wastage and losses are predicted to become a key strategic concern for water utilities seeking to increase their service coverage (Mugabi et al., 2007a,b). Hrudey et al. (2006)

Table 1. Current coverage of water supply in Tanzania (in thousands)

Service area

Population With house connection

With public water point

Served Not served

% Pop. served

Urban 11,021 1,212 3,932 8,817 2,204 80 Rural 22,496 172 8,552 9,448 13,048 42 Total 33,517 1,384 12,484 18,265 15,252 54

Source: WHO 2000b, part II page 256.

Table 2. Sources of investment (in 1000 US$) for urban and rural water supply and sanitation

Service area National Funds External Funds Total Urban water 794 2,121 2,915 Rural water 1,332 2,720 4,052

Urban sanitation 190 597 787 Rural sanitation 46 117 163

Total 2,362 5,555 7,917 Source: WHO 2000b, part II page 259.

Fig. 4. Prevalence of cholera by region in Tanzania.

0100200300400500

Dar

Mwanza

Tabora

Coast

Rukwa

Kiliman

jaroTan

ga

Mbeya

Kagera

Morogo

ro

Singida

Iringa

Lindi

Shinya

nga

Mara

Mtwara

Arusha

Ruvuma

Dodoma

Kigoma

Regions

No.

of c

ases

01020304050

No.

of d

eath

sCasesDeaths


15

also pointed out that water utilities must seek ways to maximise the availability, ser-viceability and life of their assets and mini-mise expenditure on energy, chemicals and processes. The high cost of water treatment chemicals hinders the use of appropriate dosages at treatment plants, especially during the rainy season when river water turbidity is very high. The water scarcity in Dar es Salaam city has created a social contrast whereby the poor, especially those living in squatter areas, have to pay more than the upper class residents for the same amount of water used (Mashauri & Katko, 1993; Chaggu et al., 2002). Similarly, UNICEF (1989) reported that indigent populations often pay exorbitant prices for limited quan-tities of poor quality water, with costs that can represent 20% of family budget. This situation increases the risks of contracting water-borne diseases and also supports findings by the WHO & UNICEF (2000b), which reported that the poor tend to be more vulnerable to disease and have least access to basic services.

6.1.2 Rural water supply: The case of Singida rural district About 80% of Tanzania’s estimated popula-tion of 34 million live in rural areas. Despite significant investment in the rural water supply (RWS) since the early 1970s, at pre-sent only about 50% of the rural population

has access to a reliable water supply. How-ever, over 30% of the rural water supply schemes installed are not functioning prop-erly because of lack of commitment of the beneficiaries to operation and maintenance of the facilities, which were provided with-out the active participation of the beneficiar-ies in planning and management (URT, 2002, 2004). Currently, the government is implementing Rural Water Supply and Sanitation Projects (RWSSPs) with financial assistance from the World Bank. The RWSSPs encourage people to work together to plan, develop and manage their own water and sanitation facilities (MoWLD, 2003). However, the RWSSPs face different types of problems, including financial con-straints, poor operation and maintenance and poor governance. As a result, the aver-age rural water supply coverage is only 42% WHO, 2002), with actual coverage ranging between 26.9 and 65.0% (URT, 2004). Singida Rural District is among the districts where the RWSSPs are implemented. The main water supply sources in Singida Rural District are shallow wells, small man-made reservoirs (charco dams) and medium depth boreholes (Table 3). The ratio of population served to water supply source is 1171:1, which is approximately 167 households per water source. However, about 70% of water schemes installed (of which 67% are shallow

Table 3. Number of water supply sources in Singida Rural District

Type of water sevice/Years 2001 2002 2003 June 2004 September 2005

Shallow wells (0 -10 metres)

495 495 495 507

507

Small dams or reservoirs (charco dams)

130 130 132 135 139

Medium depth boreholes (0-60 metres)

56 56 59 59 59

Deep wells - (60 metres & above)

36 36 37 39 39

Springs 22 22 22 22 22

Rainwater harvesting 16 16 16 16 16

Large dams 15 15 16 16 16

Boreholes fitted with windmill 14 14 14 14 14

Source: Singida District Water Dept., 2006.


16

wells) are impaired, leaving a large popula-tion without water supply. Effective institu-tional arrangements for community man-agement and user education are crucial for sustaining water supply systems. In addition, project planners have an obligation to rem-edy the rural water supply situation by de-signing projects that are not only technically feasible but that will also continue to func-tion for a long period of time. 6.2 Rural water supply and traditional treatment (Paper II)

6.2.1 Water supply sources Specific studies into water supply problems and the use of locally available materials for treatment of turbid waters were conducted in the four villages in Singida rural district. The population of Msange, Mipilo, Mangida and Sifunga is 4728, 4442, 928 and 3314 respectively, with an average of 5 persons per household. Most households (99%) are occupied in cropping activities, while 22.5% keep livestock. The main sources of potable water supply include a windmill and a bore-hole while alternative sources, which are

non-potable, include Suke dam, charco dams and traditional wells or water holes (Fig. 5 and also detailed in Table 3 in Paper II). Seasonal streams and ponds supplement the existing sources during the rainy season. Despite the joint efforts of the government, donor agencies and local communities in installing new water schemes, such schemes are poorly distributed in the area and most of them are malfunctioning. This situation is caused by the poor economic basis of villag-ers, which renders them unable to afford to maintain the village water funds, poor opera-tion and maintenance of water schemes due to lack of technical skills and a free water attitude that cause villagers to lack commit-ment to maintenance of the facilities. As a consequence, more than 80% of the villagers lack access to a reliable water supply source. Such lack of access to water supply and sanitation facilities constrains the opportuni-ties of rural people to escape poverty. In order to increase the water security of rural households, multiple sources should be utilised depending on the season and geo-graphical location (Kahinda et al., 2007).

Fig. 5. Location of case study villages and the main water supply sources.


17

Women have multiple preferences for water supply sources, which are influenced by availability of water from the sources during different seasons, distance to the water source and cost of buying water from pota-ble sources. About 95-100% of the house-holds interviewed obtain water from Suke dam and charco dams, 20-60% fetch water from traditional water holes and 10-22% from the borehole located in Msange village and windmill located in Mipilo village. Al-most every day women are obliged to walk from 2 km to more that 7 km to different water sources (Fig. 5). The impacts of de-manding household chores for women on the socio-economic and health development of rural communities have been reported by Eliufoo & Marobhe (1998). Similarly, due to acute scarcity of water supply in rural Nepal, women are also compelled to walk with pitchers on their backs from about 2 or 3 am to 10 or 11 pm every day to water sources (Kathmandu Post, 2000). The use of pol-luted water sources has been reported in other rural areas such as in Trinidad and Tobago, where households may or may not treat such water before consumption (Welch et al., 2000). The World Bank Water De-mand Research Team (1993) mentioned factors that influence a household’s willing-ness to use or pay for an improved supply, which are also relevant to rural villages in developing countries. These include the socio-economic and demographic character-

istics of the household; the characteristics of the existing or traditional source of water versus those of the improved water supply; households’ attitudes toward government policy in the water supply sector and their sense of entitlement to government services. 6.2.2 Traditional water coagulation: Proce-dures and performances Women purify water fetched from charco dams, which has average turbidities between 2250 and 5250 NTU and bacteriological counts between 900 and 1500 faecal coli-forms (FC) per 100 mL, using powdered seeds from plants such as Vigna unguiculata (VU), Voandzeia subterranea (VS), Arachis hypogaea, Vicia faba, Parkinsonia aculeata (PA), Zea mays and so on. The processes involved in traditional water treatment (Fig. 6a and b) are more or less similar to those performed at conventional treatment plants and in jar test experiments, in which rapid mixing follows the dosing of water with the seed powder or extract. The coagulated water is then allowed to settle for about 25 minutes before the clarified water is decanted and stored cold in clay pots. The performance of the various seeds as traditional natural co-agulants including other traditional uses of the seeds is shown in Table 4. The coagulation efficiencies of different seeds ranged from 83 to 89% and the treated water was appealing for drinking by

Fig. 6a. Left- Preparation of seed powder on a grinding stone. Fig. 6b. Right -Treating turbid water with seed powder


18

the local people. Of the seeds used in purifi-cation of turbid water, PA seem to be a suitable natural coagulant for appraisal because unlike other types of seeds, it is not a staple food for the people and it has mul tiple beneficial uses. Diverse communities in the world traditionally use different natural materials extracted mainly from plant sources as raw water additives to produce drinking water (Suarez et al., 2005). System-atic studies have shown that among the different plant-derived materials tested, Moringa oleifera (MO) seeds are the most effective natural coagulants (Jahn, 2001). Laboratory studies have shown that MO seeds are highly effective in coagulating and removing suspended solids from water containing medium to high initial turbidity (Sutherland et al., 1989). They are also capa-ble of aggregating bacteria (Madsen et al., 1987; Ghebremichael et al., 2005) and there-fore the flocculated microorganisms may be removed by settling in the similar manner as colloidal particles in properly coagulated and flocculated water (Casey, 1997).

6.3 Effect of varying physical parame-ters on coagulation of river water with crude seed extracts (Paper III)

6.3.1 Optimum dosage and seed powder grain size effect The optimum dosage of CSEs from seeds for coagulation of river water with low (50 NTU), medium (205 NTU) and high (500 NTU) turbidity was 10, 30 and 60 mg/L for VU and VS and 10, 20 and 40 mg/L for PA (Fig. 7a and b). The CSEs demonstrated the best performance with high turbidity water, in which a turbidity removal efficiency of 92-98% was observed. The restabilisation of destabilised colloidal particles, which was associated with higher residual turbidities, occurred at dosages above the optimal. Similarly, Bhuptawat et al. (2007) observed restabilization phenomenon during coagula-tion of synthetic turbid water using crude extracts of MO seed as did Huang et al. (2000) when using chitosan as a coagulant. The dosages used traditionally by rural women are very high, about 40-125 times higher than the optimal dosages established in this work. The proper application of seed powder in purification of turbid water at

Table 4. The use pattern of seeds and water purification potential of seed powder

Traditional coagulant Other local use apart from treatment of drinking water

Dosage of seed powder (table-spoon/kg)

Residual turbidity (NTU)

Common name

Botanical name

Family name

Field bean Vicia faba Fabaceae Food stuff and cash crop

3 382 (83%)

Bambara groundnut

Voandzeia subterranea

Fabaceae Occasional food stuff 3 270 (88%)

Green gram or cow peas

Vigna ungui-culata

Fabaceae Rarely grown cash crop 4 225 (90%)

Groundnuts

Arachis hypogaea

Fabaceae Food seasoning; cash crop

2 247 (89%)

Jerusalem thorn/jerry bean/wonder tree, mkeketa*

Parkinsonia aculeata

Fabaceae Seed husks and branches are fuel wood; green beans are eaten; shade tree

4 295 (87%)

Wood or charcoal ashes

Any tree species

- Clarification of wash water and rarely for drinking

0.5 – 1 kg 247 (89%)

*Vernacular name


19

household level will help villagers to save up to 80% of the seeds per annum, and these can be used for other purposes. Moreover, coagulation of medium turbidity water with CSEs of VU prepared from seed powder with different grain sizes showed that the finest (0.200mm<GS<0.212mm) and the largest grain size (0.425mm<GS<0.850mm) gave the lowest residual turbidity of 19, and 20 NTU respec-tively, which comply with the Tanzania Drinking Water Quality Standard (TDWS) of 30 NTU. Similar trends were observed for the finest and largest grain sizes of VS and PA seed powder, for which the residual turbidity was 21 and 25 NTU; and 23 and 26 NTU respectively. The best performance of the crude extracts from the finest seed powder could be due to its large total sur-face area, whereby most of the water-soluble proteins are at the solid-liquid interface

during the extraction process (Hart, 2000). This might have increased the concentration of active coagulation polymer in the extract, which improved the coagulation process.

6.3.2 Effect of raw water pH The effect of pH on coagulation of raw water samples with initial turbidity of 205 FTU using CSEs is shown in Figure8. It was revealed that the performance of CSEs improved significantly at acidic pH of raw water samples regulated with C. aurantifolia solution. The minimum residual turbidities of 5 and 7 FTU were observed at pH 4.6 and 4.9 in raw water coagulated with optimal dosages of CSEs of VU and VS respectively. Similarly, the minimum residual turbidity of 10 FTU was seen at pH 4.6 of raw water coagulated with CSEs of PA. Such residual turbidities are very low compared with the TDWS of 30 FTU. It has been observed

Fig. 7a. Top - Effect of seed extract dosage on removal of turbidity from water with initial turbidity of 50 FTU. Fig. 7b. Bottom - Effect of seed extract dos-ages on removal of turbid-ity from water with initial turbidity values of 205 and 500 FTU.

050

100150200250300350400

0 10 20 30 40 50 60 70 80

Dosage of coagulants (mg/L)

Res

idua

l tur

bidi

ty (

FTU

) V.unguiculata (205 FTU)V.subterranea (205 FTU)P.aculeata (205 FTU)V.unguiculata (500 FTU)V.subterranea (500 FTU)P.aculeata (500 FTU)

1520253035404550

0 2 4 6 8 10 12 14 16 18

Dosage of coagulants (mg/L)

Res

idua

l tur

bidi

ty (

FTU)

V.unguiculataV.subterraneaP.aculeata


20

that the net surface charge of colloidal particles is reduced at low pH and hence the electrostatic repulsion between the colloids and the thickness of the double layer is also reduced (Bhuptawat et al., 1999). This sug-gests that efficient particle destabilization and optimal flocculation occur at lower pH. In contrast, Ndabigengesere & Narasiah (1996) showed that pH did not influence coagulation of kaolinite turbidity using MO seed extract. Such an observation might be due to the fact that synthetic kaolinite water does not have other natural river pollutants such as organic matter that influence the coagulation process (Gregor et al., 1997). It has been reported that the role of organic polymers is to coagulate the organic-laden particles in water (Bolto et al., 2001), hence improving the floc formation process and its settlement. Since C. aurantifolia fruit juice has a bactericidal effect on gastrointestinal pathogens (Dalsgaard et al., 1997), its appli-cation for treatment of drinking water by rural households will help to minimise the risks of contracting water-borne infections.

6.3.3 Mixing speed and duration In the studies on the effects of mixing speed and duration on coagulation of raw water samples with initial turbidity of 205 FTU using CSE of VU, the intensity of rapid and slow mixing was varied from 60 to 300 and 10 to 60 RPM while the mixing duration was varied from 0.5 to 20 and 10 to 60 min respectively. The results revealed that inten-sity and duration of rapid and slow mixing when applied separately in water coagulation

did not influence the removal of turbidity. High residual turbidities were observed almost throughout the range of intensities and durations of rapid and slow mixing investigated. However, minimum residual turbidities that ranged between 47 and 51 FTU were observed at the rapid and slow mixing velocities of 150 RPM (G, 200 s-1) and 40 RPM (G, 28 s-1), as well as at the rapid and slow mixing durations of 5 min (equivalent to the product, GT value of 7.5x104) and 30 min (GT 5x104). In conven-tional water treatment, rapid mixing and slow mixing are essential to achieve com-plete mixing of the chemical coagulants and to promote collisions between particles so that flocs are formed (AWWA, 1989; Kiely, 1997). These residual turbidities were much higher that those observed in the initial coagulation experiments (29 FTU) when both rapid and slow mixing speeds were used sequentially for coagulation of raw water with initial turbidity of 205 FTU using VU. This suggests that both mixing condi-tions are essential for coagulation of turbid water using natural coagulants. However, it has been observed that the mixing intensity needed for flocculation depends on the particle concentration, which for tropical river water has large variations. At low initial turbidities, a higher turbulence level is re-quired as particle spacing is relatively high. Therefore, where large variations in the turbidity of the water occur, adjustment to the turbulence is necessary (McConnachie et al., 1999).

020406080

100120140160

4 4,5 5 5,5 6 6,5 7 7,5pH of raw water

Res

idua

l tur

bidi

ty (

FTU

)

No coagulant addedV.unguiculata (40 mg/L)V.subterranea (30 mg/L)P.aculeata (20 mg/L)

Fig. 8. Effect of raw water pH regulated by C.aurantifolia on coagula-tion (dosage rates of seed extracts shown in brackets).


21

6.4 Purification and characterisation of coagulant proteins (Paper IV)

6.4.1 Protein purification The chemical composition of the seed powder is presented in Table 5. The protein contents in VU, PA and VS seeds were 23.7, 39.7 and 16.2% respectively. The presence of such significant amounts of proteins explains why these seeds are effective natu-ral coagulants. The low fat content in the seeds shows that no preliminary treatment is required to remove oil from the seeds before preparing the CSEs. Coagulant proteins were purified from the CSEs of VU (VUCE) and PA (PACE) by simplified IEX chromatography. The crude extraction of the coagulant agents from the seed powder was carried out using water as a solvent. Most coagulation studies, for exam-

ple using MO (Gassenschmidt et al., 1995; Ndabigengesere & Narasiah, 1998; McCon-nachie et al., 1999); okra and nirmali seeds (Al-Samawi & Shokralla, 1996) have used water extraction methods and the CSEs have high coagulation activities. The crude extract of MO seed using salt solution showed better coagulation activity at dosages much lower than those needed for coagula-tion with crude water extract (Okuda et al., 1999). However, Ghebremichael et al. (2006) noted that crude salt extract of MO seed significantly affected the IEX process and very high dilution of crude salt extracts was necessary to regulate the ionic strength to the level of the equilibrating buffer. Coagulation activities of protein samples from the purification process are shown in Table 6. The presence of coagulation activi-ties in water samples treated with unbound

Table 5. Proximate chemical composition of the seed powder (%) Taxon Moisture Fibre Ash Protein Carbohydrate Fat

Vi. unguiculata 10.8 5.1 4.0 23.7 56.4 1.4

P. aculeata 7.4 ND 4.6 39.73 ND 18.9

Vo. subterranea 9.2 4.9 2.8 16.2 60.7 4.9

ND denotes not determined.

Table 6. Purification of coagulation active components from VU and PA seeds by IEX matrix and their coagulation activities

Coagulant name VU PA

Protein fractions Average concentration

(µg /mL ) Coagulation activity (∆ O.D)*

Average concentration (µg /mL )

Coagulation activity (∆ O.D)*

Unbound proteins

720 0.63 960 0.58

Bound proteins eluted by 0.3 M NaCl

840 0.91 480 0.85

Bound proteins eluted by 0.6 M NaCl

420 0.79 380 0.77

*(∆ O.D)= O.D at time 0 – O.D at time 90 min.


22

proteins of VU and PA shows that there are different types of proteins or other organic compounds in the seed that possess coagula-tion properties. Although the protein con-centration in VU samples eluted by 0.3 and 0.6 M NaCl was higher than the similarly eluted proteins in the PA samples, their coagulation activities did not differ signifi-cantly. Therefore, for low-cost treatment of drinking water for poor communities, a single-step elution using a 0.3 M or 0.6 NaCl could be appropriate. This simplifies the purification procedure and avoids loss of

coagulant protein during two-step elution of proteins. Further investigations to identify the exact nature of VUP and PAP and the mechanism of particle destabilisation are necessary.

6.4.2 Characterisation A rapid and simplified method that em-ployed a small volume of sample was applied for characterisation of purified proteins of VU (VUP), PA (PAP) and CSEs in terms of coagulation properties, thermal resistance, floc settling rates and pH coagulation zones.

Fig. 9a – top; 9b bottom. a. Coagulation activity of crude seed extracts and alum at different dosages. b. Coagulation activity of purified protein samples of VU and PA at different dosages.

00,20,40,60,8

11,21,41,6

0 2 4 6 8 10 12 14 16Dosage (µg/mL)

Abs

orba

nce

(500

nm

)

V.unguiculata P.aculeataAlum Control

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

0 0,5 1 1,5 2 2,5 3Dosage (µg/mL)

Abs

orba

nce

(500

nm

)

VU - 0.3 M NaCl elutedVU - 0.6 M NaCl elutedPA - 0.3 M NaCl elutedPA - 0.6 M NaCl elutedControl


23

Compared to the standard jar tests, this method reduced the sample volume and coagulant dosage and also allowed a large number of experiments to be conducted simultaneously, which reduced the time needed for data acquisition. 6.4.2.1 Coagulation and floc settling It was revealed that maximum coagulation activities of VUP and PAP eluted by 0.3 M and 0.6 M NaCl were achieved at the dosage of 1 and 2 µg/mL, and 0.75 and 1.5 µg/mL respectively. These dosages are 5 to 10 times lower than those of the respective CSEs (Fig. 9a and b), which suggests that the use of pure proteins as coagulants may reduce the costs of treating water as well as avoid the release of organic and nutrient loads to the treated water. The resulting coagulation efficiencies after 90 min of settling time of water treated with VUP and PAP were 88 and 87% respectively, compared with water treated with CSEs, which was only 80%. Similarly, alum demonstrated 84% coagula-tion efficiency at the optimum dosage of 7.5 µg/mL. The optimal dosages for coagula-tion of turbid water (250-300 NTU) with MO crude extracts and MO coagulant pro-tein (MOCP) are 15 and 5 µg/mL (Ghe-bremichael et al., 2004), which are quite similar to those obtained here for purified proteins and CSEs of VU and PA. The floc settling studies of water coagulated with purified proteins, CSEs and alum showed that about 2-2.5 hr are required to achieve 89-92% turbidity reduction of water coagulated with VUP and PAP eluted by 0.3 and 0.6 M NaCl. Moreover, maximum turbidity removal efficiency of 86% and 90% was observed after 2 hr of settling time of water treated with CSEs and alum respec-tively. The observed floc settling patterns of VUP and PAP are very similar to those reported for MOCP (Ghebremichael et al., 2005). The observed long settling time of water coagulated with the seeds and alum possibly supports our previous findings that both rapid and slow mixing are needed for an efficient coagulation process. In protein characterisation studies, only rapid mixing

Fig. 10a. top, b. bottom

a. SDS–PAGE of PA seed crude extracts, unadsorbed proteins and purified pro-teins. Lane 1 shows molecular weight of marker (4-148 kDa); lane 2 MO pro-teins;lane 3 CSE of PA; lane 4 unad-sorbed proteins of PA; lane 5 pooled desalted fractions eluted by 0.3 M NaCl from PA; lane 6 pooled desalted fractions eluted by 0.6 M NaCl from PA. b. SDS-PAGE of VU purified proteins applied at different dosages of pooled desalted fractions eluted by 0.3 and 0.6 M NaCl. Lane 1 represents marker proteins; lanes 2 and 3 show proteins eluted by 0.3 M NaCl; lanes 4, 5 and 6 show proteins eluted by 0.6 M NaCl.


24

was employed to disperse the applied coagu-lants throughout the raw water to be treated. 6.4.2.2 Electrophoretic patterns The SDS-PAGE revealed that pure proteins of VU and PA seeds eluted by 0.3 M and 0.6 M NaCl were eluted in an almost pure state and gave single bands in the region corresponding to a molecular weight of about 6 kDa, which is closely similar to the molecular weight of the purified MO protein (Fig. 10a and b). From the general results of electrophoretic mobilities and coagulation properties of CSEs, unbound proteins and purified proteins, it could be envisaged that

VU and PA seeds have two or more coagu-lating proteins. Studies have also revealed that more than one protein family with coagulation activity is present in MO seeds (Gassenschmidt et al., 1995). Current re-search on plant genomes show that plants express many closely related proteins during different developmental stages (Dong et al., 2004. It is thus possible that VU and PA produce variant peptides that are effective coagulants. For treatment of water for rural populations and other communities in de-veloping countries, a highly pure protein is not necessary and therefore a single-step elution using 0.6 M NaCl would be suffi-

0

0,2

0,4

0,6

0,8

1

1,2

0 2 4 6 8 10 12 14 16Dosage of proteins (µl/mL)

Coa

gula

tion

activ

ity

40 deg. cel. - 0.3 M eluted (VU) 40 deg. cel. - 0.6 M NaCl eluted (VU)80 deg. cel. - 0.3 M NaCl eluted (VU) 80 deg. cel. - 0.6 M NaCl eluted (VU)95 deg. cel. - 0.3 M NaCl eluted (VU) 95 deg. cel. - 0.6 M NaCl eluted (VU)40 deg. cel. (MO proteins) 80 deg. cel. (MO proteins)95 deg. cel. (MO proteins)

00,2

0,40,6

0,81

0 2 4 6 8 10 12 14 16Dosage of proteins (µl/mL )

Coa

gula

tion

activ

ity

40 deg. cel. - 0.3 M NaCl eluted 40 deg. cel - 0.6 M NaCl eluted80 deg. cel. - 0.3 M NaCl eluted 80 deg. cel - 0.6 M NaCl eluted95 deg. cel - 0.3 M NaCl eluted 95 deg. cel. - 0.6 M NaCl eluted

Fig. 11a. Top - Coagulation activity of VU purified proteins heated at different tempera-tures: (40oC for 4 hr, 80oC for 2 hr and 95oC for 1 hr). Fig. 11b. Bottom - Coagulation activity of PA purified proteins heated at different tempera-tures: (40 ºC for 4 hr, 80ºC for 2 hr and 95ºC for 1 hr.)


25

cient to recover almost all proteins bound to the matrix. 6.4.2.3 Protein thermal tolerance Extreme temperature is one of the most important physical factors that denature proteins. However, the CSEs of VU and PA heated at 40oC for 5 hr retained coagulation activities of 95% and 100% respectively. Furthermore, heating the CSEs at 80oC for 2 hr and 95oC for 1 hr did not significantly affect their coagulation activities. The puri-fied proteins of VU (Fig. 11a) and PA (Fig. 11b) eluted by 0.6 M NaCl and pure protein of MO seed retained coagulation activity of 87, 89 and 87; 94, 92 and 91; and 87, 82 and 78%, respectively after heating at 40ºC for 4

hr, 80oC for 2 hr and 95oC for 1 hr. These results are analogous to those of Conte et al. (2007), which showed that the polycationic proteins retained antibacterial activity on several bacterial strains after autoclaving. Therefore, it was suggested that the struc-ture of the protein molecule rather than its charge is important for the antimicrobial activity and hence could be used in food preservation. The observed heat tolerance of our CSEs and purified proteins makes the extraction and purification process of coagu-lant proteins much easier. In addition, the storage conditions for the coagulant proteins may be simple and convenient for rural people in hot countries such as Tanzania, where proper storage facilities are rare.

a. Pseudomonas spp.

(i) Control (ii) VUP (iii) PAP (iv) CF b. Aeromonas spp.

(i) Control (ii) VUP (iii) PAP (iv) CF c. Bacillus spp.

(i) Control (ii) VUP (iii) PAP (iv) CF

d. Acinetobacter spp.

(i) Control (ii) VUP (iii) PAP (iv) CF Fig. 12. Flocculation of (a) Pseudomonas spp.; (b) Aeromonas spp.; (c) Bacillus spp. and (d) Acinetobacter spp. by minimum effective dosages of 0.47 mg/mL VUP; 0.32 mg/mL PAP; and 0.5% CF.


26

0,1

0,15

0,2

0,25

0,3

0,35

0 50 100 150 200 250 300 350 400 450 500 550 600Time (min)

Abs

orba

nce

(620

nm

)

Control VUP (0.38 mg/L)PAP (0.51 mg/L) CF (0.4%)

0,1

0,2

0,3

0,4

0,5

0,6

0 50 100 150 200 250 300 350 400 450 500 550 600Time (min)

Abs

orba

nce

(620

nm

)

ControlVUP (0.75 mg/L)PAP (0.88 mg/L)CF (0.5%)

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 50 100 150 200 250 300 350 400 450 500 550 600

Time (min)

Abs

orba

nce

(620

nm

)

ControlVUP (0.56 mg/mL)PAP (0.38 mg/mL)CF (0.2%)

Fig. 13. Effect of pure proteins of VU (VUP), PA (PAP) and Citrus fruit juice (CF) on growth of differ-ent bacterial species: 13a – top - Pseudo-monas spp. 13b - center - Aero-monas spp. 13c – bottom - Bacill-lus spp. (c).


27

Another merit of VU and PA coagulant proteins is their wide pH range for coagula-tion activity. Significant coagulation activity of VU and PA proteins occurred in the pH range 5.5 to 8.5, compared with 6.5 to 9.5 for MO proteins.

6.5 Antimicrobial activity on tropical river water bacteria (Paper V)

Water-borne diseases are widespread in urban and rural areas of developing coun-tries due to drinking water of inferior qual-ity. Analysis of the 16s rRNA gene sequence together with morphological and biochemi-cal characterisation of 126 bacterial strains isolated from river water showed that the river is polluted with pathogenic and oppor-tunistic bacteria including strains that belong to Vibrio, Pseudomonas, Bacillus and Acinetobac-ter spp. Similarly, Zamxaka et al. (2004) found that the domestic water supplies in rural areas of the Eastern Province, South Africa, are polluted mainly with human intestinal patho-gens. The effects of flocculation on Pseudo-monas, Aeromonas, Bacillus and Acinetobacter cells after incubation with VUP, PAP and CF in comparison to the control in which

the cells were incubated without proteins are shown in Figure 12. Unlike the cells of the other bacterial spp., the cells of Acinetobacter flocculated even in the absence of the pro-teins. Broin et al. (2002) observed that re-combinant protein from MO seeds aggre-gated both Gram-positive and Gram-negative bacteria. Casey (1997) reported that in properly coagulated-flocculated water, flocculated microorganisms may be removed by settling in a similar manner to colloidal particles. Therefore, the ability of bacteria to aggregate enhances the settlement and the clarification of turbid water treated with coagulants (Finch & Smith, 1986). The flocculent activity of coagulant proteins and CF could be attributed to antimicrobial properties of the peptides that are present in almost all plant organs (Broekaert et al., 1997). The results of the growth inhibition studies on Pseudomonas, Aeromonas and Bacillus cells by VUP, PAP and CF are shown in Figures. 13a, b and c respectively. The minimum dosages of 0.38 and 0.51 mg/L; 0.75 and 0.88 mg/L; and 0.56 and 0.38 mg/L inhib-ited the growth of Pseudomonas, Aeromonas and Bacillus cells after 2-4 hr of incubation

Table 7. Counts of faecal coliforms in river water coagulated with pure proteins of PA (PAP) and Citrus fruit juice (CF).

Time

(a) coagulation: PAP - (3 mg/L)

[7.0]

(b) pH regulation prior to coagula-

tion: PAP - (2 mg/L) + 0.3% lime juice

[6.46]

Settled and decanted water (b) [pH 6.46] + different CF dosages

log10 FC count/100 mL Dosage of CF added (% solution, V/V)/log10 FC count/100 mL

0.1% [5.90]

0.2% [5.60]

0.3% [5.21]

0.4% [4.90]

0.5% [4.79]

0.6% [4.52]

0 min 4.9 4.9 3.39 3.39 3.39 3.39 3.39 3.39

30 min 3.9 3.39 2.23 2.23 2 1.65 1.41 0

2 hr 3.4 2.71 2.05 2 1.64 1.04 0 0

24 hr 3.5 1.83 1.72 1.49 1.23 0.78 0 0

Note: Values in square brackets are the pH levels produced after adding different dosages of lime juice to water samples. The initial turbidity of water sample was 45-70 NTU, pH 7.25 and alkalinity 61 mg /L as CaCO3.


28

with VUP and PAP respectively. These results are in agreement with Izadpanah & Gallo (2005), who demonstrated that the molecules with antibacterial activity are mainly positively charged peptides. The pure proteins used in this study were also charac-terised as cationic proteins. Resumption of growth of Bacillus cells after 4 hr of incuba-tion could be attributed to the resistance of bacterial endospores to seed proteins. Dif-ferences in cell wall composition could be

among the reasons for the variation in an-timicrobial activity of VUP and PAP on various bacterial spp. (Ghebremichael et al., 2005). Moreover, the growth of bacterial cells was inhibited remarkably after incuba-tion with CF at the minimum dosage of 0.2-0.5%. This could be due to the fact that acidic consumables possess protective prop-erties against enteric pathogens (Mujica et al., 1994). This shows that CF could be very useful for post-disinfection of water treated

Fig. 14a. Top - Effect of crude extracts of VU(VUCE) and PA (PAP) seeds on Fe2+, NO3-

and PO43- concentra-tions in treated water. Fig. 14b. Center - Effect of proteins of VU (VUP) and PA (PAP) and alum on Fe2+, NO3-and PO43- concentrations in treated water. Fig. 14c. Bottom - Appearance of charco dam water coagulated with different dosages of crude extract of PA (PACE). From left to right are raw water samples (NTU) treated with increasing dosage of PACE.

05

10152025303540

0 2 4 6 8 10 12 14 16Dosage of coagulants (mg/L)

Con

cent

ratio

n (

mg/

L)

PAP (ferrous ion) PAP (phosphate) PAP (nitrate)

ALUM (ferrous ion) ALUM (phosphate) ALUM (nitrate)

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120Dosage of coagulants (mg/L)

Con

cent

ratio

n (m

g/L )

VUCE (ferrous ion)

VUCE (phosphate)

VUCE (nitrate)

PACE (ferrous ion)

PACE (phosphate)

PACE (nitrate)


29

with proteins prior to drinking, especially in poor households where drinking water is stored under unhygienic conditions. Moreover, it was of interest to study antim-icrobial effects of PAP and CF on faecal coliforms (FC) during flocculation-coagulation of the river water. Table 7 shows the faecal coliforms counts in Ruvu River water samples coagulated with PAP and CF. The results showed that a marked reduction in faecal coliform count occurred in water samples in which the pH was low-ered to less than 5.6 using CF prior to co-agulation. When PAP was used as coagulant without lowering the pH of raw water sam-ples, it reduced faecal coliforms by 89% and 96% after 30 min and 2 hr of settling time respectively. For the water samples in which the pH was regulated to pH 6.46 before coagulation with PAP, the reduction in faecal coliforms count was 97% and 99.3% after 30 min and 2 hr of settling time respec-tively. Exposure of faecal coliforms to water samples with pH between 4.79 and 4.52 for more than 2 hr was accompanied by com-plete destruction of faecal coliforms in the treated water. The treated water samples were all palatable and the pH levels in the settled water remained stable throughout the

experiment. Studies by Dalsgaard et al. (1997) showed that exposure of V. cholera strains to 1% Citrus juice (pH 4.4 and 4.5) for two hours caused a 5- log reduction to less than 100 CFU/mL of tap water.

6.6 Physico-chemical quality (Paper VI)

Coagulation of charco dam water with coagu-lant proteins reduced most of the water pollutants analysed to levels that complied with Tanzanian local standards for drinking water and in some cases the WHO standards were achieved. The residual turbidity of water treated with purified proteins and CSEs was reduced from 800 NTU to the range of 3-13 and 3-19 NTU respectively. The CSEs and purified proteins did not affect the pH of the treated charco dam water, which was main-tained within the range 6.9-7.4. This implies that the use of natural coagulant proteins in water treatment processes avoids the need for extra additives to regulate the pH of the treated water. The concentration of Fe2+, NO3

- and PO43- in water treated with CSEs

(VUCE and PACE) and purified proteins (PAP) were reduced from an initial 21.1, 33.3 and 7.5 mg/L by 98.1%, 99.6% and 94.4%; 98.5%, 99.2% and 91.5% and 89.6, 87.7 and 45.3 % respectively (Fig. 14a and b). The removal of iron in the treated water is very important for the villagers because water from most charco dams contains high con-centration of Fe2+, which cause the water to appear reddish brown (Fig. 14c). These re-sults are analogous to those reported by Sajidu et al. (2005), which demonstrated that treatment of wastewater with MO seed kernel removed up to 92.14% of Fe2+. The most probable mechanism for removal of Fe2+ is complete aeration of water samples during coagulation–flocculation process, resulting in the formation of iron precipitates that settled along with the flocs of colloidal particles. Unlike CSEs, the purified proteins reduced organic load in the treated water by 71-75%, which improved the shelf-life of the treated water. Although the proteins eluted with 0.6 M NaCl increased the conductivity of the treated water significantly, the water was palatable.

0123456

VUCE PACE ALUMCoagulants

Slud

ge v

olum

e (m

l/L )

Fig. 15. Sludge volumes produced by VUCE, PACE in comparison to alum at optimal dosages for coagulation of river water (205 NTU). The optimal dosages were 30 mg/L for VUCE; 20 mg/L for PACE and 25 mg/L for alum. The drop lines are SD based on three analy-ses.PACE and 25 mg/L for alum. The drop lines are SD based on three analyses


30

However, elution of proteins with low concentration of salt solution could be advantageous. The use of these traditional coagulants, especially the purified proteins, for treatment of turbid water sources will help to improve health and hygiene condi-tions of rural populations. It is estimated that less than 2 USD would be required by poor communities to purify 1 m3 of turbid water using PAP. The volumes of sludge produced from river water samples with initial turbidity of 205 NTU coagulated with optimal dosages of VUCE, PACE and alum are shown in Fig. 15. VUCE and PACE produced 1.6 and 2.1 ml/L of sludge respectively, while alum produced 4.5 ml/L, which is about 2 to 3 times higher than the volume produced by the natural coagulants. Ndabigengesere and Narasiah (1998) also observed that the crude extracts of MO seeds produced a sludge that was 4-5 times lower than that produced by conventional alum. Moreover, using natural polyelectrolytes in the drinking water treat-ment process can increase the settling rate, reduce costs for sludge handling and im-prove the finished water quality (Kawamura, 1991). From the rural population point of view, the sludge could be used as a fertiliser. Since the seeds that we used as natural coagulants are also food materials, we as-sume that the sludge will be suitable for plant growth.

7 CONCLUSIONS AND

RECOMMENDATIONS

Despite significant investment in water supply projects, a large proportion of the population in urban and rural areas of Tan-zania are facing acute water supply prob-lems. This is due inter alia to lack of coherent action by water authorities and communities to address social, technical, operation and maintenance, management and financial issues. The urban water supply is character-ised by ageing and worn-out structures, intermittent and inadequate supply, poor water quality, low tariffs, poor customer management and uncontrolled water leak-ages. The rural areas suffer most from water supply problems and a large population use

polluted sources that are far from the home. The drinking water is purified using locally available plant seed powder. Rural women lack knowledge for improving the perform-ance of the seed powder and hence they rarely obtain good clarification results. Endemic cholera in the country is partly attributed to consumption of water of infe-rior quality. Laboratory studies on performance of crude seed extracts (CSEs) and purified seed proteins from Parkinsonia aculeata (PA), Vigna unguiculata (VU) and Voandzeia subterranea (VS) plants as natural coagulants and Citrus fruit juice (CF) juice as either an additive or a biocide during coagulation of water sam-ples confirmed their effectiveness in water clarification. The PA and VU seed proteins were purified from CSEs using simple and straightforward ion exchange (IEX) chroma-tography that could be easily scaled up using locally available facilities. Characterisation of purified proteins by SDS-PAGE showed that they are small proteins with molecular weight closely similar to that of purified Moringa oleifera (MO) coagulant protein. In addition, the purified proteins, CSEs and MO proteins are thermotolerant and have a wide pH range for coagulation, properties that simplify the extraction and handling of the purified proteins. The CSEs, purified proteins, and aluminium sulphate (alum) showed high coagulation activity although purified proteins were effective at much lower dosages than CSEs and alum. The removal of turbidity by co-agulant proteins was associated with a sig-nificant reduction in concentration of other inorganic pollutants to levels in line with Tanzania drinking water quality standards. Unlike CSEs, the purified proteins did not impart an organic load to the treated water and produced less sludge than alum. The use of coagulant proteins avoids the need of application of other water additives to regu-late pH and alkalinity, which is not the case for alum. The pure proteins and CSEs of VU and PA showed antimicrobial effect against bacteria isolated from river water. They flocculated and inhibited the growth of both Gram-


31

positive and Gram-negative bacterial cells. The CF showed high antibacterial effect and hence could be useful for post-disinfection of water treated with proteins prior to drink-ing in rural areas and urban areas where people who do not have a safe drinking water supply. It is recommended that the use of natural coagulants for water treatment should not be taken as a panacea for water supply problems in Tanzania, but rather as one of several means for improving the water supply services in Tanzania. Rural women could improve the performance of the CSEs by using minimum dosages, adequate mixing and settling time of treated water for maxi-mum turbidity removal. Lime or lemon juice could also be used for disinfecting clarified water prior to consumption. Furthermore, the low cost water lifting facilities that do not involve significant human energy and time should be developed. To increase the water security of rural households, multiple water sources should be utilised depending on the season and geographical location, provided proper treatment is applied.

8 AREAS FOR FURTHER

RESEARCH

The results from this study are far from complete. The implications of this study could be better understood through further research into Tanzanian natural coagulants at a large scale for the benefit of rural house-holds and the water industry at large. This implies that: - Large-scale production of coagulant pro-teins using the simple protein purification method and locally available facilities is necessary. Other simple extraction methods for coagulant proteins also should be devel-oped. - Specific antimicrobial studies of VU and PA proteins on other enteric pathogens including the mechanism of action are an important area for further investigations. Further characterisation of coagulant pro-teins might also provide new dimensions for understanding the nature and functions of coagulant proteins. - The potential for large-scale production of the seeds and lime or lemon for domestic use and water treatment is necessary. In particular, the potential of Parkinsonia acu-leata, as a useful multipurpose tree for dry areas of Tanzania should be explored.


32

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