45
Coagulant Protein from plant materials: Potential Water Treatment Agent Ida Bodlund M.Sc. Royal Institute of Technology Stockholm 2013

Coagulant Protein from plant materials: Potential Water ...575557/FULLTEXT01.pdf · Coagulant Protein from plant materials: Potential Water Treatment Agent ... Coagulant Protein from

Embed Size (px)

Citation preview

Coagulant Protein from plant materials: Potential Water Treatment Agent

Ida Bodlund M.Sc.

Royal Institute of Technology Stockholm 2013

© Ida Bodlund 2013

School of Biotechnology

Royal Institute of Technology (KTH)

AlbaNova University Center

Stockholm

Sweden

ISBN 978-91-7501-593-4

ISSN 1654-2312

TRITA-BIO Report 2013:1

Printed at E-print AB 2012-13

“ You must not lose faith in humanity. Humanity is an ocean; if a few

drops of the ocean are dirty, the ocean does not become dirty. ”

Mahatma Gandhi

Ida Bodlund (2013): Coagulant Protein from plant materials: Potential Water

Treatment Agent. School of Biotechnology, Royal Institute of Technology (KTH),

AlbaNova University Center, Stockholm, Sweden.

Abstract Access to fresh water is a human right, yet more than 780 million people, especially in rural areas, rely on unimproved sources and the need for finding ways of treating water is crucial. Although the use of natural coagulant protein in drinking water treatment has been discussed for a long time, the method is still not in practice, probably due to availability of material and limited knowledge. In this study, about hundred different crude extracts made from plant materials found in Southern India were screened for coagulation activity. Extracts of three Brassica species (Mustard, Cabbage and Cauliflower) were showing activity comparable to that of Moringa oleifera and were further investigated. Their protein content and profile were compared against each other and with coagulant protein from Moringa. Mustard (large) and Moringa seed proteins were also studied for their effect against clinically isolated bacterial strains. The protein profiles of Brassica extract showed predominant bands around 9kDa and 6.5kDa by SDS-PAGE. The peptide sequence analysis of Mustard large identified the 6.5kDa protein as Moringa coagulant protein (MO2.1) and the 9kDa protein band as seed storage protein napin3. Of thirteen clinical strains analysed, Moringa and Mustard large were proven effective in either aggregation activity or growth kinetic method or both in all thirteen and nine strains respectively. To my knowledge this is the first report on the presence of coagulant protein in Brassica seeds. Owing to the promising results Brassica species could possibly be used as a substitute to Moringa coagulating agent and chemicals in drinking water treatment. Keywords: antimicrobial effect; Brassica; coagulant protein; MO2.1; Moringa oleifera; Mustard; napin; thermo-resistance; plant material; protein extraction; salt extract.

List of publications: This thesis is based on the following publications, which are referred to by their

roman numerals:

I. Bodlund I, Sabarigrisan K, Chelliah R, Sankaran K, Rajarao G K.

Screening of Coagulant Proteins from Plant Materials in Southern India (Submitted to Water Science and Technology: Water Supply, under review)

II. Bodlund I, Pavankumar A R, Chelliah R, Sabarigrisan K, Sankaran K, Rajarao G

K. Coagulant Proteins Identified in Mustard: A Potential Water Treatment Agent (Submitted to Int. J. Env. Sci. Technol., revised)

III. Chelliah R, Bodlund I, Sankaran K, Rajarao GK. Antibacterial activity of

Mustard and Moringa seed extracts against pathogenic organisms (Manuscript) Related publication:

I. Bodlund I, Marobhe N J, Rajarao G K. A preliminary evaluation of drinking water treatment with locally available plant seeds in Tanzania (submitted to International Water Association Publishing)

Contribution to papers:

I. Principal author, performed minor part of the experimental work. Writing of the manuscript.

II. Principal author, took part in outlining the experiments, performed most of the

experimental work and writing of the manuscript.

III. Participated in planning of the experiments, prepared seed extracts and isolated coagulant proteins and took part in the preparation of the manuscript.

Related publication I: Principal author, took part in outlining the experiments and performed all experimental work and writing of the manuscript.

Table of contents 1 Introduction ............................................................................................. 1

1.1 Background ....................................................................................... 1 1.2 Conventional water treatment processes .......................................... 3 1.3 Water treatment with natural coagulants .......................................... 7

1.3.1 Moringa oleifera coagulant protein ............................................................... 7 1.3.2 Other natural coagulants .......................................................................... 11

1.4 Objective ......................................................................................... 12

2 Experimental technique ........................................................................ 14 2.1 Preparation of coagulant protein .................................................... 14 2.2 Protein analysis ............................................................................... 15

2.2.1 Coagulation activity ................................................................................... 16 2.2.2 Effect against microbial strains ............................................................... 16

3 Present investigation ............................................................................. 17 3.1 Screening for coagulants (Paper I) ................................................. 17 3.2 Coagulants from Brass i ca species (Paper I & II) ........................... 18

3.2.1 Protein content and profile ...................................................................... 18 3.2.2 Coagulation activity before and after heating ....................................... 19

3.3 Mass Spectrometric analysis of protein (Paper II) ......................... 20 3.4 Effect against clinical isolated strains (Paper III) .......................... 23 3.5 Potential applications of natural coagulants .................................. 25

3.5.1 Effect of coagulant protein on turbid water from pond (Paper II) .. 25 3.5.2 Water treatment with plant seeds in Tanzania ...................................... 26

4 Concluding remarks .............................................................................. 28

5 Further studies ....................................................................................... 29

6 Acknowledgements ............................................................................... 30

7 References ............................................................................................. 32

Abbreviations BSA bovine serum albumin Cab Cabbage Cau Cauliflower CE crude extract DBP disinfection by-product HWT household water treatment MALDI-TOF matrix-assisted laser desorption/ionization-time of flight MDG Millennium Development Goal MO Moringa oleifera MS/MS mass spectrometry/mass spectrometry MusL Mustard Large MusS Mustard Small MusY Mustard Yellow NaCl sodium chloride NOM natural organic matter NTU Nephelometric turbidity units SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SODIS solar water disinfection WHO World Health Organisation

1

1 Introduction

1.1 Background

The Millennium Development Goal (MDG) aims at halving the portion of the

population without sustainable access to safe drinking water by 2015. The

indicator for the MDG is “use of an improved drinking water source” as defined

by the World Health Organisation (WHO). Although 2 billion people have gained

access to improved water sources between 1990 and 2010, more than 780 million

people are still relying on unimproved sources such as surface water and

unprotected wells for drinking water (WHO/UNICEF 2011).

A recent report (WHO/UNICEF 2011) points out the inequities of the access to

improved drinking-water sources in the world (Figure 1).

Figure 1 Worldwide use of improved drinking water sources in 2008.

2

The increase of accessibility of potable water has been lowest in the least

developed countries and amongst the poorest people and there are great

disparities between the regions of the world. The situation is worst in sub-Saharan

Africa, where 40% still do not have improved water and in this area the

population growth exceeds the number of people that have gained access to

improved water sources.

There is also a great inequity between genders in the sustentation of water. In

households without access to drinking water on the premises the responsibility for

collecting water falls on women in 64% of the cases, the tribute of men, girls and

boys are 24, 8 and 4% respectively. This is a significant burden and very time-

consuming, in some cases girls have to stay home from school to help their

mothers in the household.

Although a water source is considered as improved, this does not take into

account if the water is of good quality. In Southern-Asia, the number of people

that have access to water from boreholes has increased by 310 millions over the

last 20 years; nevertheless, the quality of this water is of concern and might

contain microorganisms or other contaminants (WHO/UNICEF 2011).

For instance, in India, rapid population growth and other factors such as industrial

discharge, agricultural run-off and poor sanitation practices put the long-term

availability and quality of the potable water at stake. Water from boreholes is often

too hard and although most people in the urban area have access to water treated

by the municipality through tap or protected pumps; this water is rarely fit for

drinking unless treated first. Microbial contamination through faecal

contamination in water is the major reason for the poor water quality, transmitting

3

a large number of diseases. The pathogens present in the drinking water include:

Shigella species, Salmonella species, Klebsiella species, Escherichia coli, Enterobacter

species, and parasites such as Giardia lamblia and Entamaeba histolytica.

Unsafe drinking water, along with poor sanitation and hygiene, accounts for

around 10 % of diseases worldwide including 4 billion cases of diarrhoea disease

annually, causing 1.8 million deaths (WHO 2005). In particular, children, elderly

and people suffering from chronic diseases are affected.

Microbial contamination can be removed by household water treatment (HWT)

and the most commonly used is boiling of the water. HWT is more likely to be

put in practice by people who do have access to improved water sources than

those who do not. This is believed to be due to economical, social and educational

reasons. HWT also reduce the risk of post-collection contamination during

transport (Wright et al., 2004).

Water treatment processes exists in many parts of the world to provide safe

drinking water and most commonly the municipalities carry out this service

through physical and chemical processes.

1.2 Conventional water treatment processes

Drinking water treatment involves a number of combined processes based on the

quality of the water source such as turbidity, amount of microbial load present in

water and the others include cost and availability of chemicals in achieving desired

level of treatment.

4

Generally drinking water treatment protocols consist of two major steps:

coagulation/flocculation and disinfection (Figure 2). Commonly alum (aluminum

sulfate) is used as a coagulation agent, as it is efficient and relatively cost-effective

in developed countries; while, disinfection is achieved by the addition of chemical

disinfectants like chlorine-based compounds.

Sludge

Coagulation/Flocculation Sedimentation Disinfection Inlet Outlet Filtration

Figure 2 Schematics over conventional water treatment process.

Coagulation/flocculation and sedimentation

Turbidity is caused by suspended particles and natural organic matter (NOM)

present in the water. These particles are negatively charged and are therefore

repelling each other, making them unable to aggregate and settle. The particles are

carriers of unwanted contaminants and pathogenic organisms. In order to

decrease the turbidity of the inlet water some positively charged chemical is added.

This will destabilize the particles by neutralization of the negative charges.

Flocculation is the agglomeration of these particles into large size particles known

as flocs, which will settle by gravity, i.e. sedimentation (Figure 3). If the water is

highly turbid, a flocculant aid is necessary in addition to the coagulant. Turbidity

of water is most commonly measured by turbidimeter and expressed in

Nephelometric Turbidity Units (NTU).

5

Figure 3 Schematics over the coagulation/flocculation process with chemicals.

Prolonged usage of such chemical substances cause adverse health effects like

autonomous and central nerve systemic disorders including Alzheimer’s disease

(Crapper et al., 1973; Martyn et al., 1989; Gupta et al., 2005; Domingo, 2006) and

inhibits bone mineralisation (Mjöberg et al., 1997). Moreover, if proteolytic

additives are required along with alum it becomes an expensive process.

In the sedimentation process, the settled particles (sludge) is removed and the

water filtered before entering the disinfection step. The sludge is unstable and

contains accumulated pathogenic organisms and should therefore be treated.

Dewatering is one of the most important steps in reducing sludge volume and has

a very big impact on total sludge treatment costs. It is desirable if the sludge

volume is as low as possible, i.e. the flocs are really tight hence the sludge more

compact as this will facilitate the sludge disposal.

6

Disinfection

The chemicals most commonly used for disinfection of the water are chlorine,

ozone, chlorine dioxide, and chloramines, and these chemical disinfectants are all

removing microorganisms present in the water. However, they are powerful

oxidants and will react with NOM and contaminants in the water, and form

disinfection by-products (DBPs). These DBPs have been associated with

increased risk for cancer and other health related issues (Morris et al., 1992;

Cantor et al., 1998; Villanueva et al., 2006; Richardson et al., 2007).

In addition to the health concerns, such complicated methods are difficult to

adopt, especially in poor or developing countries, where cost-effective and simple

drinking water treatment methods are needed. Therefore, usage of safe, traditional

water treatment agents from natural sources becomes essential.

Although the technology and the efficiency of treating water are rapidly increasing

it will take time before these techniques will be available for all. This is an urgent

matter and it is very important to find alternative means of treating water in areas

where the advanced treatment methods are not yet available. There is no universal

solution for solving the problem with water supply in the world, every place and

village is unique, and hence it is desirable to find alternative methods for water

treatment to access potable water. Moreover, due to the emerging threat of

climate changes WHO is now recommending small-scale water treatment rather

than relying on large-scale treatment plants that constitute a more fragile system.

7

1.3 Water treatment with natural coagulants

Traditionally, natural coagulants of plant origin have been used at household level.

Women in rural areas of Sudan (Jahn & Dirar, 1979), Tanzania (Marobhe et al.,

2007a) and India (Saif et al., 2012) treat their water with Moringa seed powder

prior to use as drinking water.

1.3.1 Moringa oleifera coagulant protein

The genus Moringa is a tropical plants belonging to the family of Moringaceae, 14

species have been identified so far and all possess coagulant properties in varying

degrees (Jahn, 1988). The most extensively studied, as water treatment agent are

seed extracts of Moringa oleifera (MO), commonly called Moringa. Various parts of

this plant such as the leaves, roots, seed, bark and fruit, possess many beneficial

qualities such as antitumor, anti-inflammatory, antibacterial and antifungal

activities, and are being employed for the treatment of different ailments in the

indigenous system of medicine, particularly in South Asia (Anwar et al., 2007).

Preparation of Moringa seed extracts

Extraction of MO coagulant agent prepared with sodium chloride (NaCl) solution

is more efficient than extraction with distilled water due to the salting-in

mechanism in proteins, wherein a salt increases protein-protein dissociations and

protein solubility as the salt ionic strength increases (Okuda et al., 1999).

Coagulation activity

Crude extract (CE) of MO have been reported to show coagulation activity (Jahn,

1988; Muyibi & Evison, 1995; Ndabigengesere et al., 1995) similar to that of alum

for samples with high turbidity (Ghebremichael et al., 2005). MO is biodegradable,

and unlike alum, does not significantly affect the pH and conductivity of the water

8

after the treatment (Ndabigengesere et al., 1995; Yarahmadi et al., 2009). Further it

produces significantly less sludge volume than alum (Ndabigengesere et al., 1995).

Moringa has also been shown efficient for conditioning of sewage sludge (Wai et

al., 2009) and as a natural adsorbent for the treatment of dairy industry wastewater

(Vieira et al., 2010). The concerns are that MO CE may not be an efficient

coagulant for low turbid water (Muyibi & Evison, 1995) and that concentration of

organic matter in the treated water increases considerably with the dosage of MO

CE. This leads to changes in colour, odour and taste of the treated water if it is

stored for a long time. Therefore it is suggested that the extracts should be

purified before treating the water, in order to decrease the amount of organic

matter added (Ndabigengesere, 1998). A simple methods for purification of the

coagulating proteins has been developed by Ghebremichael et al (Ghebremichael

et al., 2005).

Mechanisms of action

The suggested mechanism of coagulation property of MO protein is supposed to

be that positively charged proteins bind to part of the surface of negatively

charged particles through electrostatic interactions. This leads to the formation of

negatively and positively charged areas of the particle surface. Due to particle

collision and neutralization, formation of flocs with a net-like structure take place

(Figure 4) (Ndabigengesere et al., 1995; Gassenschmidt et al., 1995). The poor

performance of CEs in low turbid water could be explained by low rate of

contacts between particles in such water.

9

Raw water Flocculation Settlement Figure 4 Coagulation/flocculation mechanism of Moringa oleifera protein.

Protein structure

The coagulating agent in Moringa seed has been identified as a protein with a

molecular mass of about 6.5 kDa and the isoelectric point is above pI 10. Amino

acid analysis and sequencing showed a total of 60 residues and this peptide has

been reported to the protein database and given the name MO2.1 (SwissProt

ID:P24303) (Gassenschmidt et al., 1995). A 3D structure of the protein has been

suggested by Okoli et al. (Okoli, 2012). The peptide has eight positively charged

amino acids (7 arginines and 1 histidine) and 15 glutamine residues. It has been

shown that glutamine-rich regions in proteins cause their aggregation (DePace et

al., 1998).

There are significant homologies between MO2.1 and common seed storage

proteins such as 2S albumin and a part of the large chain of both napin and

10

mabinlin (Broin et al., 2002; Suarez et al., 2005). Napins are initially synthesized as

a precursor protein, which is proteolytically cleaved to generate a smaller A chain

of 4,000 Da and a larger B chain of 9,000 Da held together by disulfide bonds

(Ericson et al., 1986). Such homologies may be useful for explaining the flocculent

activity of other seeds that have been traditionally used in water clarification.

Thermo-resistance of MO coagulant protein

The protein is found to be thermo-resistant and posses coagulation activity after 5

hours of heat treatment at 95°C (Ghebremichael et al., 2005). Since this property

makes them less susceptible to deterioration, it can be seen as a prerequisite for

natural coagulants to be thermo-resistant. Due to the high average temperatures

(35-40°C) in tropical countries, they would not be suitable if they were heat

sensitive.

Effect of MO against microorganisms

MO extract has flocculating properties towards bacteria (Broin et al., 2002;

Ghebremichael et al., 2005) and bacteriostatic effect has been observed against

several human pathogens. However, the inhibition of Escherichia coli growth was

found to be transitory, with resumption of growth after 3–6 hours (Suarez et al.,

2003). In addition, MO were able to reduce the number of the parasite Schistosoma

mansoni cercariae (Olsen, 1987) and helminth eggs (Sengupta et al., 2012). Moringa

extract has been suggested as a complement to solar water disinfection (SODIS)

due to its bacteriostatic effect and ability to clarify and decolorize water before

SODIS treatment (Wilson & Andrews, 2011).

11

1.3.2 Other natural coagulants

Coagulation activity has also been reported from other plant materials such as:

Cactus (Opuntia spp.) (Zhang et al., 2006; Miller et al., 2008), common bean

(Phaseolus vulgaris (!!iban & Antov, 2006), red bean (Phaseolus vulgaris sp.), sugar

maize and red maize (Zea mays sp.) (Gunaratna et al., 2007), chestnut and acorn

(!!iban et al., 2009) Cactus latifaria and seeds of Prosopis juliflora (Diaz et al., 1999),

Cassia angustifolia (Sanghi et al., 2002), Grape seeds (Jeon et al., 2009), Nirmali

seeds (Strychnos potatorum) (Babu & Chaudhuri, 2005), Jatropha curcas and Guar gum

(Pritchard et al., 2009), Vigna unguiculata and Parkinsonia aculeate (Marobhe et al.,

2007b), Tannins (Ozacar & Sengil, 2000; Ozacar & "engil, 2003; Beltrán-Heredia

et al., 2010; Sánchez-Martín et al., 2010), Enteromorpha extract (green algae)

(Zhao et al., 2012) and Algal alginate (Devrimci et al., 2012).

Thermo-resistance of coagulant protein was also confirmed in seeds from V.

unguiculata, P. aculeate (Marobhe et al., 2007b), red bean, sugar maize and red maize

(Gunaratna et al., 2007). Further, a coagulant protein with a molecular mass of

around 6.0 kDa, similar to that of MO, was identified in both V. unguiculata and P.

aculeate (Marobhe et al., 2007b).

Although many natural coagulants have been identified, most of them are not

feasible due to cost and availability worldwide, thus difficult to implement for

water treatment. Moreover, the availability throughout the year is an important

factor to take into consideration. Some of them are competing with food-crops or

do not show effect against microorganisms. This calls for a systematic study in

order to identify other natural coagulants that might be more applicable for water

treatment.

12

1.4 Objective

The creative idea of women in villages in Asia and Africa for household water

treatment using natural plant materials deserves to be acknowledged. Therefore,

the potential natural coagulants should be assessed for its application in HWT.

These findings should be evaluated for potential use in advanced water treatment

technology and knowledge for improved HWT should be transferred back to the

villages.

Southern India was selected as a good location to perform these experiments, as it

constituted a good representative for tropical climates and the results and findings

can be applicable to other parts of the world. This area is having both water

scarcity and limited water treatment hence, there is an urgent need for efficient

processes.

The aim of the work presented in this thesis was to identify new plant materials

with coagulating properties. These should have competitive characteristics such as

being cost effective, easily available and having an effect against microorganisms.

This was done by an extensive screening study with many various plant materials

from fruits, vegetables, nuts and pulses. The materials showing the best potential

coagulating agent were further examined with respect to physico-chemical

properties such as protein content, protein profile and thermo-

resistance/activation and the active agent was identified in order to better

understand the molecules responsible for coagulation property. This information

could add knowledge to the field of natural coagulants and help in understanding

the specific seed storage protein responsible for coagulating ability.

13

In addition to the coagulation activity of the seed extract, it is desirable to find out

whether the extract possesses antibacterial activity and can be used as a water

treatment agent. This was tested by aggregation of bacterial cells and growth

inhibition of pathogenic microorganisms causing waterborne diseases. The active

coagulant from the newly identified plant materials was further analysed in pond

water for their potential application in water treatment.

14

2 Experimental technique

2.1 Preparation of coagulant protein

The seeds were removed from the dried pods or from fruits and ground to fine

powder, other plant material such as leaves were dried and ground to a paste.

Lipid extraction was thereafter conducted with ethanol as described by

Ghebremichael (Ghebremichael et al., 2005). All the experiments were performed

at least three times in replicates and the results are discussed.

Extraction of soluble proteins from all dried cake solids was prepared (5% w/v)

using distilled water or 0.5 M sodium chloride (NaCl) solution. A flowchart of the

coagulant extracting process is depicted in Figure 5.

Coagulant (Crude Extract)

Re-suspend solid particles in water/salt

solution

Dry plant material

Powdered seed in ethanol

(remove oil content)

Grind with pestle and mortar

!

Oil for lamp, cooking, cosmetics

etc.

Animal fodder

By-products

!

!

!

Figure 5 Schematic of coagulant protein extraction from plant material.

15

The CEs were further purified by Sepharose ion exchange method as described by

Ghebremichael et al. (2005). Sepharose ion exchanger was prepared by using

10mM Ammonium acetate solution. The aqueous crude extract was added to the

equilibrated sepharose ion exchanger and mixed gently for 30 minutes. Finally the

protein was eluted using NaCl. The obtained liquid is referred to as coagulant

protein (CP).

2.2 Protein analysis

Protein content of the extracts was estimated using protein-dye binding method

(Bradford, 1976) and a standard curve was plotted using known concentrations of

Bovine Serum Albumin (BSA). Before the antimicrobial experiments, the protein

content of the extracts was estimated by Lowry’s method (Lowry et al., 1951).

The protein profile and the molecular mass of the protein were determined by

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

(Laemmli, 1970; Fling & Gregerson, 1986).

In order to analyse the stability of coagulant proteins, the CEs were heated at

95ºC for 5 hours. Protein content and protein profiles, before and after the heat

treatment, were compared and coagulation activity was tested.

Protein bands from SDS-PAGE were excised and processed according to

Ghebremichael et al. (2005) with some modifications. The mass spectra were

acquired (after calibration with standard peptides) using Mass Spectrometer

(Waters, USA). The proteins were identified by GPS explorer software using the

16

Mascot software program; sequence information and other details were obtained

from SwisProt and UniProt.

2.2.1 Coagulation activity

Water sample for coagulation activity tests was prepared as 1% kaolin solution

using tap water. Coagulation activity test was carried out with kaolin synthetic clay

solution as described by Ghebremichael (Ghebremichael et al., 2005). Crude

extract (10µl) was added to the clay solution, mixed instantly and measured the

initial absorbance at 500 nm (A500) using a spectrophotometer. The solution was

allowed to settle and thereafter A500 was measured again. The percentage of

coagulation activity was calculated using the following formula: ((t0-t)/t0)*100,

were t0 is the A500 measured instantly after the sample has been homogenized and

t is the A500 measured after 60 or 90 minutes.

2.2.2 Effect against microbial strains

The bacteria were grown in nutrient broth at 37˚C in shaking incubator overnight

and diluted to an initial absorbance of 0.1 at 600 nm. The two characteristics to be

investigated were: i) the ability of the extracts to aggregate bacterial strains, and ii)

the effect of the extracts on the growth of the bacterial strains. See paper III for

detailed description.

17

3 Present investigation

3.1 Screening for coagulants (Paper I)

Around 100 plant materials from 75 different plant species were collected in and

around Chennai region, India. From these plant materials, both water and salt

(NaCl) crude extracts were prepared for coagulation activity test, i.e. 200 extracts

in total. Among the samples tested, only seven extracts other than Moringa oleifera

(MO), showed more than 50 % activity as can be seen in Figure 6.

Figure 6 Coagulation activity of various CEs after 60 minutes, prepared with water and salt solution.

In all cases there was higher activity in salt extracts than in the water extracts,

implying the salting-in phenomenon (Okuda et al., 1999). The coagulation activity

of Mustard, Cabbage, and Cauliflower crude seed extracts showed more than 80%

activity and the results are comparable to that of MO. Moreover, Mustard and

Cabbage were the only water CEs showing more than 70% coagulation activity.

Interestingly, these plant seeds belong to the same genera, Brassica.

18

Cabbage and Cauliflower are food-crops and the availability and costs of seeds are

of concern. On the other hand, Mustard is a spice cultivated abundantly for oil

production and different Mustard varieties are cultivated in various parts of the

world. It would be of interest to test the coagulation activity from different

Mustard varieties available in Southern India.

Although the activity of the salt extracts was slightly higher for these seeds, water

extracts were used for further studies in order to develop a simple protocol for

preparing coagulant protein as a potential candidate in water treatment.

3.2 Coagulants from Brassica species (Paper I & II)

The crude seed extracts were made from Cauliflower (Cau), Cabbage (Cab) and

three types of Mustard seeds: Mustard large (MusL), Mustard Small (MusS) and

Mustard Yellow (MusY), belonging to the family Brassica, except MusY. Mustard

Yellow (Sinapsis alba) was previously in the Brassica family but is now considered as

a member of the Sinapsis family.

3.2.1 Protein content and profile

There was a reduction in protein content after heat treatment in all the crude seed

extracts (Table 1).

Table 1: Protein content in water extracts of Brassica seeds before and after heat treatment Protein content (mg/ml) Crude Extract Before heating After heating Reduction (%) Cauliflower (Cau) 2.4 2.2 7 Cabbage (Cab) 1.8 1.0 43 Mustard Yellow (MusY) 4.1 1.1 72 Mustard Large (MusL) 2.9 2.7 8 Mustard Small (MusS) 0.9 0.8 18

19

The protein profile of the CEs from different seeds before and after heat

treatment revealed by SDS-PAGE can be seen in Figure 7. There are several

protein bands visualized on samples before heat treatment, while after heating,

only the 9 kDa and 6.5kDa protein was observed with similar intensities. This

indicates that some of the proteins have been denatured during the heating

process (Table 1). The heat-stable low-molecular weight protein could possess

coagulation activity.

66 45 36 29 24 20 14 6.5 !!!!

Cau ! Cau Cab ! Cab Mus ! Mus marker 66 45 36 29 24 20 14 6.5 !!!!

Cau ! Cau Cab ! Cab Mus ! Mus marker

!!!!!"#!$!!!!%&!!!%"!!!%'!!!!!("!!!!!!!!!)#!

MusY ! MusY MusL ! MusL marker MusS ! MusS

Figure 7 Protein profile for Brassica CEs before and after heating (marked with #). The loaded amount of protein was around 10 µg/well. Marker used was low molecular weight marker from Sigma (6.5-66 kDa).

3.2.2 Coagulation activity before and after heating

MusS and MusL, which had the highest activity before heating showed only a

slight increase in coagulation activity after heating whereas the other extracts

clearly gained activity upon heating (Figure 8). This suggests some partial

hindrance that was removed upon heating. MusL and MusS, whose coagulation

activity did not increase dramatically, on the other hand did not lose much protein

upon heating. The possible mechanism of thermo-activation of coagulant protein

remains to be clarified. The thermo-resistance of coagulant protein are in

accordance with previous findings from other natural coagulants (Ghebremichael

et al., 2005; Marobhe et al., 2007b) and since only two protein bands of 9 and 6.5

20

kDa ( marked with arrows in Figure 7) are remaining in heated extracts these are

likely to be responsible for the coagulation property. Hence it is interesting to

investigate the peptide sequences of the low molecular weight molecules.

Figure 8 Coagulation activity for various crude extracts before and after heat treatment (90min). The kaolin solution as blank is marked with a line.

3.3 Mass Spectrometric analysis of protein (Paper II)

The purification protocol developed for MO (Ghebremichael et al., 2005) was not

found equally efficient for MusL, after purification two protein bands with a

molecular weight of 6.5 and 9 kDa were still visible on SDS-PAGE gel (Figure 9).

Hence, after SDS-PAGE the protein bands of 6.5 and 9 kDa were excised and

digested from peptide analysis by MALDI-TOF to disclose their identity.

21

1 2 3 4 5

66 45 36 29 24 20 14 6.5

The MS/MS analysis of these two trypsin digested protein bands from SDS-

PAGE revealed totally six peptide fragments (Table 2). From the 6.5 kDa protein

band, only one sequence was obtained whereas five peptide fragments were

obtained from the 9 kDa protein band.

Table 2: Identified peptide sequences from Mustard large seed extract

No.

Mol. weight

of protein

Sequence

1 6.5 kDa QAVQLTHQQQGQVGPQQVR

2 9 kDa GPQGPQQRPPLLQQCCNELHQEEPLCVCPTLK

3 9 kDa GQQGQQLQQVISR

4 9 kDa QQQGQQGQQLQQVISR

5 9 kDa QQVRQQQGQQGQQLQQVISR

6 9 kDa KVCNIPQVSVCPFQK

Figure 9 SDS-PAGE gel of Mustard and Moringa extracts. Lane 1: MusL CE, 2: MusL CP, 3: Marker Sigma-Aldrich 6.5-66 kDa, 4 MO CP, 5 MO CE. The CPs shown on the gel are crude extracts first eluted with 0.3 M NaCl followed by elution with 0.6 M NaCl. The box marks the two protein bands from MusL CP.

22

Peptide sequence 1 (Table 2) obtained from 6.5 kDa protein band showed a

sequence coverage of 37% with flocculent protein of Moringa oleifera (MO2.1;

SwissProt ID: P24303) (Figure 10).

The other five peptides from 9 kDa band correlated with napin3 protein

(SwissProt ID: P80208) (Figure 10). Napin3 consists of two chains, small (4.2

kDa) and large (9.8 kDa) and the identified peptides and the size of the band are

matching the large chain. Peptide sequences 3, 4 and 5 were found to overlap each

other.

P24303|MO2X_MOROL Flocculent-active proteins MO2.1 and MO2.2 QGPGRQPDFQRCGQQLRNISPPQRCPSLRQAVQLTHQQQGQVGPQQVRQMYRVASNIPST 1QAVQLTHQQQGQVGPQQVR P80208|2SS3_BRANA Napin-3 Small chain: 1SAGPFRIPKCRKEFQQAQHLRACQQWLHKQAMQSGSG37 Large chain: 38PQGPQQRPPLLQQCCNELHQEEPLCVCPTLKGAS61 2GPQGPQQRPPLLQQCCNELHQEEPLCVCPTLK 62RAVKQQVRQQQGQQGQQLQQVISRIYQTATHLPKVCNIPQVSVCPFQKTMPGPS125 5QQVRQQQGQQGQQLQQVISR 6KVCNIPQVSVCPFQK 4QQQGQQGQQLQQVISR 3GQQGQQLQQVISR Figure 10 Sequence alignment of identified peptides in Table 2.

The homology of MO2.1 with other seed storage proteins like 2S albumin, napin

and mabinlin has been reported earlier (Broin et al, 2002; Suarez et al., 2005).

Sequence alignment between MO2.1 and napin3 heavy chain showed 23 identical

and 22 similar residues out of 60 and 88 amino acids respectively (Figure 11).

23

P24303|MO2X_MOROL| -1QGPG-RQPDFQRCGQQLRNISPPQRCPSLR29 1*** * * :*:* ::*:: .* **:*: P80208|38-125|2SS3_BRANA 338PQGPQQRPPLLQQCCNELHQEEPLCVCPTLK68 30 QAVQLTHQ----QQGQVG---PQQVRQMYRVASNIPST60------------------ * : .:* **** * * : ::*:.*:.:*.. 69 GASRAVKQQVRQQQGQQGQQLQQVISRIYQTATHLPKVCNIPQVSVCPFQKTMPGPS125 Figure 11 Sequence alignment of MO2.1 and Napin3 heavy chain. (*) indicates position with fully conserved residue, (:) and (.) indicate conservation between groups of strongly and weakly similar properties respectively.

Similar to MO2.1, napin3 heavy chain is also rich in positively charged and

glutamine residues, which could explain the coagulating properties. Out of 88

amino acids, there are 10 positively charged, only 3 negatively charged and 20

glutamines residues. It could be speculated that these proteins might have

synergistic coagulation activity in Mustard since both 6.5 and 9 kDa protein was

found. The coagulant property of the 9 kDa protein from Mustard remains to be

evaluated.

In the present investigation, MusL seed extract was found to posses 6.5 and 9kDa

protein having coagulation activity and these peptide sequences are homologous

with MO coagulant protein. Therefore, it is desirable to find out their antibacterial

properties and evaluate the potential use as water treatment agent.

3.4 Effect against clinical isolated strains (Paper III)

The effect of coagulant protein from MO and MusL were analysed for bacterial

aggregation and reduction in bacterial growth. Table 3 summarises the results of

thirteen clinical strains against crude and isolated coagulant proteins. Different

24

concentrations were tested to find out the optimum dosage for cell aggregation

and growth reduction of bacteria. The optimum concentration of Moringa and

Mustard seed extracts and the effect of cell aggregation can be seen in Table 3.

Table 3: Comparative analysis of Moringa and Mustard soluble seed protein towards thirteen different clinical pathogens

Nr. Species Moringa Mustard

Protein concentration (mg/ml) CE/CP 0.016/0.031 0.021/0.056

1 Escherichia coli + +

2 Enterococcus faecalis + +

3 Shigella flexneri + +

4 Shigella dysenteriae - -

5 Salmonella typhi + -

6 Salmonella paratyphi A - -

7 Salmonella paratyphi B + +

8 Salmonella typhimurium + -

9 Serratia marcescens + -

10 Klebsiella pneumoniae - +

11 Enterobacter species + + 12 Proteus mirabilis + + 13 Staphylococcus aureus + +

+ indicates effect

- indicates no effect

In conclusion, based on the above findings, both Moringa and Mustard seeds

appear to have potential to be used for the removal of microorganisms, which can

be readily applied for water treatment. Both these seed extracts showed good

aggregation property; Mustard seed extract needs higher dosage level when

compared with seed extract of Moringa. The studies showed that Moringa and

25

Mustard seed protein have properties such as aggregation and reduced bacterial

growth (Paper III). Similar results were observed with P. aculeata and V. unguiculata

seed protein showing aggregation and inhibition of bacterial growth (Marobhe et

al., 2007b).

Since Mustard coagulant proteins possess similar coagulation and heat resistant

properties to that of Moringa, it is interesting to investigate the application of

Mustard seed extract for water treatment.

3.5 Potential applications of natural coagulants

3.5.1 Effect of coagulant protein on turbid water from pond (Paper II)

To further confirm the coagulation activity of Mustard seed extract, highly turbid

water was collected from a pond in Maduravoil, Chennai, India and treated with

MusL and MO CE. Both extracts showed good coagulation activities. In fact,

MusL showed better coagulation activity than MO (Figure 12) indicating the

possible variability in efficiencies of plant coagulant proteins according to the

nature of clay and other suspended particles.

26

Figure 12 Coagulation activity of Moringa and Mustard Large CE in turbid pond water. Samples for measurement of turbidity is taken after 60, 120 and 180 minutes. Pond water alone was used as control.

3.5.2 Water treatment with plant seeds in Tanzania

In a similar experiment conducted in Tanzania (Related publication I), two plant

seeds were investigated regarding their coagulation activity and reduction of faecal

coliforms. i) Azadirachta indica, commonly known as Neem, a tropical, fast

growing, evergreen tree (Girish & Shankara, 2008) with many biological and

pharmacological activities attributable to different parts of the plant (Atawodi &

Atawodi, 2009). ii) The plant called Groundnut, a wild nut that is called

“karanga”, which means Groundnut in Swahili.

27

The preliminary results with highly turbid water from Ruvu River clearly indicate

that Groundnut CE is very efficient. As an example it can be mentioned that 75

mg of Groundnut seed powder (3.3 mg protein) in one litre of river water

managed to reduce the turbidity from 1000 to 2 NTU in 30 minutes, thus 99.8 %

reduction. Moreover, it was observed that water treated with Groundnut gave

visibly smaller and more compact flocks than the water treated with alum (Figure

13).

Figure 13 Water with initial turbidity 1000 NTU treated with Groundnut CE (to the left) and alum (to the right).

Jar test experiments showed that both Neem and Groundnut CEs have the ability

to reduce the turbidity of river water with the initial turbidity of 200 and 450 NTU

by 95% or more. The removal efficiencies of faecal coliforms by Neem and

Groundnut CEs were 81 and 61 % respectively. It is difficult to state the

antibacterial mechanism of CEs, although bacterial flocculation and settlement

along with other coagulated colloidal particles could have been involved.

However, the Neem CE seem to possess antimicrobial properties due to its

remarkable reduction in bacterial count while the residual turbidity remained

higher than that observed for Groundnut CE.

28

4 Concluding remarks Among hundred different materials tested for coagulation activity, the present

study revealed the presence of coagulant proteins in Brassica seed extracts. The

coagulant proteins are remained active even after heat treatment at 95°C for 5

hours. Coagulant proteins with molecular mass of approximately 6.5 and 9 kDa

were identified in all Brassica extracts and peptide sequence analysis of Mustard

large confirms the presence of coagulant protein with approximately 6.5kDa

protein similar to MO2.1 and a 9kDa protein similar to napin3 seed storage

protein. The napin3 peptide sequence has homology with MO2.1 suggesting that

both 6.5 and 9kDa protein could be involved in coagulation activity. This research

has further established that Mustard large seeds have an effect against

microorganisms. To extend the study, some experiments were conducted with

water from pond and river, and the results were promising. To my knowledge this

is the first report on coagulating agent present in Brassica species.

The low cost of the raw material and the availability of Mustard throughout the

year are the added advantages and Mustard large coagulating agent might be a

good complement to Moringa and chemicals in drinking water treatment. The

technology of using natural coagulants for treatment of water is most appropriate

in developing countries, especially in rural areas, where they cannot afford the

high cost of conventional coagulants.

29

5 Further studies The prospect of using Brassica species for water treatment is dependant on

availability of the raw material and the costs of the extraction process. In order to

really make it a viable option it would be most preferable if residues from other

processes such as oil industry could be used. In a preliminary study, Mustard cake

from oil industry and seed extracts from Brassica napus (Rapeseeds) provided by

Lantmännen SW Seed AB showed promising coagulation property. This could

constitute additional materials in large-scale production of coagulants for water

treatment processes. Furthermore, it could reduce the cost of production and

increase the availability of the material. Moreover, it is interesting to find out the

differences in coagulation property of 6.5 and 9 kDa protein in Mustard seed

extracts. Based on these findings large-scale production of coagulant protein will

be studied for water treatment. It is of interest to test the potential coagulant in a

pilot study with different water quality and contaminants from different localities.

The results of these findings will lead to the development of household water

treatment methods and a transfer of scientific knowledge to the rural people who

are using natural coagulants.

30

6 Acknowledgements First and foremost I wish to thank my excellent supervisor, Dr. Gunaratna

Kuttuva Rajarao for accepting me as her student, her endless patience and great

discussions. I have learned so much during these years and I really can’t thank her

enough for all she has done for me! I want to thank my co-supervisor Professor

Gunnel Dalhammar for good support and encouraging me to travel.

I would like to thank the Erasmus Mundus India4EU scholarship program, for

funding the work presented in this thesis. I would also like to thank Alphonsa

Lourdudoss at the International Relations Office. Further, I would like to thank

Lantmännen SW Seed AB for providing seeds.

At Anna University in India I would like to express my gratitude to Professor

Sankaran, Head of Department at Centre for Biotechnology, for good advices and

letting me use the lab facilities. I would also thank Professor Baskar and the rest

of the staff at Centre for International Affairs for assistance with all the

paperwork. Further, I wish to express my warmest ‘thank you’ to my friends and

colleagues in the lab at Taramani campus. Most of all to Ramachandran Chelliah

for all the help but the rest of you as well, I had so much fun with you guys! Also

Ia, Montse, Monica and Maite that gave me a home away from home.

At Ardhi University in Tanzania I am deeply grateful to Dr. Nancy Marobhe for

nice discussions and really making me feel welcome. I wish to express my

gratitude towards Professor Kassenga, dean at the School of Environmental

Science and Technology, for the letter of invitation and for allowing me to use the

31

lab facilities. I wish to thank Mr. Ndimbo and Ramadhani Mbulume for great

assistance in, and outside the lab. I wish to express thanks to the Moshi family,

Hanna and all other nice people I met during my stay.

Of course I also want to thank all my friendly and very helpful colleagues in our

group Dr. Chuka Okoli, Dr. Pavankumar and Ramnath Lakshmanan, we had a

great time in the office! There’s not much we haven’t discussed. I would also like

to extend this gratitude to Dr. Sofia Andersson and all the Diploma workers.

Thanks to all the co-workers at floor 1 for fika-time, Friday beer and providing a

nice work environment, especially to Marita for all the help over the years.

I would like to thank Miriam for assistance with the proofreading.

Finally, I want to thank Andreas and my fantastic friends for all the fun you bring

to my life! Last, but not least, my beloved family for their great support and not

complaining too much while I’ve been away for a long time.

Tack! ¡Gracias! Thank you! Asante!

32

7 References Anwar F, Latif S, Ashraf M & Gilani AH (2007) Moringa oleifera: a food plant with

multiple medicinal uses. Phytotherapy research 21:17–25.

Atawodi SE & Atawodi JC (2009) Azadirachta indica (Neem): a plant of multiple biological and pharmacological activities. Phytochemistry Reviews 8:601–620.

Babu R & Chaudhuri M (2005) Home water treatment by direct filtration with natural coagulant. Journal of water and health 3:27–30.

Beltrán-Heredia J, Sánchez-Martín J & Gómez-Muñoz MC (2010) New coagulant agents from tannin extracts: Preliminary optimisation studies. Chemical Engineering Journal: 1–7.

Bradford MM (1976) Rapid and Sensitive Method for Quantitation of Microgram Quantities of Protein Utilizing Principle of Protein-Dye Binding. Analytical Biochemistry 72:248–254.

Broin M, Santaella C, Cuine S, Kokou K, Peltier G & Joet T (2002) Flocculent activity of a recombinant protein from Moringa oleifera Lam. seeds. Applied Microbiology and Biotechnology 60:114–119.

Cantor KP, Lynch CF, Hildesheim ME, Dosemeci M, Lubin J, Alavanja M & Craun G (1998) Drinking Water Source and Chlorination Byproducts I. Risk of Bladder Cancer. Epidemiology 9:21–28.

Crapper DR, Krishnan SS & Dalton AJ (1973) Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science (New York, N.Y.) 180:511–513.

DePace AHA, Santoso AA, Hillner PP & Weissman JSJ (1998) A Critical Role for Amino-Terminal Glutamine/Asparagine Repeats in the Formation and Propagation of a Yeast Prion. Cell 93:12–12.

Devrimci HA, Yuksel AM & Sanin FD (2012) Algal alginate: A potential coagulant for drinking water treatment. Desalination 299:16–21.

33

Diaz A, Rincon N, Escorihuela A & Fernandez N (1999) A preliminary evaluation of turbidity removal by natural coagulants indigenous to Venezuela. Process Biochemistry:391–395.

Domingo JL (2006) Aluminum and other metals in Alzheimer's disease: a review of potential therapy with chelating agents. Journal of Alzheimer's Disease 10:331–341.

Ericson ML, Rödin J, Lenman M, Glimelius K, Josefsson LG & Rask L (1986) Structure of the Rapeseed 1.7 S Storage Protein, Napin, and Its Precursor. The Journal of Biological Chemistry 261:14576–14581.

Fling SP & Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Analytical Biochemistry 155:83–88.

Gassenschmidt U, Jany KD, Tauscher B & Niebergall H (1995) Isolation and characterization of a flocculating protein from Moringa oleifera Lam. Biochimica et biophysica acta 1243:477–481.

Ghebremichael KA, Gunaratna KR, Henriksson H, Brumer H & Dalhammar G (2005) A simple purification and activity assay of the coagulant protein from Moringa oleifera seed. Water Research 39:2338–2344.

Girish K & Shankara B (2008) Neem–A Green Treasure. Electronic journal of Biology 4:102-111

Gunaratna KR, Garcia B, Andersson S & Dalhammar G (2007) Screening and evaluation of natural coagulants for water treatment. Water science and technology: water supply 7:19–25.

Gupta VB, Anitha S, Hegde ML, Zecca L, Garruto RM, Ravid R, Shankar SK, Stein R, Shanmugavelu P & Jagannatha Rao KS (2005) Aluminium in Alzheimer's disease: are we still at a crossroad? Cellular and molecular life sciences 62:143–158.

Jahn SAA & Dirar H (1979) Studies on Natural Water Coagulants in the Sudan, with Special Reference to Moringa Oleifera Seeds. WATER SA 5:90–106.

34

Jahn S (1988) Using Moringa Seeds as Coagulants in Developing Countries. Journal American Water Works Association 80:43–50.

Jeon J-R, Kim E-J, Kim Y-M, Murugesan K, Kim J-H & Chang Y-S (2009) Use of grape seed and its natural polyphenol extracts as a natural organic coagulant for removal of cationic dyes. Chemosphere 77:1090–1098.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.

Lowry OH, Rosenbrough NJ, Farr AL & Randall RJ (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265–275.

Marobhe N, Renman G & Jacks G (2007a) The study of water supply and traditional water purification knowledge in selected rural villages in Tanzania. Indigenous knowledge systems and development: Relevance for Africa. 1:11–120.

Marobhe NJ, Dalhammar G & Gunaratna KR (2007b) Simple and Rapid Methods for Purification and Characterization of Active Coagulants from the Seeds of Vigna unguiculata and Parkinsonia aculeata. Environmental technology 28:671–681.

Martyn CN, Osmond C, Edwardson JA, Barker DJP, Harris EC & Lacey RF (1989) Geographical Relation Between Alzheimer's Disease and Aluminium in Drinking Water. The Lancet 333:61–62.

Miller SM, Fugate EJ, Craver VO, Smith JA & Zimmerman JB (2008) Toward Understanding the Efficacy and Mechanism of Opuntia spp. as a Natural Coagulant for Potential Application in Water Treatment. Environmental Science & Technology 42:4274–4279.

Mjöberg B, Hellquist E, Mallmin H & Lindh U (1997) Aluminum, Alzheimer's disease and bone fragility. Acta Orthopaedica 68:511–514.

Morris R, Audet A, Angelillo I, Chalmers T & Mosteller F (1992) Chlorination, Chlorination by-Products, and Cancer - a Metaanalysis. American Journal of Public Health 82:955–963.

35

Muyibi S & Evison L (1995) Optimizing Physical Parameters Affecting Coagulation of Turbid Water with Moringa-Oleifera Seeds. Water Research 29:2689–2695.

Ndabigengesere A (1998) Quality of water treated by coagulation using Moringa oleifera seeds. Water Research.

Ndabigengesere A, Narasiahn K & Talbot B (1995) Active Agents and Mechanism of Coagulation of Turbid Waters Using Moringa-Oleifera. Water Research 29:703–710.

Okoli C (2012) Development of Protein-Functionalized Magnetic Iron Oxide Nanoparticles: Potential Application in Water Treatment. Doctoral Thesis.

Okuda T, Baes A, Nishijima W & Okada M (1999) Improvement of extraction method of coagulation active components from Moringa oleifera seed. Water Research 33:3373–3378.

Olsen A (1987) Low technology water purification by bentonite clay and Moringa oleifera seed flocculation as performed in Sudanese villages. Water Research 21:517-522

Ozacar M & Sengil A (2000) Effectiveness of tannins obtained from Valonia as a coagulant aid for dewatering of sludge. Water Research 34:1407–1412.

Ozacar M & "engil $A (2003) Evaluation of tannin biopolymer as a coagulant aid for coagulation of colloidal particles. Colloids and Surfaces A: Physicochemical and Engineering Aspects 229:85–96.

Pritchard M, Mkandawire T, Edmondson A, O’Neill JG & Kululanga G (2009) Potential of using plant extracts for purification of shallow well water in Malawi. Journal of Physics and Chemistry of the Earth 34:799–805.

Richardson SD, Plewa MJ, Wagner ED, Schoeny R & Demarini DM (2007) Occurrence, genotoxicity, and carcinogenicity of regulated and emerging disinfection by-products in drinking water: a review and roadmap for research. Mutation research 636:178–242.

36

Saif MMS, Kumar NS & Prasad MNV (2012) Binding of cadmium to Strychnos potatorum seed proteins in aqueous solution: adsorption kinetics and relevance to water purification. Colloids and Surfaces B: Biointerfaces 94:73–79.

Sanghi R, Bhatttacharya B & Singh V (2002) Cassia angustifolia seed gum as an effective natural coagulant for decolourisation of dye solutions. Green Chemistry 4:252–254.

Sánchez-Martín J, Beltrán-Heredia J & Solera-Hernández C (2010) Surface water and wastewater treatment using a new tannin-based coagulant. Pilot plant trials. Journal of Environmental Management 91:2051–2058.

Sengupta MEM, Keraita BB, Olsen AA, Boateng OKO, Thamsborg SMS, Pálsdóttir GRG & Dalsgaard AA (2012) Use of Moringa oleifera seed extracts to reduce helminth egg numbers and turbidity in irrigation water. Water Research 46:3646–3656.

Suarez M, Entenza JM, Doerries C, Meyer E, Bourquin L, Sutherland J, Marison I, Moreillon P & Mermod N (2003) Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities. Biotechnology and Bioengineering 81:13–20.

Suarez M, Haenni M, Canarelli S, Fisch F, Chodanowski P, Servis C, Michielin O, Freitag R, Moreillon P & Mermod N (2005) Structure-function characterization and optimization of a plant-derived antibacterial peptide. Antimicrobial Agents and Chemotherapy 49:3847–3857.

!!iban M & Antov M (2006) Extraction and partial purification of coagulation active components from common bean seed. Acta periodica technologica 37:37-43.

!!iban M, Kla"nja M, Antov M & !krbi! B (2009) Removal of water turbidity by natural coagulants obtained from chestnut and acorn. Bioresource Technology 100:6639–6643.

Vieira A, Vieira M, Silva G & Araújo Á (2010) Use of Moringa oleifera seed as a natural adsorbent for wastewater treatment. Water Air Soil Pollution 206:273–281.

37

Villanueva CM, Cantor KP, Grimalt JO, Malats N, Silverman D, Tardon A, Garcia-Closas R, Serra C, Carrato A, Castano-Vinyals G, Marcos R, Rothman N, Real FX, Dosemeci M & Kogevinas M (2006) Bladder Cancer and Exposure to Water Disinfection By-Products through Ingestion, Bathing, Showering, and Swimming in Pools. American Journal of Epidemiology 165:148–156.

Wai KT, Idris A, Johari MMNM, Mohammad TA, Ghazali AH & Muyibi SA (2009) Evaluation on different forms of Moringa oleifera seeds dosing on sewage sludge conditioning. Desalination and Water Treatment 10: 87–94.

WHO (2005) World Health Report 2005: Make Every Mother and Child Count. Geneva: World Health Organization.

WHO/UNICEF Joint Monitoring Programme for Water Supply and Sanitation (JMP) (2011) Drinking Water Equity, Safety and Sustainability: Thematic report on drinking water.

Wilson SA & Andrews SA (2011) Impact of a natural coagulant pretreatment for colour removal on solar water disinfection (SODIS). Journal of Water, Sanitation and Hygiene for Development 1:57-67.

Wright J, Gundry S & Conroy R (2004) Household drinking water in developing countries: a systematic review of microbiological contamination between source and point-of-use. Tropical medicine & international health: TM & IH 9:106–117.

Yarahmadi M, Hossieni M & Bina B (2009) Application of Moringa Oleifer Seed Extract and Polyaluminum Chloride in Water Treatment. World Applied Sciences Journal 7 (8): 962-967.

Zhang J, Zhang F, Luo Y & Yang H (2006) A preliminary study on cactus as coagulant in water treatment. Process Biochemistry 41:730–733.

Zhao S, Gao B, Li X & Dong M (2012) Influence of using Enteromorpha extract as a coagulant aid on coagulation behavior and floc characteristics of traditional coagulant in Yellow River water treatment. Chemical Engineering Journal 200-202:569–576.