INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, Copyright by the authors - Licensee IPA- Under Creative Commons license 3.

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1 INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 3, No 5, 2013 Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article ISSN Purification of Charco dam water by coagulation using purified proteins from Parkinsonia aculeata seed Nancy J. Marobhe 1, Gunno Renman 2 1- Department of Environmental Engineering, Ardhi University (ARU). P.O Box 35176, Dar es Salaam, Tanzania. 2- Department of Land and Water Resources Engineering, Brinellvägen 28, Royal Institute of Technology (KTH), SE Stockholm, Sweden. nancymarobhe@yahoo.com doi: /ijes ABSTRACT Large rural populations in arid areas of Tanzania use polluted water from man-made water reservoirs (charco dams) for drinking with minimal or without treatment. The ability of coagulant proteins purified from Parkinsonia aculeata seed to purify water from charco dam was studied. The P. aculeata coagulant proteins were purified from P. aculeata crude seed extract by simplified ion exchange chromatography (IEX). The Purified proteins are cationic in nature with molecular mass of about 6 kda, very similar to that of Moringa oleifera proteins. Jar tests were used to investigate the coagulation performance of the purified proteins in comparison to that of a conventional chemical coagulant, which is aluminium sulphate (alum). Coagulation efficiencies of the purified proteins and alum varied slightly. The optimum coagulation dosages for the purified proteins and alum were 6 mg/l and 14 mg/l, which reduced turbidity from 880 NTU (initial raw water turbidity) to 3 and 13 NTU, respectively. The purified proteins reduced turbidity along with Fe 2+, NO 3- and PO 4 3- in coagulated water samples to levels that complied with Tanzania Drinking Water Quality Standards, and, in some cases with WHO standards. Contrary to alum, PAP did not affect the ph and alkalinity of treated water hence; coagulant proteins do not need additives to regulate the ph. The IEX method used for seed protein purification is simple and easily scalable, and hence, it is recommended for production of potable water for poor communities. Keywords: Coagulant proteins, polluted water, ion exchange chromatography, Parkinsonia 1. Introduction Contamination of drinking water sources by fecal matter and inorganic and organic substances represents a major health hazard in many parts of the developing world (Pokhrel and Viraraghavan, 2004). The removal of turbidity is of paramount importance in water treatment because suspended particles represent transport vehicles for undesirable organic and inorganic contaminants, taste, odor and color causing compounds and pathogenic organisms (Raghuwanshi, et al., 2002). Although aluminium salts and synthetic organic polymers are the most widely used coagulants in water treatment works, cost implications and deleterious environmental effect of these chemicals has triggered interest in research for natural coagulants (Fatoki and Ogunfowokan, 2002; Martyn et al., 1989). Like many other rural areas in tropical countries, large rural populations in Tanzania rely on shallow man-made reservoirs (charco dams), rivers and unprotected traditional dug wells for drinking and other domestic purposes. Large variations in annual rainfall, the few perennial Received on January 2013 Published on April

2 rivers and lack of natural lakes throughout much of the semi-arid zones of Tanzania make the dependence on charco dams for domestic water supply and livestock production in rural areas inevitable (SWMRG, 2005). However, the water in the reservoirs is turbid and grossly contaminated due to poor planning and designs, improper management and poor operation and maintenance (Mwaura, 2007; Marobhe, 2008). Traditionally, clarification of turbid surface water sources in rural areas of developing countries is done at household level using plant seeds (Jahn, 1984; Al-Samawi and Shokralla, 1996; Leite et. al., 2007). The seeds of Parkinsonia aculeata are among the most commonly used natural coagulants for treatment of turbid charco dam and river water sources in rural areas of Tanzania where potable water supply is unavailable. Parkinsonia belongs to the family Fabaceae (a peas family, also known as legumes), has a wide range of uses including food (low-cost protein), folk medicine, fodder, building material, shade tree, agroforesty and rehabilitation of degraded land in dry areas (Leite et. al., 2007; Foroughbakhch et al., 2001; Prakash et. al., Laboratory studies have been conducted on effectiveness of crude extract of P. aculeata seeds has revealed that the seeds are effective polyelectrolyte coagulants for clarifying natural and synthetic turbid water (Marobhe et al., 2007 a ; Marobhe et al., 2007 b ). Several research papers have been published on the performance of natural coagulants of which, Moringa oleifera has been studied extensively (Muyibi and Evison, 1995; Ndabigengesere and Narasiah, 1996; Okuda et. al., 2001; Ghebremichael et al., 2006). Such studies have observed that crude seed extracts introduce a large amount of organic matter into the treated water and hence limit its shelf-life (Ghebremichael, et al., 2005; Ndabigengesere and Narasiah, 1998). In the present study, a simple method was used for purification of active coagulating proteins from P. aculeata seeds and thereafter investigated their performance in improvement of various quality parameters of charco dam water. To the best of our knowledge, this is the first investigation about the performance of purified coagulating protein from P. aculeata seed for purification of water for drinking purposes. 2. Material and methods 2.1 Source of water samples Coagulation experiments were conducted using turbid water collected from Nunguru charco dam in Singida rural district of Tanzania during dry season. Approximately ten liters of water samples were collected in plastic buckets and transported to the laboratory for analysis. Physico-chemical characteristics of water samples from charco dam were determined according to procedures detailed in APHA (1998). 2.2 Preparation of crude seed extracts and protein purification and quantification Dry seeds of P. aculeata (PA) were obtained from Tanzania and transported to the Applied Environmental Microbiology laboratory at the Royal Institute of Technology (KTH), Sweden, for extraction of coagulating proteins. A five percent (5%, W/W) solution was prepared from the fine powder of PA seed in Milli Q water. The solutions were first filtered through a fine glass fiber material followed by centrifugation at 3000 RPM for 2 min and the resulting supernatant is termed crude seed extract (CSE). 1750

3 Coagulant proteins were purified from the PACE using a cation exchange resin, followed by elution of bound proteins using 0.3 M and 0.6 M sodium chloride (NaCl) (Marobhe a et al., 2007) with slight modifications. In this study, protein samples eluted with 0.6 M NaCl without prior desalting were used in all coagulation experiments to study the physicochemical quality parameters of treated water. Protein concentration in samples collected during purification process was measured using dye binding method (Bradford, 1976). The purified P. aculeata proteins were abbreviated as PAP. For comparison purposes, alum was prepared as 5% solution (W/V), while the purified M. oleifera protein (MOCP) was provided by the Department of Environmental Microbiology at KTH, Sweden. The SDS - Polyacrylamide Gel Electrophoresis (SDS-PAGE) to check the purity and molecular masses of the purified proteins was carried out on 10% acrylamide gel using a Mini PROTEN 2 apparatus (Bio-Rad) and tris-tricine SDS-PAGE (Hultmark et al., 1983). 2.3 Coagulation experiments Screening of coagulation activity Coagulation activity of proteins samples during different steps of purification was assayed using a small volume of turbid water samples prepared from kaolin clay as detailed by Marobhe a et al. (2007). The assay employed 1 ml of kaolin turbidity that had an initial turbidity of about NTU that correspond to the absorbance or optical density (OD 500 ) of Different dosages of seed extracts were added to water samples in semimicro plastic cuvette (10x4x45 mm, Sarsted Aktiengesellschaft & Co., Germany) and homogenized instantly using a 1 ml micropipette. For control samples, different volumes of distilled water were added to water samples instead of seed extracts or purified proteins. The suspensions were allowed to settle undisturbed for 90 min and thereafter the optical density was measured at 500 nm using U.V-Visible spectrophotometer (Cary 50 Bio). The reduction in optical density relative to the control (i.e. OD) defines the coagulation activity Coagulation experiments employing large sample volume Jar tests were used for evaluating the coagulation-flocculation processes following the screening of coagulation activities of coagulating proteins. The jar tester apparatus (Model, Phipps and Bird PB-700 TM ) was used in coagulation-flocculation experiments in which, different dosages of coagulants were added separately into six glass beakers each one containing 1L of water samples collected from the charco dam. The intensity and duration of rapid and slow mixing were 150 revolutions per minute (RPM) for 5 minutes (min) and 40 RPM for 25 min, respectively. The duration of sedimentation of coagulated water was 30 min and all experiments were done at room temperature (Marobhe b et al., 2007). 2.4 Analysis of water quality Quality parameters of water coagulated with purified proteins and alum were measured following the sedimentation phase according to (APHA, 1998). The parameters analyzed were turbidity, ph, Total Dissolved Solids (TDS), conductivity, salinity, alkalinity, cation concentration (Fe 2+ ), and anion concentration (NO - 3-3, PO 4 and SO 2-4 ) and organic compounds measured as Chemical Oxygen Demand (COD). Turbidity measurements were conducted using a Hach portable turbidimeter (Model, 2100 P) while, the ph, conductivity, TDS and salinity were measured using a Hach combined ph, TDS and conductivity meter 1751

4 (model Sension 156). The cation and anions were analyzed either titrimetrically using a Hach digital titrator or a Hach spectrophotometer (Model, 21D, Milton Roy). The COD was measured with the same spectrophotometer after 2 h of digestion in a Hach tube digester at 150ºC. 3. Results 3.1 Purification of protein and its purity on SDS PAGE Parkinsonia aculeata proteins were purified from the seed crude extract and the average concentration of P. aculeata protein including their coagulation activities during different steps of purification procedure are show in Table 1. The concentration of proteins in the crude seed extract was 2400 mg/l while that of bound proteins eluted by 0.3 M and 0.6 M NaCl were 710 and 690 mg/l, respectively. Moreover, the coagulation activities of the crude seed extract and proteins eluted by 0.3 M and 0.6 M NaCl using kaolinite turbidity with initial absorbance of 1.33 and 60 min of settling time were 0.79±0.12, 0.93±0.05 and 0.8±0.06, respectively. The concentration of unbound proteins was 720 mg/l with coagulation activity of 0.60±0.12 while that in washing water was negligible. Figure 1 shows the SDS PAGE output of P. aculeata crude seed extract, unbound proteins and purified proteins. It can be observed that PAP eluted with 0.3 M and 0.6 M NaCl showed clear single bands located in the region, which is about 6 kda, very similar to the molecular weight of MOCP protein. Table 1: Purification of coagulating protein from P. aculeata seeds by IEX matrix and its coagulation activities Purification stage Average concentration (mg/l) Coagulation activity ( O.D)* Crude seed extract 2400± ±0.12 Unbound proteins 720± ±0.12 Bound proteins eluted by 710± ± M NaCl Protein in column Washings Bound proteins eluted 350± ± ± ±0.06 by 0.6 M NaCl Proteins in column washings 1.6± ±0.07 ( O.D)* = O.D at time 0 O.D at time 90 min. 3.2 Quality of water treated with different dosages of coagulant proteins The physico-chemical quality of raw water samples from Nunguru charco dam is shown in Table 2. The results revealed that the dam is very turbid and also contains very high concentration of iron and nitrate. The quality of water after coagulation and settling is presented in Figure 2 through

5 Figure 1: SDS PAGE of protein samples in P. aculeata during different stage of purification process. Lane 1 shows marker protein with molecular masses of 4 148; Lane 2 represents purified protein of M. oleifera; Lane 3 represents crude seed extract; Lane 4 shows unbound proteins and Lane 5 and 6 represent proteins eluted by 0.3 M and 0.6 M NaCl, respectively. Table 2: Selected water quality parameters of Nunguru charco dam Parameter Value* ph 7.4 ( ) Turbidity (NTU) 810 ( ) Total dissolved solids (mg/l) 37.8 (39-43) Electrical conductivity (µs cm -1 ) 72.9 ( ) Alkalinity (mg/l as CaCO 3 ) 74 (72-80) Iron (mg/l) 27 (26-28) Chloride (mg/l) 120 (99-140) Nitrate N (mg/l) 33 (28-32) Sulphate (mg/l) 205 ( ) Phosphate (mg/l) 9 ( ) Chemical oxygen demand (COD) (mg/l) 80 (70-88) Turbidity and COD *Ranges of measured values are shown in brackets The effect of PAP and alum on turbidity and COD in the treated water is presented in Figure 2. The results revealed that the reduction of turbidity increased with increasing dosage of 1753

6 PAP and alum until optimal dosage, which is the minimum dosage corresponding to the lowest residual turbidity ensued. The optimum coagulation dosages for PAP and alum were 6 mg/l and 14 mg/l, which reduced turbidity from 880 NTU (initial raw water turbidity) to 3 and 13 NTU, respectively. Similarly, the COD of the treated water dropped from 80 mg/l at zero dosage of coagulants to 23 and 15 mg/l COD at optimal coagulation dosages of PAP and alum respectively. There was no added advantage observed at dosages above optimal ones in terms of residual turbidity and COD (Figure 2). Also, specific analysis of protein concentration in treated water at 280 nm showed that the protein concentration diminished to almost zero at optimal dosage of PAP (results not shown). This suggests that most of the proteins participated in destabilization of colloidal particles. Figure 2: Effect of altering the dosages of purified protein of P. aculeata (PAP) seeds on turbidity and COD of treated water in comparison to alum ph and alkalinity The results for ph and alkalinity levels in water treated by different dosages of PAP and alum are shown in Figure 3. The ph of water treated by different dosages of PAP ranged from 7.2 to 7.4 while the alkalinity (as CaCO 3 ) ranged from 50 to 55 mg/l. The average ph and alkalinity values of raw water samples (ref. Table 2) are respectively, 7.4 and 74 mg/l. Unlike proteins, the ph and alkalinity of water treated with alum was remarkably reduced from 7.4 to 4.3 and 70.7 to 3 mg/l (as CaCO 3 ), respectively Water conductivity The conductivity of water treated with different dosages of PAP and alum is presented in Figure 4. It was revealed that, the water conductivity increased considerably with increase of the dosage of PAP and alum. At the optimum coagulation dosages of PAP (6 mg/l) and alum 1754

7 (14 mg/l), the conductivity increased from 74 µs/cm of raw water to 990 and 1270 µs/cm respectively. Figure 3: Effect of altering the dosages of purified proteins of P. aculeata (PAP) seeds on ph and alkalinity of treated water in comparison to alum. Figure 4: A comparison of conductivity of water treated with different dosages of purified proteins of P. aculeata (PAP) seeds in comparison to alum Iron, nitrate and phosphate ions The change in concentration of Fe 2+, NO 3 - and PO 4 3- in treated water as a function of PAP and alum dosages are presented in Figure 5. The results showed that the concentration of Fe 2+, NO 3 - and PO 4 3- decreased with increasing the dosage of PAP. The concentration of Fe 2+, NO 3 - and PO 4 3- at optimum dosage for turbidity removal (6 mg/l) was 2.2, 2.9 and 4.1 mg/l, which corresponds to the removal efficiencies of 89.6, 87.7 and 45.3% respectively. However, the lowest concentration of contaminants was observed at protein dosage higher than those required for turbidity removal. Similarly, the removal efficiencies of respectively, 1755

8 34.8, 83.8 and 65.3% were observed for Fe 2+, NO 3 - and PO 4 3- in water treated with optimal coagulation dosage of alum (14 mg/l). Figure 5: Effect of varying the dosages of purified protein of P. aculeata (PAP) seed on concentration of ferrous, nitrate and phosphate ions in treated water in comparison to alum. 4. Discussion Purification of proteins from the crude seed extract was carried out so as to separate active coagulating proteins from inactive ones and remove other organic and inorganic materials from coagulating protein samples. The protein purification procedure used in this study is highly simplified and it is straightforward that makes the purification process to be fast and user friendly compared to conventional processes that are complex and require special training to perform the tasks. The protein concentration in pure protein samples eluted by 0.3 and 0.6 M NaCl is about 0.6 of the total protein concentration in the crude seed extract. Such pure protein samples performed better in coagulating turbid water sample than unbound protein samples, which were also present in large concentrations. Such a discrepancy could suggest that unbound protein samples contain a high proportion of inactive coagulating proteins. The purified proteins are cationic in nature and on SDS-PAGE it showed that these proteins were eluted in pure state. This indicates that a single fractionation step is capable separating most of non-coagulating proteins from PACE to produce a sample rich in coagulating proteins. The purified proteins either as a protein concentrate or powder forms are stable and could be stored easily at different conditions. Also, the IEX procedure used in this study could be easily scaled up and hence renders it suitable for production of coagulant proteins from locally available seeds (Marobhe et al, 2007 a ). Studies conducted by Ghebremichael et al. (2006) on M. oleifera seed showed that the molecular weight of purified coagulating seed protein is less than 6.5 kda and possess remarkable coagulating activity very similar to that of Parkinsonia seed proteins purified in this work. It is possible that there is gene that codes for coagulant proteins in plant seeds. Studies for further characterization of the coagulant protein in P. aculeata and other plant seeds in particular mapping a gene that code for a coagulant protein are however necessary. Investigations on the effect of PAP on physico-chemical quality of treated charco dam water samples have shown that PAPs have the potential to remove turbidity along with other 1756

9 pollutants. The optimum coagulation dosage of PAP is almost half the dosage needed for alum to coagulate charco dam water. Moreover, PAP performed better than alum with coagulation efficiencies of 99.7 and 98.5% for PAP and alum, respectively. The residual turbidity produced by PAP complied with Tanzania Drinking Water Quality Standard (TDWS) of 30 NTU as well as the standard acceptable by World Health Organization (WHO) of 5 NTU. The good performance of PAP could be due to the fact that the coagulation potential of polyelectrolytes increases with increasing initial turbidity of raw water (Marobhe et. al, 2007; Muyibi and Evison, 1995). According to Gregory and Duan (2001), a high rate of interparticle contacts occurs in high turbid waters that influences the destabilization of particles by adsorption and interparticles bridging whereby, a along polymeric chain absorbs to more than one particle to form a particle bridge that settle efficiently. The PAP also did not introduce organic load into the treated water because most of the proteins participated in coagulation-flocculation process. These findings are in agreement to those reported by Ghebremichael et al. (2005) for protein purified from M. oleifera seed, which did not impart organic load in the treated water even at dosages above those required for maximum turbidity removal. Besides the potential health risks associated with organic matter in treated water, its presence in excessive amounts is also associated with color and objectionable tastes and odors. Most of the non-proteinous organic matter (non active organic matter) and other nutrient contents in crude seed extracts were removed during purification of proteins (Marobhe et al., 2007); therefore, there are no unaesthetic conditions that will develop upon storage of treated water. One of the drawbacks of using alum in conventional water treatment plants lies on its propensity to reduce the ph and alkalinity. The ph and alkalinity of water treated with alum increased significantly while that treated with PAP remained more or less constant irrespective of the dosage used (Figure 3). This suggests PAP is a suitable natural coagulant for use in conventional water treatment works either to substitute or supplement chemicals for turbidity removal. Its use will minimize the extra cost for adjustment of alkalinity and ph as well as for importation of synthetic coagulant aids. The high treatment costs at water works have often culminated into improper dosages at and hence the resultant outbreaks of water borne diseases. However, thorough investigations to ascertain the potential of PAP as coagulant aid to supplement or replace synthetic coagulant aids are necessary. The increase in conductivity of water treated with purified proteins is due to ions introduced into the water from sodium chloride solution used for protein purification. In case of alum, the increased conductivity is caused by sulphate ions remaining in the treated water. Total Dissolved Solids (TDS) in treated water samples in all analyses were found to be much lower than those for conductivity (data not shown) and thus such values of TDS are definitely low compared to the guideline value of 1000 mg/l recommended for drinking water (WHO, 1996). Coagulation of turbid water using proteins eluted using a low strength salt solution (i.e. 0.3 M NaCl) produced water that had conductivity and TDS that were 2.5 to 2.9 lower than those observed in water coagulated with proteins eluted using 0.6 M NaCl (data not shown). It has been observed that, protein samples eluted by 0.3 M NaCl have very similar coagulation properties, thermo-resistance and electrophoretic mobility as proteins eluted by 0.6 M NaCl (Marobhe et al., 2007) a. This suggests that PAP is suitable for treating drinking water without prior desalting. This makes the procedure for purification of coagulant proteins very simple and cheap and can thus be scaled up to produce proteins locally for use by large rural populations. 1757

10 The coagulating PAP have the potential for producing potable water for large rural populations because, apart from removing suspended particles, it also reduced significant amount of Fe 2+, NO - 3 and PO 3-4 in treated water. The concentration of NO - 3 in the water after treatment complied with that of TDWS set at 100 mg/l, while the concentration of Fe 2+ in water treated with PAP was slightly higher than the TDWS of 1 mg/l. The removal of 3- PO 4 and perhaps other ions could be due to adsorption of ions onto the large flocs of colloidal particles and settle with flocs (Özacar and Sengil, 2003). Coagulation using alum also removed significant amount of NO - 3 and PO 3-4 although the coagulant was inefficient in removing Fe 2+ with the used dosage. As shown in Table 1, Nunguru charco dam like other charco dams is very turbid and contains high concentration of Fe 2+ that range between 2.4 to 27 mg/l which cause the water to be reddish brown in color. The water samples from Nunguru dam treated with PAP were very clear and appealing for drinking and other domestic uses. The removal of up to 92 % of Fe 2+ in wastewater treated with Moringa seed kernel was also reported by Sajidu (2005). The removal of Fe 2+ from water is necessary not only on health reasons but also for its effects on laundry. The probable mechanism for removal of Fe 2+ is complete aeration of water samples during coagulation flocculation process that resulted into the formation of iron precipitates that settled along with the - flocculated colloidal particles. The removal of NO 3 is necessary for limiting the risk of gastric cancer mortality as well as occurrence of methemoglobinemia in infants (Kassenga, 2009). Recent unpublished results have shown that PAP possesses flocculent and growth inhibition effect for pathogenic bacteria isolated from tropical river water. The simultaneous flocculent and antibacterial effects of Parkinsonia seed protein renders this natural coagulant ideal for production of potable water for households in rural area as well as in many urban areas where turbid and microbiologically contaminated water sources are used for drinking purposes. However, the clarified water using these natural coagulants can be made safer by disinfecting prior to drinking using locally available lime juice (Dalsgaard et al, 1997) or heat and ultraviolet (UV) radiation (Clasen and Bastable, 2003). An economic study of the purification process and use of coagulant proteins has not yet been conducted. However, many factors could help outweigh the cost of purification such as low optimal dosage, nutritional, social, economic and environmental advantages. About 2400 mg of proteins could be purified from 5 g of P. aculeata seed and about 5300 mg PAP can purify 1 m 3 of turbid water. It is estimated that, less than 2 USD will be required by poor communities to purify 1 m 3 of turbid water using PAP. These costs estimates will only be incurred at the start of purification work because; the sepharose (matrix) and desalting columns are reusable and can be easily regenerated. However, more studies are warranted to look into the feasibility of purifying larger quantities of the proteins including testing the performance at a larger pilot scale using different type of water sources. It has been pointed out by Sajidu et al. (2005) that application of natural coagulation technology is appropriate for agro-based developing countries like Tanzania because local production of these coagulants will contribute to the rural and national economy at large. 4. Conclusion This study has revealed that coagulant protein from P. aculeata (PAP) seeds are cationic proteins that can be purified using simple technique that can be scaled up to purify coagulant proteins for treatment of water for drinking purpose in poor communities. The PAP reduced 1758

11 turbidity and most other pollutants in coagulated charco dam water samples to levels that comply with TDWQS. The turbidity removal efficiency of PAP and alum was very similar. PAP reduced organic load in treated water by 71% and this enables the treated water to be stored for long period without deterioration. PAP did not affect the ph and alkalinity of the treated water. Unlike alum, the application of coagulant proteins avoids using additives for regulating ph and alkalinity of the treated water. The aesthetic quality of charco dam water treated with both coagulants improved significantly due to the removal of substantial amounts of ions especially ferrous ions that imparted reddish brown color to the dam water. Acknowledgements We gratefully acknowledge the financial support provided by Sida-SAREC to carry out this study. We also extend our gratitude to the Department of Environmental Microbiology at the Royal Institute of Technology (KTH), Sweden in particular Prof. Gunnel Dalhammar for allowing us to use the facilities for protein purification and Associate Prof. Gunaratna Rajarao for supervision during protein purification and fruitful discussions. 5. References 1. Al-Samawi, A.A., Shokralla, E.M., (1996), An investigation into an indigenous natural coagulant, Environmental Science and Health, A31, pp (APHA), (1989). Standard Methods for Examinations of Water and Wastewater, American Public Health Association American, American Water works Association (AWWA), and Water Environment Federation (WEF), 20 th Edition, Washington DC. 3. Bradford, M.M., (1976), A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, pp Clasen, T.F., Bastable, A., (2003), Faecal contamination of drinking water during collection and household storage: the need to extend protection to the point of use, Water and Health, 1, pp Dalsgaard, R., Reichert, P., Mortensen, H.F., Sandström, A., Kofoed, P.E., Larsen, J.L., MØlbak, K, (1997), Application of lime (Citrus aurantifolia) juice to drinking water and food as cholera preventive measure, Food Protection, 609, pp Fatoki, O.S., gunfowokan, A.O., (2002), Effect of coagulant treatment on the metal composition of raw water. Water SA, 28, pp Foroughbakhch, R., Háuad, L.A., Cespedes, A.E., Ponce, E.E., González, N., (2001), Evaluation of 15 indigenous and introduced species for reforestation and agroforestry in North eastern Mexico, Agroforestry System, 51, pp Ghebremichael, K.A., Gunaratna, K.R., Dalhammar, G., (2006), Single-step ion exchange purification of the coagulant protein from Moringa oleifera seed, Applied Microbiology and Biotechnology, 70, pp

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