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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
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