Comparative study of flocculation and adsorption behavior of Moringa peregrina and Moringa oleifera seeds proteins Shirin Nouhi1, Habauka M. Kwaambwa2, Philipp Gutfreund3 and Adrian R. Rennie1* 1. Centre for Neutron Scattering, Uppsala University, Box 516, 751 20, Uppsala, Sweden. 2. Namibia University of Science and Technology, Faculty of Health and Applied Sciences, Private Bag 13388, 13 Storch Street, Windhoek, Namibia. 3. Institut Laue - Langevin, 71 avenue des Martyrs, F-38000 Grenoble, France * Corresponding author, E-mail address: [email protected] Abstract Trees of Moringa oleifera are the most widely exploited species of Moringa and proteins extracted from its seeds have been identified as the most efficient natural coagulant for water purification. Largely for climatic reasons, other Moringa species are more accessible in some regions and this paper presents a comparative study of the adsorption to different materials of the proteins extracted from seeds of Moringa peregrina and Moringa oleifera as a means to explore their use as flocculating agents in regions where each are more readily accessible. Results showed that Moringa peregrina seed proteins had higher adsorption to alumina compared to silica, in contrast to the opposite behavior for Moringa oleifera. Both species provide proteins that can act as effective coagulants for the various impurities in water with different surface potential. Despite considerable similarity in the amino acid composition, Moringa peregrina seed proteins have significantly different adsorption to those from Moringa oleifera and this presents the opportunity to improve processes by choosing the optimal species or combination of species depending on the type of impurity and the possible development of separation processes. Keywords: Alumina, Coagulation, Colloidal particles, Flocculation, Moringa oleifera, Moringa peregrina, Silica, Water purification, 1 Introduction pH whereas cationic polymers are regarded as environmental pollutants that are toxic to fish (Liber, Challenges in water purification 2005). For these reasons, there has been increasing attention to find alternative flocculants particularly Remediation and purification of water remains a from natural products. A range of biopolymers have major challenge for environmental engineering in been identified that include polysaccharides and many areas: there is a continuing pressure to provide proteins (Bodlund, 2013; Muthuraman and Sasikala, safer water supplies. Costs must be carefully 2014; Teixeira et al., 2017; Verma et al., 2012; Choy controlled, and it is highly desirable to avoid the use et al., 2015) as potentially useful, however, feasibility of treatment chemicals that could be toxic if used of use of particular products is affected by lack of incorrectly or in excess. Furthermore, there is a need availability in some areas. to develop processes that do not require the supervision of trained technical personnel Background particularly in rural areas in developing countries. These reasons are some of the powerful drivers to The most widely exploited natural material for water investigate the exploitation of non-traditional treatment has been crushed seeds from Moringa technology in water purification. oleifera trees. This was first observed in use as a In treatment of water for human consumption or for traditional process in the valley of the Nile river but remediation of wastewater, a common first step is work by Broin et al. (2002), for instance, identified clarification by coagulation or flocculation of that the protein from the seeds is the effective particulate impurities that can then settle or float coagulant. Adsorption to various materials has been according to their density. This step is important both reported (Santos et al., 2012; Araujo et al., 2010; to remove potentially toxic material but also to enable Kwaambwa et al., 2010; Kwaambwa et al., 2015) and further processes such as disinfection where the it was shown that although the molecules are small, required amounts of oxidizing agents such as chlorine they bind effectively to many potential impurities as or ozone would be substantially increased if there well as associating so that flocculation occurs. The were too much organic residue. Even ultraviolet material effectively clarifies water with impurities irradiation requires adequate water clarity. Many that include mineral particles, bacteria and algae specifications for water treatment therefore impose (Hellsing et al., 2014; Lürling et al., 2010; Barrado- standards of sufficiently low turbidity not only Moreno et al., 2016). Engineering tests have been because discolored water is unattractive. made with various scales of process equipment from The process of flocculation of the impurities has been small scale jar-tests (Muyibi and Evison, 1995) up to described extensively (Chong, 2012) and involves plant trials (e.g. de Paula, 2014; Beltran-Heredia and controlled stirring and reduction the stability of Sanchez-Martin, 2009). particles. Often particulate impurities are negatively Although other seed proteins could be exploited to charged. In brief, colloidal stability is usually some extent in a similar manner, systematic reduced by adding polyvalent salts such as aluminum comparison of over a hundred natural coagulants sulfate or iron (III) chloride (Duan and Gregory, have shown that those of Moringa oleifera trees are 2003), or by adding synthetic polymers that are often the most effective coagulant (Bodlund et al., 2014; cationic (Gregory and Barany, 2011). The Bodlund, 2013; Conaghan, 2013; Kwaambwa, 2013; mechanism in the first case depends on screening the Kansa and Kumari, 2014; Muthuraman and Sasikala, charged repulsion that imparts colloidal stability to 2014). Seeds from Moringa trees are also a rich the particles. In the latter case, the polymers are source of oil and the widespread use of different parts chosen to adsorb readily and are usually sufficiently of the tree for nutrition and medicine in many large that they may link to several particles. Choice countries gives ready acceptance for use in the of flocculating agents and mixing conditions that treatment of drinking water. Moringa seed proteins produce large flocs that are compact or can be have been proposed for wider use as a biodegradable, dewatered readily is valuable for efficient processing. low-toxicity, low-cost and sustainable replacements There are several difficulties with conventional to the usual synthetic materials and they can be used treatment that arise from potential toxicity if high in a simple way without trained technical supervision concentrations of metal ions remain in the water. For (Stohs and Hartman, 2015). example, aluminum has been implicated in There are thirteen known species of Moringa trees Alzheimer’s disease (Flaten, 2001; Mirza et al., 2017; that are native to Africa, Asia, Middle East and Exley, 2014). In the case of iron salts, it is often Madagascar (Olson, 2002; Price, 1985; Shindano and necessary to precipitate excess them by increasing the Kasase, 2009) but some are grown in various regions 2 of the world, including regions with little rainfall. study provides an example of a heterogeneous Although Moringa oleifera is the most widely used, interface with both hydrophobic regions and some it is interesting to consider the possibility to exploit groups that carry negative charge. It provides a other varieties. There is much less known about the model for typical particles dispersed in water. alternative species and there are few comparative Neutron reflectometry was chosen as a technique that studies showing whether other species can be as allows determination of the structure and effective as Moringa oleifera in water treatment. The composition of interfacial layers as well as the species native to East Africa, Moringa stenopetala, amount of material bound to the surface (Thomas and has been investigated (Hellsing et al., 2014). In Penfold, 1996). The reflected intensity measured as respect of the Moringa oleifera, there have been a function of wavelength and angle at low grazing several reports that the proteins are a complex angles can be analyzed to provide the molecular mixture. Moulin (2018) has reported extensive mass- composition profile. Neutron reflection is valuable spectrometry studies of the material that was obtained for studies of adsorption as it is straightforward to from Africa and the study clarified that certain apply, and the results are simple to interpret. components may bind differently to various However, it requires the use of large, flat substrates materials. that are transparent to neutrons so that the signal Moringa peregrina is another widely grown species reflected at low grazing angles of incidence can be of Moringa and it is native to various regions such as measured is sensitive to the adsorbed layer onto the Iran, the Arabian Peninsula, India, parts of Africa and surface (see Figure S1 and S2 in supporting as far north as the Dead Sea (Moustafa et al., 2012). information). Both silicon, which forms a native This geographical spread includes many areas where silica oxide layer on top, and alumina have the population suffer from limited access to clean insignificant absorption of neutrons and are readily water supplies. In the present study, we have chosen available as large and flat substrates. This provides seeds from two different varieties of Moringa trees another motivation to use silica and alumina for the from different regions, to