THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF AGRICULTURAL AND BIOLOGICAL ENGINEERING

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1 THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF AGRICULTURAL AND BIOLOGICAL ENGINEERING TURBIDITY REMOVAL FROM KAOLIN SUSPENSIONS AND WASTEWATER USING MORINGA OLEIFERA ANDREW NEAL SPRING 2013 A thesis submitted in partial fulfillment of the requirements for a baccalaureate degree in Biological Engineering with honors in Biological Engineering Reviewed and approved* by the following: Stephanie Velegol Instructor of Environmental Engineering Thesis Supervisor Herschel Elliott Professor of Agricultural and Biological Engineering Thesis Supervisor Ali Demirci Professor of Agricultural and Biological Engineering Honors Adviser * Signatures are on file in the Schreyer Honors College.

2 i ABSTRACT Moringa oleifera (Moringa or MO) is a tropical tree containing seeds that possess a cationic protein (MOCP) that has both antimicrobial and coagulation properties potentially useful in water treatment in developing countries. Since pathogen removal is correlated with turbidity removal in water supplies, this research investigated turbidity removal with time from kaolin suspensions and primary municipal wastewater effluent using extracted MOCP contained in the Moringa seeds. This research also focused on what effect seed maturity had on turbidity removal with time. In an effort to save seeds, micro-scale jar tests were used to evaluate the coagulation effectiveness of MOCP as exhibited by turbidity removal in two kaolin suspensions (initial turbidities of 100 and 500 NTU) and one 100 NTU primary effluent wastewater. For the 100 NTU kaolin suspensions, the presence of MOCP moderately increased turbidity removal compared to the control samples after 1 hour of sedimentation(mocp absent). This finding was consistent with previous studies documenting the less effective coagulation ability of MOCP for low turbidity waters. However, for 500 NTU kaolin, turbidity removal after 1 hour was 86.4% in the presence of MOCP compared to 19.1for the control samples. Assuming turbidity was proportional to the concentration of particles in solution, the data was analyzed for conformance to perikinetic flocculation which assumes that the rate of change of the concentration of particles with time is a second-order process. Plots of the integrated form of a secondorder process (1/turbidity versus time) were highly linear (R 2 = 0.97 and 0.99 for 100 NTU and 500 NTU kaolin, respectively). Perikinetic flocculation dictates that the rate of

3 ii turbidity removal depends on the initial particle concentration and explains the observed greater rate of turbidity removal for the 500 NTU versus the 100 NTU kaolin suspensions. The same micro-scale jar tests were performed using 100 NTU primary effluent wastewater. After one hour, the turbidity removal was 60.6% in the presence of MOCP and 20.1% in the control samples. Compared to the 100 NTU kaolin suspension, the wastewater initially had greater turbidity removal, but after 24 hours regrowth in the wastewater began to increase the turbidity. After 24 hours the turbidity removal in 100 NTU kaolin was 98.2% whereas the turbidity removal in wastewater was 73.3%. Regrowth of the wastewater over time competed with turbidity removal, and perikinetic flocculation theory alone was not sufficient in describing the wastewater results. Micro-scale jar tests were also conducted using less mature medium-sized MO seeds. Contrary to current Moringa literature, a major finding was that the less mature, partially green seeds still were effective at reducing turbidity. Further research is needed to clarify the relationship between seed maturity and effectiveness as a natural coagulant for water treatment. Using a newly developed percent turbidity removal calculation, the turbidity removal after 24 hours for the medium and large seeds was 79.9% and 92.7%, respectively. The research demonstrates that extracts of MO can serve as natural coagulants for treatment of turbid waters, although the effectiveness depends on many factors including the initial turbidity of the water, sedimentation time, and maturity of the MO seeds. Further research is needed to define the conditions under which MO should be

4 implemented as a low cost, point-of-use water treatment method in communities lacking access to clean drinking water. iii

5 iv TABLE OF CONTENTS Abstract... i-iii Table of Contents... iv-v List of Figures... vi-vii List of Tables... viii Acknowledgements... ix Epigraph...x Chapter 1 Introduction and Background Knowledge... 1 Motivation... 1 Moringa oleifera as a Solution... 2 Other Moringa Research... 5 Research Hypothesis and Objectives... 6 Introduction to Coagulation Science... 7 Moringa oleifera s Methods of Coagulation Chapter 2 Materials and Methodology Moringa oleifera Seed Picking and Collection Preparation of Moringa oleifera Serum Synthetic Turbid Water Preparation Wastewater Collection and Preparation Process Micro-Scale Jar Test Procedure Summary of the Micro-Scale Jar Test Procedure Turbidity Versus Time Procedure Chapter 3 Results and Discussion Micro-Scale Jar Test Experimental Procedure Verification of the Developed Procedures New Data Analysis Technique Explanation of Negative Percent Turbidity Removal Effect Initial Turbidity had on Percent Turbidity Removal Comparing Turbidity Removal for the Three Systems Comparison of 100 NTU Kaolin and 100 NTU Primary Effluent Wastewater Effect of Seed Size on Percent Turbidity Removal Mass Distribution of Chiang Mai Moringa Seeds... 40

6 v Percent Seed Data for Large and Medium Chiang Mai Seeds Effect of Seed Size on Percent Turbidity Removal-Graphs Chapter 4 Conclusions and Future Work Major Findings Implications for the Developing World Suggestions for Future Research Appendix A Additional Collected Data Turbidity Versus Time Data Micro-Scale Jar Test Experiments Seed Mass Distribution Data Percent Seed Data REFERENCES... 67

7 vi LIST OF FIGURES Figure 1-1. World water scarcity and locations Moringa grows Figure 1-2. Moringa oleifera tree, seed pods, and seeds... 4 Figure 1-3. Electric Double Layer diagram Figure 1-4. DLVO theory diagram... 9 Figure 2-1. Dr. Bates collecting the MO seed in Thailand Figure 2-2. Moringa seed pods drying Figure 2-3. Moringa seeds with their shells Figure 3-1. Turbidity vs. time data for 500 NTU kaolin Figure 3-2. Residual turbidity vs. dosage graph Figure 3-3. Turbidity vs. time data for 500 NTU kaolin Figure 3-4. Percent turbidity removal data for 500 NTU kaolin showing the new and old analysis techniques Figure 3-5. Turbidity vs. time data for 100 NTU kaolin Figure 3-6. Percent turbidity removal data for 100 NTU kaolin showing the new and old analysis techniques Figure 3-7. Scattering light intensity vs. particle diameter Figure 3-8. Perikinetic flocculation theory with 100 and 500 NTU kaolin Figure 3-9. Comparison of turbidity removal in 100 and 500 NTU kaolin Figure Turbidity vs. time data for 100 NTU primary effluent wastewater Figure Percent turbidity removal data for 100 NTU primary effluent wastewater showing the new and old analysis techniques Figure Comparison of turbidity vs. time data for 100 NTU kaolin and 100 NTU primary effluent wastewater Figure Perikinetic flocculation theory with 100 NTU kaolin, 500 NTU kaolin, and 100 NTU primary effluent wastewater Figure Comparison of turbidity removal in 100 NTU kaolin and 100 NTU primary effluent wastewater

8 vii Figure Comparison of turbidity vs. time data for 100 NTU kaolin using medium and large MO seeds Figure Percent turbidity removal using the new analysis for 100 NTU kaolin using medium and large seeds after 24 hours Figure Percent turbidity removal using the old analysis for 100 NTU kaolin using medium and large seeds after 24 hours Figure A-1. Mass distribution of large Chiang Mai MO seeds Figure A-2. Mass distribution of medium Chiang Mai MO seeds... 60

9 viii LIST OF TABLES Table 3-1. Mass distribution of large Chiang Mai MO seeds Table 3-2. Mass distribution of medium Chiang Mai MO seeds Table 3-3. Percent seed data for large Chiang Mai seeds Table 3-4. Percent seed data for medium Chiang Mai seeds Table A-1. Turbidity vs. time data for 500 NTU kaolin with new and old analysis data Table A-2. Turbidity vs. time data for 100 NTU kaolin with new and old analysis data Table A-3. Turbidity vs. time data for 100 NTU primary effluent wastewater with new and old analysis data Table A-4. Micro-scale jar test-turbidity data for 100 NTU kaolin with large seeds Table A-5. Micro-scale jar test-turbidity data for 500 NTU kaolin Table A-6. Micro-scale jar test-turbidity data for 100 NTU primary effluent wastewater Table A-7. Micro-scale jar test-turbidity data for 100 NTU kaolin with medium seeds Table A-8. Mass distribution of large Chiang Mai seeds-all data Table A-9. Mass distribution of medium Chiang Mai seeds-all data Table A-10. Percent seed data for large Chiang Mai seeds-all data Table A-11. Percent seed data for medium Chiang Mai seeds-all data

10 ix ACKNOWLEDGEMENTS I would like to sincerely thank both Dr. Stephanie Velegol and Dr. Herschel Elliott for serving as my thesis supervisors. Your consistent support and encouragement allowed me to learn and grow throughout this entire process. I look up to both of you for the people you are, and for the work you are accomplishing in your respective fields. I would also like to thank Dr. Darrell Velegol and the entire Velegol research group in Chemical Engineering for their guidance and use of the lab. Special thanks to Toni Bechtel for helping me get started in the lab. Thanks to Dr. Ricky Bates for collecting the Moringa oleifera seeds used in this research. I would also like to thank Ben Kutz and Krista Liguori for their friendship and assistance with data collection. Both Ben and Krista are fellow undergraduate researchers on this project. Finally, I would like to thank my honors advisor, Dr. Ali Demirci, and the Department of Agricultural and Biological Engineering for their ongoing support.

11 x EPIGRAPH Jesus replied, Everyone who drinks some of this water will be thirsty again. But whoever drinks the water I give them will never thirst. Indeed, the water I give them will become in them a spring of water welling up to eternal life. John 4:13-14

12 1 Chapter 1 Introduction and Background Knowledge Motivation Worldwide, there are about 890 million people who do not have access to an improved water supply (UN, 2013). The United Nations claims that every 20 seconds a child dies because of poor sanitation. More specifically, diarrhea kills one in five children worldwide. That is more than AIDS, malaria and measles combined (UN, 2008). Also, 3.4 million people die each year from water-related diseases (UN, 2008). This lack of improved water supplies is found in developing nations primarily in Africa, and Southern Asia. Figure 1-1 shows what areas thought out the world lack access to improved water sources. Right below this image shows where Moringa oleifera can grow through-out the world. The red dots on the figure shows contacts that the Velegol research group has where Moringa trees are currently growing.

13 2 Access to Moringa oleifera Figures 1-1. The top image shows areas in the world that lack access to safe drinking water. The bottom image shows areas that Moringa oleifera can grow; with the red dots on current contacts that the Velegol research group has. Moringa oleifera as a Solution One solution to improve water supplies is utilizing a locally available seed of the Moringa oleifera (MO) tree. Moringa is the single genus of the Moringaceae and has thirteen species. It is

14 3 native to the sub-himilayan areas of northwestern India, and can be grown in many regions worldwide including Africa, Arabia, Southeast Asia, the Pacific Islands, the Caribbean Islands, and South America (Fuglie, 2001; Palada, 1996; Crosby, 2007). Moringa has many uses and is commonly referred to as the drumstick tree for its seed pods, the ben oil tree for the oil from the seeds, and the horseradish tree for the flavor of its roots (Crosby, 2007). Moringa is also a highly nutritious crop with nearly every part of the plant containing some food value. The leaves, pods, flowers and seeds of Moringa oleifera are valuable in the nutritional, medicinal, cosmetic, and water areas (Palada, 1996; Crosby, 2007). Ounce for ounce, Moringa leaves contain more betacarotene than carrots, more iron than spinach, more potassium than bananas, more Vitamin C than oranges, and more protein that peas (Palada et al., 2003; Crosby, 2007). The tree is fast growing, can live for 20 years, contains a deep root system, and reaches heights of 7-12 meters tall (Foidl et al., 1999). One beneficial aspect of Moringa is that it grows in semi-arid regions and produces leaves in times of drought when other crop growth is limited (Marcu, 2005; Senall, 2007). Generally inadequate food supplies are found in areas with inadequate water supplies. Water clarification, using natural occurring coagulants has been in practice for hundreds of years, and Moringa seeds have been one of the best natural coagulants (Marcu, 2005; Senall, 2007). Utilizing Moringa seeds to treat water is a naturally occurring, cost effective, safe solution to improve water supplies in the developing world. Currently, the use of naturally occurring materials like Moringa oleifera has been suggested to be used in water treatment to help reduce the high cost associated with conventional water treatment (Ghebremichael 2006; Katayon et al. 2007; Nkurunziza, 2009). In figure 1-2, images of a Moringa oleifera tree from Chiang Mai, Thailand is shown. Also, in figure1-2 the tree s seeds and the pods containing seeds are shown.

15 Figures 1-2. These are all images that Dr. Ricky Bates took while collected the MO seeds in Chiang Mai, Thailand. The upper left image is an MO tree with pods attached. The lower image is of the pods drying after being taken off of the tree, and the upper right image is of the individual MO seeds still in their shells. 4

16 5 Other Moringa Research Multiple studies have been conducted on the performance of MO seeds as an alternative coagulant (Jahn, 1988; Muyibi and Okuofu, 1995; Ndabigengesere et al., 1995; Muyibi and Evison, 1996). In fact, MO was first observed and reported to help clarify turbid water by (Jahn, 1981 cited by Nkurunziza et al. 2009) after observing Sudan women use the seeds to clean water from the Nile. Since then, MO has been studied in water treatment as both a coagulant and a disinfectant (Okuda et al. 1999; Katayon et al. 2004; Farooq et al. 2007). Moringa oleifera s seeds contain cationic proteins (MOCP) that act as a natural coagulant. This coagulant has been shown to work best for high turbidity waters (Muyibi and Evison, 1995). The dimeric cationic protein responsible for coagulation has a molecular weight of 13kDa and an isoelectric point between 10 and 11 (Ndabigengersere, 1995). The mechanism of coagulation appears to be adsorption and neutralization of the colloidal charges (Ndabigengersere, 1995). MOCP has also been studied compared to alum, and the purified proteins are a more effective coagulant than alum (Ndabigengesere, 1995). Previous research was also done in the Velegol research group to show that MOCP can be adsorbed onto sand granules and used as a sand filter to reduce turbidity and kill certain bacteria while reducing the release of BOD (Velegol, 2012). The EPA states that turbidity removal is important because, turbidity provides food and shelter for pathogens, and can promote regrowth of pathogens that leads to outbreaks of waterborne diseases. Turbidity itself is not a direct indicator of health risk, but numerous studies show a correlation between protozoa removal and turbidity removal. Also, the EPA claims that controlling turbidity is a competent safeguard against pathogens in drinking water ( 2013).

17 6 Research Hypothesis and Objectives This thesis focused on the development of a micro-scale jar test procedure to find the percent turbidity removal in different media over time due to the addition of MOCP. This procedure is the first micro-scale jar test of Moringa, and this method uses less MO seeds than the traditional jar test. Micro-scale jar turbidity tests were completed to determine Moringa s effectiveness at reducing the turbidity in 100 NTU kaolin, 500 NTU kaolin, and 100 NTU primary effluent wastewater. Analysis of the different media was completed, and the major research questions were: What effect do different media have on percent turbidity removal? What effect does initial turbidity and sedimentation time have on percent turbidity removal? What effect does seed maturity have on percent turbidity removal? Each of these questions is important when thinking about how to practically use Moringa for water treatment in the developing world.

18 7 Introduction to Coagulation Science Briefly, an introduction to the science behind coagulation and flocculation will be presented. Much of the theory presented was found in Dr. Darrell Velegol s book titled Colloidal Systems (unpublished book, University Park, PA.: Penn State University, Department of Chemical Engineering). This is presented for a basic understanding of how Moringa oleifera is functioning as a coagulant. There are two main competing forces acting on suspended particles in a liquid media. They are electrostatic repulsions forces and Van der Waals (VDW) attractive forces. VDW forces occur naturally due to slightly more electrons in one region of an atom at any time. This causes temporary electric dipoles to result, and this induces dipoles on neighboring atoms or particles. This interaction between neighboring atoms causes an attractive force responsible for aggregation. A basic equation that estimates the VDW attractive energy between two particles is shown below: Where a is the radius of the two spherical particles, δ is the distance the particles are separated by, and A is the Hamaker constant, specific to the particle and media. Electrostatic repulsion refers to the positive potential energy caused by the repulsion of two similarly charged particles. Electrostatic forces in aqueous solutions will have an electric double layer (EDL). The EDL occurs because in solutions ions are mobile, and generally the positive ions in solution will naturally move toward the negatively charged surfaces to neutralize the surface charge. This results in a thin layer of mostly positively charged particles around the negatively charge surface and this layer is called the electric double layer (EDL). An image of the double layer can be seen below.

19 8 Figure 1-3. Electric double layer surrounding a negatively charged particle in an aqueous solution. (Velegol, 2013, personal communication) κ -1 is the thickness of the EDL and is called the Debye screening length. The electric repulsion potential decays away from the particle at a rate approximately equal to the Debye-Huckel equation: Where ψ o is the surface potential of the particle, and x is the distance from the surface. Combining these different theories of electrostatic repulsion, the electrostatic energy of two particles is equal to: Where a is the radius of the two particles, δ is the distance between the particles, and ε is the electrical permittivity describing the fluid. To approximate the total energy between particles, the electrostatic and VDW energies can be summed to obtain the Derjaguin-Landau-Verwey-Overbeek or DLVO energy between two particles.

20 9 Substituting the original equations for the potential energy of electrostatic repulsion and Van der Waals energy gives the above equation for total energy between two particles. A basic graph shown in figure 1-4, shows the energy between two particles where the dashed ES line shows the repulsive electrostatic energy, the dashed VDW line shows the attractive Van der Waals energy, and the solid blue lines shows the total energy or DLVO energy. (Velegol, 2012) Figure 1-4. Energies effecting two particles in an aqueous solution. The top dashed line show the repulsive electrostatic energy and the bottom dashed line shows the attractive Van Dar Waal energy. The solid blue line is known as the DLVO energy or total energy between the particles. By decreasing the electrostatic energy particles can aggregate together. (Velegol, 2013, Colloidal Systems, personal communication) The primary idea behind coagulation is reducing the electrostatic repulsion between particles so that the VDW attractive forces can cause flocculation. The time that it takes for these particles to aggregate is also of interest. In order to aggregate, the particles must collide and in this thesis s current data collection method the primary form of motion is described as Brownian motion. The aggregation time(τ) is the time it takes to allow roughly half of the suspended particles to aggregate together and is described by:

21 10 Where a is the particle radius, η is the fluid viscosity, ϕ is the volume fraction of particles, W is the stability ratio which is defined as the average number of times particles must collide before they adhere. Once these particles aggregate, the basic governing equation for the sedimentation process is Stokes Law. The velocity (v) of a particle settling in laminar flow conditions is described by the following equation: Where r is the particle radius, g is the acceleration due to gravity, ρ is the density of the corresponding substance, and µ is the fluid s absolute viscosity. Knowing the velocity of the settling particle and the depth of the fluid can give the time it takes for the particle to settle out of the fluid. Moringa oleifera s Methods of Coagulation According to Ndabigengesere et al.(1995) the coagulation properties of Moringa are due to the water soluble cationic proteins (MOCP) found in the seeds. These proteins are densely charged cationic dimers that cause coagulation by adsorption and charge neutralization (Ndabigengesere, 1995). Essentially, MOCP is reducing the electrostatic energy of the particles by neutralizing the negatively charged surface, so Van der Waals attractive forces can dominate and cause coagulation.

22 11 Chapter 2 Materials and Methodology Moringa oleifera Seed Picking and Collection The Moringa oleifera seeds used in the experiments in this thesis were collected by Dr. Bates on a trip to Chiang Mai, Thailand specifically in the San Sai district. Moringa seeds were primarily collected from a group of six trees from late January until early April. Since these trees are in a tropical environment, on each tree there is a variety of seed sizes and maturities. On the tree, the seed kernels are contained in long pods. The pods were picked and then allowed to air dry for about one week. After the seeds were dry, the pods were broken open and the seed kernels collected in plastic bags according to their size. Please see the images below that show the picking and drying process. Figure 2-1. Dr. Bates in Chiang Mai, Thailand with the Moringa oleifera trees that the seeds were picked from.

23 12 Figure 2-2.The picked MO seed pods air drying. Figure 2-3. The seeds from inside the pods with shells still on. Note the green color on the outside of the shells, indicating that these seeds are not fully mature yet.

24 13 Preparation of Moringa oleifera Serum The cationic protein (MOCP) responsible for coagulation properties that Moringa contains was extracted with the following procedure. First, just over 1.0 gram of whole Moringa oleifera seeds was weighted out. The seeds were grinded with a coffee grinder or crushed with a mortar and pestle into fine pieces. Note that the seeds were crushed with the shells still on. The crushing should only take a few minutes. Next, 1.0 gram of the crushed MO seed was measured out and was added to 15mL of DI water. This serum rolled on roller bars for one hour at a speed setting of 100%, which was roughly 60rpms. After the serum has rolled for one hour, it was removed from the roller bars and filtered through 11 µm Whatman filter paper (catalog number: ). Then the serum was further filtered by using a 0.2 µm syringe filter. After the serum was filtered through the 11 µm filter paper and the 0.2 µm syringe filter, it was ready to be added to the media of interest. The cationic protein responsible for coagulation was extracted from the MO seeds during the rolling process and was now in the filtered serum. In the turbidity experiments completed in this research, the prepared serum was used right after the filtration process. If the MO serum was not used immediately, it was refrigerated. Synthetic Turbid Water Preparation Synthetic turbid water was made using kaolin powder from Sigma-Aldrich product K7375. This suspension was used for the purpose of having controlled laboratory turbidity experiments. We closely followed the procedures used by Ndabigengesere et al. (1995) to make the synthetic turbid water. For our experiments, 5.0 grams of kaolin was added to 1 Liter of DI water. The

25 14 kaolin and DI water were then stirred for 1.5 hours at setting 8 on a Thermolyne Nuova II stir plate using a 1.5 inch stir bar on a magnetic stir plate. The mixture was then allowed to sit undisturbed for 24 hours. After 24 hours of sedimentation, the supernatant was decanted into another 1 liter bottle for storage. The stock solution was diluted down to 500 NTU and 100 NTU for the turbidity experiments done in this thesis. Wastewater Collection and Preparation Process The wastewater used in these turbidity experiments was collected from the primary effluent of the University owned and operated wastewater treatment plant. Wastewater was used as a better representation of the type of surface waters that would be treated in the developing world. To mimic water treatment in the developing world, the wastewater samples were collected the same day the micro-scale jar turbidity tests were completed. Before the micro-scale jar turbidity tests were started, the wastewater was allowed to sit for 10 minutes to allow sedimentation of the bigger particles. After sitting, the supernatant was collected and mixed thoroughly for at least 10 minutes. The supernatant averaged a turbidity of about 100 NTU depending on the wastewater collected. This was used as the wastewater media for the micro-scale jar turbidity experiments. Micro-Scale Jar Test Procedure The micro-scale jar turbidity test procedure used in this thesis was developed by Walker et al., 2012 (personal communication). In this thesis, the experiments completed followed this basic procedure. The following procedure was followed to measure the percent turbidity removal effects of Moringa oleifera on various media.

26 15 To begin, MO serum was prepared by following the procedure described above in the Preparation of Moringa oleifera serum section. Typically, the seeds were crushed using 20 seeds at one time. In summary, the MO serum preparation included crushing of seeds, adding 15mL of DI water, mixing the solution for one hour, filtering with 11 µm filter paper, and finally filtering with a 0.2 µm syringe filter. While waiting for the serum to filter, the kaolin or wastewater solution was mixed for at least 10 minutes on a magnetic stir plate to ensure that the solution was mixed evenly. For these experiments, the media s turbidity was checked to obtain a turbidity close to 100 NTU for primary effluent wastewater, and 100NTU kaolin, and 500 NTU for the 500 NTU kaolin media. Be sure to dilute down the kaolin solution as necessary. Next, the well mixed media solution was poured into five separate 100mL beakers. 100mL of the media was poured into one 100mL control beaker and 97.5mL of kaolin or wastewater media was poured into four separate 100mL sample beakers. Then these beakers, filled with media, were placed on a magnetic multi-stir plate. A 0.5 inch magnetic stir bar was added to each 100mL beaker and the media begin mixing at 150rpm. These five beakers (1 control with 100ml and 4 samples with 97.5ml) were stirred at 150rpm while the MO serum finished filtering. After the Moringa serum has been filtered, 2.5mL of the filtered MO serum was added to each of the four kaolin or wastewater samples. Note that the samples and control were already mixing at 150rpms. One minute after adding the MO serum, the stir plate was turned off. Then sufficient volume (approximately 13mL) was pipetted out from the control and each sample into five different Turbidimeter vials. Pipetting was done carefully from the center of each beaker having the pipette tip 0.5 cm below the surface of the media. The turbidity reading was recorded for the control and all four samples using the standard procedures for the HACH 2100Q Turbidimeter (catalog number: 2100Q01).

27 16 Next, the control and each sample were stirred for 30 min at 60rpms to allow flocculation. After 30 minutes, the stir plate was turned off. The control and samples then sat for one hour to allow sedimentation. After that hour, sufficient volume was again pipetted out from the control and each sample into the five Turbidimeter vials. The turbidity of the vials was measured using the HACH 2100Q Turbidimeter. Finally, the control and samples sat for 23 additional hours (24 hours total) before the last turbidity measurement was taken. After 24 hours the turbidity was again measured using the HACH 2100Q Turbidimeter. The results were recorded and will be discussed later. Summary of the Micro-Scale Jar Test Procedure A summary of the basic procedure followed to complete the Micro-Scale jar turbidity experiments was: Make the Moringa serum and filter it. Fill four sample beakers with 97.5mL of media and one control beaker with 100mL of media. Mix these beakers at 150rpms. Add 2.5mL of this serum to each of the four sample beakers. Let all the beakers stir for one minute at 150 rpms. Then turn off stir plate and immediately take a turbidity measurement of each beaker. Next, let all five beakers stir for 30 min at 60 rpms. After stirring, let the beakers sit for 1 hour of sedimentation. Record the turbidity after 1 hour of sedimentation. Record the turbidity after 24 hours of sedimentation.

28 17 Turbidity Versus Time Procedure Three turbidity versus time graphs are presented in the results and discussion portion of this thesis. There is one graph for each of the following media: 100 NTU kaolin, 500 NTU kaolin, and 100 NTU primary effluent wastewater. These three media were all used in the micro-scale jar turbidity test experiments. For the turbidity versus time experiments, the motivation was to determine how long the media needed to sit to obtain a certain reduction of turbidity. The procedures for the turbidity versus time experiments are identical to the micro-scale jar turbidity test procedures except additional turbidity measurements were taken at times in between the 1 hour and 24 hours of sedimentation. For example, the turbidity was measured after 2, 4 and 6 hours of sedimentation for the 100 NTU kaolin turbidity versus time experiments.

29 18 Chapter 3 Results and Discussion Micro-Scale Jar Test Experimental Procedure A portion of this thesis work consisted of developing a standard procedure to be used to properly measure the reduction of turbidity due to MO seeds. It is the hope that future researchers will be able to use the standard procedure developed in this thesis to run turbidity experiments using the micro-scale jar test procedures. Using the micro-scale jar test procedure from Walker et al. (personal communication) as a starting point, the developed procedure are described in the Materials and Methodology section. The MOCP extraction procedure was similar to other procedures found in literature (Ndabigengesere et al., 1995). Filtering the serum through 11µm filter paper and then further filtering with a 0.2µm syringe filter was to ensure that the MO serum added to the sample beakers did not add any turbidity to the samples. The turbidity of the filtered MO serum was also checked using the HACH 2100Q Turbidimeter, and was found to be approximately 1.5 NTU. Next, 2.5mL of MO serum was added to four sample beakers containing 97.5mL of media each. One control beaker was used with 100mL of media, so all five beakers ended with 100mL of media in them. The stirring speeds and times were consistent with that found in the micro-scale jar test procedure from Walker et al. However, after the one minute of mixing at 150rpms, our procedure had a flocculation period of 30 minutes stirring at 60rpms instead of 30rpms. This was due to the limitations of the multi-stir plate used in these experiments. Turbidity measurements were taken after one hour and after 24 hours of sedimentation, and whenever these measurements were made 13mL of media, with the added MO

30 19 serum, was pipetted out of the sample beaker and placed into a HACH 2100Q Turbidimeter vial. The 13mL was pipetted out from the center of the beaker at a depth of 0.5cm. This was to ensure that the collection of the sample was at a set distance from the water surface and that no surface water was included in the turbidity measurements. Verification of the Developed Procedures It was important to verify that the procedures developed and used in this thesis were consistent with the current literature on Moringa s ability to remove turbidity. Comparison of the 500 NTU kaolin data to that of Ndabigengesere et al. (1995) shows similar results. On figure 3-2, a blue triangle is placed at the dosage of 25mL/L which was the dosage for the experiments completed in this thesis. It can be seen that at this dosage the resulting final turbidity was around 10 NTU and the resulting turbidity for our experiments was 3.07 NTU. This shows that the procedure used in this thesis gave results consistent with those found in literature. In figures 3-1 and 3-2 below, a comparison of this thesis s data and current literature is shown.

31 20 Figure 3-1. Turbidity vs. time data for 500 NTU kaolin control and samples. The four samples had 2.5mL of MO serum added to the 97.5mL of 500 NTU kaolin. Turbidity measurements were taken over time. Figure 3-2. Turbidity vs. dosage data found in Ndabigengesere et al. (1995). A blue triangle indicates the dosage used in this thesis s turbidity experiments. The resulting turbidity obtained is consistent with turbidities obtained in this thesis.

32 21 It is important to acknowledge that figure 3-2 was in a paper published by Ndabigengesere et al. (1995). That paper utilized the jar test for evaluating coagulation, and they made their recorded turbidity measurements after 20 minutes of slow mixing and then 30 minutes of sedimentation. The final turbidity results in this thesis utilized the micro-scale jar test and were recorded after 24 hours of sedimentation. New Data Analysis Technique Another aspect of this thesis was developing a new way to analyze the obtained turbidity data. In the literature today, almost every paper compares the turbidity of the sample back to the initial turbidity of the sample in order to calculate percent turbidity removal. When doing this, two key pieces of information are left out. First, the initial turbidity spike due to the increase in the effective particle size is not considered. Second, there is no control data; meaning the particles that naturally settle out with time are included in the turbidity removal calculations. In this thesis, percent turbidity removal was calculated based on a control that was allowed the same amount of sedimentation time as the samples. With this calculation, a more accurate percent turbidity removal value is obtained which is due to only the coagulation of the MOCP. In this thesis, percent turbidity removal was calculated with the new analysis technique defined by the following formula: In other literature, calculating percent turbidity removal is done using the old analysis technique defined by the following formula:

33 22 Other researchers are calculating percent turbidity removal based solely on the current (after a certain sedimentation time) and initial turbidity of the sample. Throughout this thesis, both the new and old analysis techniques will be utilized in order to compare obtained data. Also, an illustration of a control and sample turbidity versus time graph in shown in figure 3-3. In figure 3-3 sample and control turbidity data can be seen for a 500 NTU kaolin media. Figure 3-3. Turbidity vs. time data for 500 NTU kaolin control and samples. The four samples had 2.5mL of MO serum added to the 97.5mL of 500 NTU kaolin. Turbidity measurements were taken over time. Using the above data in figure 3-3, the below figure 3-4 is obtained using both the new and old analysis techniques for calculating percent turbidity removal discussed above.

34 23 Figure 3-4. Percent turbidity removal of MOCP for 500 NTU kaolin media using the new and old analysis methods. The green diamonds and red circles represent the new and old analysis technique respectively. The blue triangles show the control data s percent turbidity removal calculated with the old analysis technique. In the appendix in table A-1, each specific point is shown. Again, the new analysis percent removal is calculated using this equation So in order to calculate percent turbidity removal with the new analysis technique, the control turbidity at the same time needed to be measured. Since the micro-scale jar test was used and the control beaker only had 100mL of media, there was a limit on how many turbidity measurements

35 24 could be taken. This is why the new analysis technique only has four points represented on the graph. The old analysis is calculated using this equation: After just one hour of sedimentation, the removal of the sample with the new analysis 75.2%, the old analysis gave a percent removal of 82.9%, and the control only reduced the turbidity by 19.1% due to particles naturally settling out with time. In figures 3-5 and 3-6 the same data is shown for 100 NTU kaolin. Defining percent turbidity using the new analysis, gives a very interesting result for media with a lower initial turbidity. Figure 3-5. Turbidity vs. time data for 100 NTU kaolin control and samples. Four samples were averaged to obtain the samples data point.

36 25 After one and two hours of sedimentation, the percent turbidity removed is a negative value using the new analysis technique. This is because the turbidity of the control is less than the turbidity of the samples that MO serum was added to. Whenever the MO serum was added to the media, there was always a turbidity spike seen in the initial turbidity measurement of the samples. This initial turbidity spike is due to the cationic protein within the Moringa seeds beginning to flocculate particles. Right after adding the MO serum to any solution studied, a turbidity measurement was taken, and this measurement was always higher in turbidity than the control turbidity measurement. The average turbidity spike one minute after addition of the MO serum was 52 NTU for the 100 NTU turbidity kaolin media. In 100 NTU kaolin, two hours of sedimentation does not allow enough time for this turbidity spike to decrease below the turbidity of the control. That is why a negative percent turbidity removal value is obtained. In figure 3-5, it is seen that after four hours of sedimentation, the average sample turbidity is well below the control turbidity. Figure 3-6 displays the percent turbidity removal of the samples and control using the new and old analyses.

37 26 Figure 3-6. Percent turbidity removal of MOCP vs. time for 100 NTU kaolin media using the new and old analysis methods. The green diamonds and red circles represent the new and old analysis technique respectively. The blue triangles show the control data s percent turbidity removal calculated with the old analysis technique. In table A-2 found in Appendix A, each specific point is shown. The 100 NTU kaolin media did not have percent turbidity removals as high as the 500 NTU kaolin media. It also took a longer amount of sedimentation time to see positive turbidity removal results. This will be discussed in detail later. It is expected that the samples turbidity decrease to below the control turbidity in between the 2 and 4 hours of sedimentation period for the new analysis technique. Again, concerning the old analysis technique, it is impossible to have a negative turbidity. That technique does not take into account the initial spike caused by the introduction of the MOCP coagulant or the particles that naturally settle out over time. Throughout this thesis, both the new and old analysis

38 27 techniques will be used because there are limitations with each one. Generally, the new analysis technique will be used, but when the new analysis is returning negative percent turbidity removals, the old analysis will be used for additional clarification. Explanation of Negative Percent Turbidity Removal The negative percent turbidity removal result from the new analysis technique focused the research on a few other questions. They were how does the percent turbidity removal change with time and initial turbidity. These questions will be discussed more later. First, to address the question why did the turbidity spike in the 100 NTU kaolin and not decrease as fast as it did in the 500 NTU kaolin. It was initially ruled out that the MO serum was adding turbidity to the samples by verifying that the turbidity of the MO serum was negligible (1.5 NTU). There are a few reasons why this spike is expected. This spike in turbidity is primarily due to coagulation of particles that is already taking place one minute after the addition of the MO serum. Immediately after the MO serum is added to the sample media, the kaolin particles begin to aggregate together due to the introduction of the cationic protein acting as a coagulant. This aggregation creates larger particle diameters which in turn scatter more light. Turbidity is a measurement that is directly related to light scattering. With increasing particle diameter, light scattering will increase (Behrens et al., 1999). This is only true for particles less than about 0.3µm in diameter though. As seen from figure 3-7, the light scattering increases proportional to the diameter cubed for particles below 0.3 µm, but decreases at a rate of 1/diameter for particles bigger than 0.3 µm in diameter.

39 28 Figure 3-7. Light scattering intensity is on the y axis and the particle diameter on the x axis. With increasing particle diameter up to 0.3µm the light scatter increases in proportion to the diameter cubed. The light scattering decreases in proportion to 1/diameter if particle diameters are greater than 0.3µm ( This increased light scattering from the particles smaller than 0.3 µm, directly gives a higher turbidity reading. It is likely then that the higher initial turbidities right after the addition of the MO serum is due to the coagulation of the particles smaller than 0.3 µm. It is also of interest to ask why the average turbidity spike was 52 NTU for the 100 NTU kaolin and 151 NTU for the 500 NTU kaolin. The answer to this question can be solved by looking into the aggregation time equation presented in the Introduction to Coagulation Science section of this thesis found in Chapter 1. The equation is also shown below. The primary reason why the 500 NTU kaolin experiences a larger initial turbidity spike is due to the fact that the volume fraction ϕ, is significantly larger in the 500 NTU kaolin than it is in the 100 NTU kaolin. This increase in the volume fraction will, in turn, decrease the aggregation time, τ. This means that the higher concentration of kaolin particles in the media come in contact with the MOCP faster, thus aggregating faster. This same idea will also be investigated by

40 29 looking at perikinetic flocculation. Another way to look at that question is by considering perikinetic flocculation. Perikinetic flocculation is a type of flocculation due to Brownian motion or the random collision of particles in the media. This is the dominant form of flocculation happening during the sedimentation process. Perikinetic flocculation is governed by a second order differential equation, and the corresponding second order differential equation graph for both 100 and 500 NTU kaolin is shown in figure 3-8. Figure and 500 NTU kaolin media plotted as a second order differential equation. The linear line of best fit has correlation coefficients of 0.99 and 0.97 indicating that the experimental data closely follows perikinetic flocculation theory. This figure directly relates to the aggregation rate equation. If the only difference expected from the 100 NTU and 500 NTU kaolin aggregation times is the volume fraction, then the slopes of these lines would be identical. As seen from the figure, the slope of the 500 NTU line of best fit is and the slope of the 100 NTU line of best fit is These slopes are very close especially when considering the fact that this is experimental data. There has not been work done

41 30 to determine if these slopes can be considered statistically the same, but the experimental data fits the perikinetic flocculation theory well. Another way to analyze this data is by looking at the correlation of the line of best fit. The correlation for the linear best fit line is and 0.972; meaning the collected data for these experiments closely follow second order perikinetic flocculation theory. Since the flocculation occurs as a second order differential equation, the half-life or time to reduce half the initial turbidity is equal to the following equation: Where k is the slope of the linear best fit line found in figure 3-7 and N o is the initial turbidity of the media. Since this is a second order differential equation, the initial concentration of particles (N o ) will affect the half-life as seen in the above equation. With increasing initial turbidity there will be a corresponding decrease in the half life. This result is directly related to the aggregation rate equation displayed earlier. This equation can be rearranged to obtain the following equation: Comparing this equation with the equation governing perikinetic flocculation, it can be seen that the volume fraction, ϕ and the initial turbidity N o are related. The aggregation time τ and the half-life t 1/2 are also related, and the slope k is related to 2kT/(πηa 3 W). k in the aggregation time equation is equal to Boltzmans constant = 1.38x With an increased understanding of these relationships and by determining the stability ratio (W), the experimental particle radius (a) could

42 31 be calculated. With this added understanding, it is safe to say the percent turbidity removal is a function of the initial turbidity(n o, and ϕ). This result also verifies the developed micro-scale jar test to determine turbidity because in other Moringa publications it has been suggested that Moringa works better at higher turbidities. (Okuda, 2000) This is the scientific reasoning behind the common claim that Moringa does not work well at low turbidities found in the current Moringa literature today. Effect Initial Turbidity had on Percent Turbidity Removal Wanting to know more detail about the effect initial turbidity had on percent turbidity removal, the research began to focus on comparing the removal percentages of 100 NTU and 500 NTU kaolin. In figure 3-9 the percent removal data for both 100 and 500 NTU kaolin is shown after one hour and 24 hours of sedimentation.

43 32 Figure 3-9. Percent turbidity removal values for 100 and 500 NTU kaolin after one hour and 24 hours of sedimentation using the new and old analysis techniques. Percent removal values can be seen on the graph. In figure 3-9, it can be seen that the 500 NTU kaolin media always had a higher percentage of turbidity removal than the 100 NTU kaolin. This result was predicted by the aggregation rate equation, perikinetic flocculation and others research. Interestingly, the turbidity spike one minute after the addition of MO serum was 52 NTU for the 100 NTU kaolin media and 151 NTU for the 500 NTU kaolin media. The higher initial turbidity corresponding to a higher volume fraction ϕ for the 500 NTU kaolin caused a bigger spike in turbidity initially because aggregation was occurring at a faster rate. Also, based on the perikinetic flocculation which is occurring during the sedimentation process, the higher concentration of particles N o causes particles to flocculate together faster. This result is consistent with others findings that Moringa oleifera is

44 more effective at reducing the turbidity of highly turbid solutions. The science behind this claim has been investigate and presented in this thesis. 33 Comparing Turbidity Removal for the Three Systems One aspect of this research focused on what effect the different media had on percent turbidity removal. Primarily this research aimed to compare the 100 NTU kaolin, 500 NTU kaolin, and 100 NTU primary effluent wastewater. As noted in the methodology discussion, these three media were used in the micro-scale jar test turbidity experiments. Kaolin was used because it is a well-known laboratory standard when creating a synthetic turbid solution. However, kaolin is not directly applicable to the types of water that would be treated in the developing world, so primary effluent wastewater was used as well. Primary effluent wastewater was used to better represent the different types of waters that would be treated in the developing world. 500 NTU kaolin was utilized to observe differences that having a media of higher turbidity would cause on percent turbidity removal. Below in figure 3-10, turbidity versus time data is shown for 100 NTU primary effluent wastewater.

45 34 Figure Turbidity vs. time data for 100 NTU primary effluent wastewater control and samples. Four samples were averaged to obtain the samples data point. Using this data the percent turbidity removal is shown in figure 3-11 using the new, and old analysis techniques.

46 35 Figure Percent turbidity removal of MOCP vs. time for 100 NTU primary effluent wastewater media using the new and old analysis methods. Regrowth of the wastewater over time actually increased the turbidity in the samples and control. The green diamonds and red circles represent the new and old analysis techniques respectively. The blue triangles show the control data s percent turbidity removal calculated with the old analysis technique. In table A-3 found in Appendix A, each specific point is shown. Primary effluent wastewater gave a very different result than 100 NTU kaolin did. In figure 3-12, a comparison of turbidity versus time data for 100 NTU kaolin and 100 NTU wastewater is shown.

47 36 Figure Turbidity vs. time data for 100 NTU kaolin and 100 NTU primary effluent wastewater. This graph is displayed to show the turbidity removal differences between the wastewater and kaolin over time. From figure 3-12, the smaller diamonds were used for the kaolin and wastewater control data. The larger squares were used to show the kaolin and wastewater sample data. The sample and control data for wastewater actually began to increase in turbidity over the 24 hours of sedimentation. This was due to the two competing mechanisms happening within the beakers. The particles were settling out of the media over time, but the microorganisms in the wastewater were also reproducing over time. This increase in turbidity over the 24 hours of sedimentation is due to the regrowth of the microorganisms. This will be discussed in more detail later.

48 In figure 3-13, the same graph as figure 3-8 is displayed but the primary effluent wastewater data points are added. 37 Figure and 500 NTU kaolin media and 100 NTU wastewater media plotted as a second order differential equation. The linear line of best fit has correlation coefficients of 0.99 and 0.97 for the kaolin media indicating that the experimental data closely follows perikinetic flocculation theory. However it is seen that primary effluent wastewater did not follow perikinetic flocculation theory. This was because the regrowth of microorganisms increased the turbidity over time. The wastewater data points do not fit a linear best fit line going through the origin. The 100 NTU wastewater does not appear follow perikinetic flocculation, but unlike a laboratory kaolin solution, the microorganisms in wastewater reproduce over time. This growth significantly affected the turbidity results for the 100 NTU wastewater samples. Over time, this regrowth of microorganisms adds another competing mechanism inside the beakers, so just looking at perikinetic flocculation is not sufficient in describing this system. A more detailed comparison of 100 NTU kaolin and 100 NTU wastewater is discussed next.

49 38 Comparison of 100 NTU Kaolin and 100 NTU Primary Effluent Wastewater Figure 3-14 shows a side by side comparison of the percent turbidity removal of 100 NTU kaolin and 100 NTU primary effluent wastewater using the new and old analysis techniques. The old analysis technique is useful in this instance because it is hard to interpret the negative turbidity removal value. Figure Percent turbidity removal values for 100 NTU kaolin and 100 NTU primary effluent wastewater after one hour and 24 hours of sedimentation using the new and old analysis techniques. Percent removal values can be seen on the graph. In this comparison, it would be expected based solely on initial turbidity that these two media would have similar percent turbidity removals. There are a few key differences that need to be addressed. The first is that the particle size in the 100 NTU kaolin was relatively uniform

50 39 compared to the wastewater because the kaolin solution was allowed to sit overnight. It is estimated by Stokes law that the maximum particle size in the kaolin solution is 2µm (Ndabigengesere, 1995). This is very different than the particle size in primary effluent wastewater. The wastewater was allowed to settle for about three hours in a clarifier, so the particle size in wastewater was much larger than the particles in the kaolin. This is the primary reason that after one hour of sedimentation the 100 NTU kaolin still had a negative percent turbidity removal, but the primary effluent wastewater had a 29.85% ± 15.24% (Average ± Standard Deviation calculated in Excel). There was also much more variability within the wastewater experiments because the primary effluent wastewater can be very different from day to day. The 24 hour percent turbidity removal is also very different but for another reason. Within the wastewater there is a variety of microorganisms. When the MO serum is added to the wastewater, then BOD is also being added. The microorganisms are using this as a food source to reproduce, so during the sedimentation process there is also a regrowth process happening in the samples. This regrowth process is increasing the turbidity of the sample over time, and is the primary reason that the 24 hour percent turbidity removal rate is only 37.44% ± 6.11%. It is expected that without the regrowth increasing the turbidity that the 100 NTU wastewater would have a similar percent turbidity removal as the 100 NTU kaolin. Timing is a more important issue in the developing world because microorganisms will be living in the water that will need to be treated. The water needs to sit for a certain amount of time to reduce the turbidity, but not so long that too much regrowth is taking place. More research needs to be done in this area to determine a recommendation for how long the water should sit to allow proper turbidity reduction without allowing harmful regrowth. Our research group is currently doing research on a technique to attach MOCP to sand to be used in a sand filter (Velegol, 2011). This idea will not increase the BOD in the water, so the microorganisms will not have an added food source.

51 40 Effect of Seed Size on Percent Turbidity Removal Seed size is directly related to seed maturity, and it is proposed that with increasing seed maturity there is an increase in the MOCP concentration. This increase in the MOCP concentration would lead to an increase in percent turbidity removal over time. This was the basic question that the experiments were considering. In this thesis, experiments were run on 100 NTU kaolin using extracted MOCP from large seeds and medium seeds. First, it was necessary to classify what large and medium seeds are. The average mass of a large seed was ± grams and the average mass of the medium seeds was ± grams. To see the corresponding graphs comparing percent turbidity removal of the medium and large seeds see figures 3-14 and 3-15 on page 45 and 46. Mass Distribution of Chiang Mai Moringa Seeds Large Seeds As stated previously in the materials and methodology section, the large Chiang Mai MO seeds were all picked primarily from a group of six trees in Chiang Mai, Thailand. The mass distribution of the MO seeds needed to be investigated in more detail because different large and medium seeds naturally have a distribution of mass. Due to this non uniform mass of different seeds of the same age, it can be hypothesized that there will be a distribution of MOCP within each seed. Having a mass distribution for the seeds collected was an important piece of information. In a table found in Appendix A, the mass distribution for Chiang Mai large seeds is shown. In table 3-1, there is a summary of the mass of all the individual seeds recorded. Please see Appendix A to see information about every seed measured.

52 41 grams. Average Mass (g) Standard Deviation % Confidence Interval Table 3-1. Collected large Chiang Mai seed mass s. The average mass of a large seed was ± From this mass distribution data, the average mass of a large Chiang Mai MO seed with the shell on was ± grams. Medium Seeds The medium Chiang Mai seeds were picked from the same trees as the large were picked from except the medium seeds were picked at a younger maturity. The medium seeds had a partially green shell. In Appendix A, the mass distribution for the medium Chiang Mai MO seeds can be found. Table 3-2 includes a summary of the mass data for the medium seeds. Again, please see Appendix A for all the measurements taken for the medium seed mass distribution. Average Mass (g) Standard Deviation % Confidence Interval Table 3-2. Collected medium Chiang Mai seed mass s. The average mass of a medium seed is ± grams. As seen in table 3-2, the average mass of the medium Chiang Mai seeds with the shell was ± grams.

53 42 Percent Seed Data for Large and Medium Chiang Mai Seeds Large Seeds Obtaining the mass distribution data was a useful piece of information to aid in better understanding the variability between seeds of the same age. Another aspect of variability within the MO seed is the shell mass. From previous research, it has been shown that there is no MOCP in the shell of Moringa (Ndabigengesere et al., 1995). Being primarily concerned with the concentration of the coagulant MOCP, which is contained in the seed of Moringa, another useful piece of information is the percentage by mass of the shell. Data was collected to determine the percentage of the seed s mass that is the shell. Table 3-3 shows this data for the large Chiang Mai seeds. Total Mass (g) Mass Without Shell (g) Shell Mass (g) % Seed % Shell Average % 25.6% Standard Deviation % 6.61% 95% Confidence Interval % 2.03% Table 3-3. Collected large Chiang Mai seed percent shell data. The average percent shell of a large seed is 25.6% ± 6.61%. Please note that these seeds are not all the same seeds used to collect data for the mass distribution. For the Chiang Mai large seeds, the average percentage of the total mass due to the shell was 25.6% ± 6.61%. This is useful data to know because this shows that about 26 percent of the seeds total mass does not contain any MOCP.

54 43 Medium Seeds Data was also collected to determine what percentage of the total mass of the medium seeds was due to the shell. Table 3-4 shows this data. Total Mass (g) Mass Without Shell (g) Shell mass (g) % Seed % Shell Average % 45.5% Standard Deviation % 19.21% 95% Confidence Interval % 7.17% Table 3-4. Collected medium Chiang Mai seed percent shell data. The average percent shell of a large seed is 45.5% ± 19.21%. In Table 3-4 the medium seeds average percentage of the total mass due to the shell was 45.5% ± 19.21%. This is of interest because the medium seed s shell account for a higher percentage of their weight than the large seeds. Recall that there is no MOCP in the shell of the seed, so in order to develop a set procedure to be used in the developing world more mass of the medium seeds with the shell on is needed to obtain the same mass of seeds contributing to the MOCP concentration. Effect of Seed Size on Percent Turbidity Removal-Graphs Figure 3-13 shows the experiments collecting turbidity versus time data for both medium and large Chiang Mai seeds.

55 44 Figure Turbidity vs. time data for 100 NTU kaolin using both medium and large seeds in the turbidity experiments. After 24 hours the medium seeds had reduced the turbidity to 6.68 NTU where the large seeds reduced the turbidity to 3.10 NTU. The medium seeds (partially green) were effective at reducing turbidity. The small diamonds represent the medium or large control turbidity and the larger squares represent the sample turbidities. Over time both the medium and large seeds did reduce turbidity. Both the medium and the large seeds had enough MOCP concentration to reduce turbidities over 24 hours. Previous research from Ndabigengesere et al. (1995) stated that green seeds did not have coagulation ability, but the green medium seeds used in these experiments did have coagulation ability. In figures 3-14 and 3-15, the 24 hour percent turbidity removals are shown using both the new and old analysis techniques.

56 Figure Percent turbidity removal for both medium and large seeds after 24 hours of sedimentation. Percent turbidity removal was calculated using the new analysis technique, and it is shown that the large seeds were more effective at reducing the turbidity, but the medium seeds were still effective. 45

57 46 Figure Percent turbidity removal for both medium and large seeds after 24 hours of sedimentation. Percent turbidity removal was calculated using the old analysis technique. With only this analysis it would appear that the medium and large seeds both removed turbidity at roughly the same level, but with the additional new analysis it is hypothesized that the large seeds worked a little better. The medium seeds were able to reduce the turbidity almost as effectively as the large seeds. The new and old analysis graphs appear to be different concerning the medium seeds. There are few reasons why. The first is that the turbidity spike with the medium seeds was 100 NTU and with the large seeds it was only 52 NTU. The old analysis technique compares the current turbidity of the sample back to the initial sample turbidity. So for the old analysis, it reports a greater percent removal because it is comparing the current turbidity back to the initial turbidity with the turbidity spike. The other reason is because that final average turbidity of the 100 NTU kaolin media using the medium seeds was 6.68 NTU where the average turbidity using the large seeds was 3.10 NTU. The new analysis technique accounts for the initial turbidity spike, and this 3.58

58 47 NTU difference in the 24 hour sedimentation turbidity is partially why the medium seeds reduced the turbidity by 79.90% and the large reduced the turbidity by 92.70%. The medium seeds definitely did work to reduce the turbidity of the media, but appear to not be as effective as the large seeds. The current hypothesis is that the large seeds have a higher MOCP concentration because the larger seeds have had more time to mature on the trees than the medium seeds. More research needs to be done on the concentration of MOCP in MO seeds as they grow before a definite conclusion can be made.

59 48 Chapter 4 Conclusions and Future Work Major Findings The major findings of this thesis work are bolded and bulleted below. The development and implementation of a new procedure to measure the reduction of turbidity in the presence of Moringa oleifera seeds. (See pages 14-21) The newly developed micro-scale jar test has different strengths and weaknesses. A few major strengths include the ability to do turbidity experiments with a limited amount of MO seeds. These seeds are becoming more expensive, and if lab tests can limit the number of seeds used that would be advantageous. Also, the results and repeated trials with multiple researchers produced similar results that can be backed up by previous MO research. One weakness is that the small 100 ml beakers only allow a few turbidity recordings before the volume is insufficiently low. Also, more research needs to be done to determine if the initial turbidity spike is an artifact of the micro-scale jar test. Utilization of a new analysis technique to analyze percent turbidity removal data obtained from the micro-scale jar test. (See pages 21-27, specifically figures 3-4 and 3-6) The new data analysis technique is a better representation of what percent of the turbidity is being reduced by MOCP than the old analysis. This is because the new analysis corrects for the initial turbidity spike and accounts for natural sedimentation of particles by referencing a control beaker allowed to settle for the same amount of time as the samples. This new analysis was developed

60 49 because of the observed initial turbidity spike right after the addition of the MO serum. The one downside is that this analysis method can result in initial negative percent turbidity removals. Negative removals can be difficult to understand conceptually. Percent turbidity removals were predictable based on sedimentation time and initial turbidity. This was explained based on the aggregation rate equation and perikinetic flocculation theory. (See pages 7-10 for background on the aggregation rate equation, pages for perikinetic flocculation information, and pages explaining the effects of initial turbidity. Specifically see figures 3-7, 3-8 and 3-9) Turbidity removal in primary effluent wastewater was not predictable based on these theories because of the regrowth of microorganisms. (See pages 33-39, specifically figures 3-11, 3-12, 3-13, and 3-14) A major portion of this thesis explained the science behind the turbidity versus time data obtained. It explained what effect sedimentation time and initial turbidity have on the turbidity removal by primarily looking at the aggregation rate equation and perikinetic flocculation theory. Another aspect of this thesis was recognizing that obtained wastewater data did not follow these theories because regrowth took place with time. Further research needs to be done to help eliminate regrowth, or slow the regrowth process so that the treated water can be stored longer. Also, more research needs to be done on the storage time of the treated water. Less mature green seeds can also be effective in enhancing turbidity removal. (See pages 40-47, specifically figures 3-13, 3-14, and 3-15) The results demonstrate that green MO seeds are also potentially useful as natural coagulants. A better definition other than green seeds needs to be established. Some smaller green seeds will not work to reduce the turbidity, but other more mature green seeds will work. Color alone is an unreliable guide to the MOCP content of a MO seed. It appears the medium-sized partially green seeds used in this thesis were able to reduce turbidity, but not quite as well as the more mature

61 50 large seeds. More research needs to be done in this area to determine at what age or size the presence of MOCP appears. Also, research should be done to determine what the optimal age or size of the MO seed to use for maximum turbidity removal. Implications for the Developing World There is still a considerable amount of work to be done in order to implement MO seeds as a water treatment technique in the developing world in an efficient, culturally appropriate, and costeffective manner. A few implications obtained from this research include: 1. The importance of extracting the water soluble protein (MOCP) from the MO seeds. 2. The finding that green seeds can work to reduce turbidity if they are mature enough. 3. Seeds need to be selected based on size or maturity characteristics, not color alone. 4. The user must allow a certain amount of time for sedimentation before using the water. 5. MOCP is more effective at reducing turbidity in waters with a higher turbidity. 6. The user needs to be careful when determining how much mass of seed to use. This is because the percent of the total mass consisting of the seed changes as the seeds mature. The MO seeds are more effective at reducing the turbidity if the protein is extracted in water before it is added to the media. This would mean that the user in the developing world should use a similar process that was used in this thesis to extract the protein. They would need to grind the seeds, add the crushed seed to a specific amount of water, and agitate the mixture. Another implication for the developing world would be to know that a certain age seed is required so that enough MOCP is present within the seed for coagulation. Green seeds can work, but a process of sorting seeds by their size needs to be used and further researched. One thought would be to do more research to determine at what size a seed contains enough MOCP to

62 51 effectively reduce the turbidity to a certain level. Then a sieve can be constructed and used to collect all the seeds bigger than that size. Another takeaway from this research would be to know that a certain amount of sedimentation time is needed to reduce the turbidity. Another competing mechanism is the regrowth of microorganisms in suspensions containing biodegradable organic matter. The MO serum adds BOD to the media and the microorganisms use this to reproduce, thus increasing the turbidity of the media. There needs to be a balance between allowing enough sedimentation time which does not allow excessive regrowth, or another technique needs to be used to prevent regrowth. It has been well documented in the literature and this work that Moringa works better in higher turbidity waters. Users must be aware that the initial turbidity of the water will have an effect on the amount of sedimentation time needed to achieve a desired level of turbidity reduction. Finally, the user should be aware that the shell can represent different mass fractions for different size seeds. The medium seeds analyzed here had a higher percent shell than the larger seeds. Dosing needs to be adjusted so that the seed mass, which contains the MOCP, is at a specific level. If the seed and shell weight together is used, then the user needs to be aware that the mass will need to be adjusted depending on the percent shell of that specific seed size. Suggestions for Future Research The following would be my suggestions for additional research on Moringa oleifera as it relates to water treatment. 1. I would recommend that more work be done to determine the concentration of MOCP in a seed throughout the growing season.

63 52 2. I would also recommend that a standardized seed collection process be used so that the age of each seed is known. Moringa oleifera is a tree that grows in a tropical environment, so the same tree could have seeds of different maturities and sizes. I would suggest tagging a certain number of flowers and picking a portion of the tagged flowers throughout the growing season. This way, seed maturity is known and could be compared to percent turbidity removal. 3. Directly related to this thesis, research should be done to further validate the micro-scale jar test. It would be interesting to determine if the initial turbidity spike is an artifact of the micro-scale jar test. The jar test results would be beneficial to compare to the microscale jar test results. Also directly related, more research should be done with a synthetic turbid solution that has a narrow particle size distribution. That way, a better relationship can be formed between the aggregation rate equation and perikinetic flocculation theory. 4. More studies need to be completed with surface and waste waters using Moringa. This needs to be done to be able to practically implement this water treatment technique into the developing world. 5. Also, further studies on the antimicrobial effects of Moringa should be conducted with wastewater and other specific pathogens. The relationship between turbidity removal and pathogen removal needs to be quantified. 6. Along the same lines, more research needs to be completed to try to eliminate or overcome the problem of regrowth. One idea from the Velegol research group would be to attach the MOCP to sand particles (f-sand) and allow the turbid water to run through the sand filters. This is one way of eliminating the BOD from the MO serum while still obtaining the coagulation and antimicrobial properties of MOCP (Velegol, 2011). 7. Further development should be completed to make and test a set procedure for the use of MO as a coagulant in the developing world.

64 53 8. Also, collecting seeds from different parts of the world and comparing turbidity removal results would be of interest. There are many other future research endeavors concerning Moringa, but these are just a few major issues that need to be addressed. With ongoing research and proper implementation, Moringa oleifera has real potential to help those in the developing world access cleaner water using this safe, natural, and culturally appropriate treatment technique.

65 Appendix A Additional Collected Data Turbidity Versus Time Data Turbidity vs. time data for 500 NTU kaolin data comparing new and old analysis techniques. Time (hrs.) Control Turbidity (NTU) Average Sample Turbidity (NTU) Percent Turbidity Removal (New Analysis) Percent Turbidity Removal (Old Analysis) Percent Turbidity Removal Control (Old Analysis) % % 82.85% 19.12% % % % % % 99.52% 89.44% % 99.45% 90.60% Turbidity vs. time data for 100 NTU kaolin data comparing new and old analysis techniques. Time (hrs.) Control Turbidity (NTU) Average Sample Turbidity (NTU) Percent Turbidity Removal (New Analysis) Percent Turbidity Removal (Old Analysis) Percent Turbidity Removal Control (Old Analysis) % 0.00% 0.00% % 27.96% 13.08% % % % % 96.88% 78.82% % 97.17% 82.55%

66 Turbidity vs. time data for 100 NTU primary effluent wastewater data comparing new and old analysis techniques. Time (hrs.) Control Turbidity (NTU) Average Sample Turbidity (NTU) Turbidity Removal (New Analysis) Turbidity Removal (Old Analysis) Turbidity Removal Control (Old Analysis) % 0.00% % 65.21% 20.16% % 76.93% 23.95% % % % % 72.22% 47.98%

67 Micro-Scale Jar Test Experiments This is all the collected micro-scale jar test turbidity removal data for large Chiang Mai seeds at 1 hour and 24 hours of sedimentation in 100 NTU kaolin media. 100 NTU Kaolin-Large Date of Experiment Sample Number 1 hr sedimentation 24 hr sedimentation 2/8/ % 92.06% 2/8/ % 90.60% 2/8/ % 91.62% 2/8/ % 89.19% 2/11/ % 95.88% 2/11/ % 93.95% 2/11/ % 93.51% 2/11/ % 95.16% 2/11/ % 92.43% 2/11/ % 95.30% 2/11/ % 93.80% 2/11/ % 94.14% 2/22/ % 92.96% 2/22/ % 90.61% 2/22/ % 91.69% 2/22/ % Average % 92.70% Standard Deviation 12.20% 1.97% 95% Confidence Interval 6.76% 1.05% This is all the collected micro-scale jar test turbidity removal data for large Chiang Mai seeds at 1 hour and 24 hours of sedimentation in 500 NTU kaolin media.

68 500 NTU Kaolin-Large Date of Experiment Sample Number 1 hr sedimentation 24 hr sedimentation 1/27/ % 98.75% 1/27/ % 98.88% 1/27/ % 99.14% 1/27/ % 98.63% 2/1/ % 99.11% 2/1/ % 99.01% 2/1/ % 99.01% 2/1/ % 99.21% 2/22/ % 98.62% 2/22/ % 97.98% 2/22/ % 99.07% 2/22/ % 2/26/ % 96.78% 2/26/ % 97.60% 2/26/ % 95.66% 2/26/ % 95.62% Average 80.86% 98.17% Standard Deviation 2.65% 1.20% 95% Confidence Interval 1.47% 0.64% This is all the collected micro-scale jar test turbidity removal data for large Chiang Mai seeds at 1 hour and 24 hours of sedimentation in 100 NTU primary effluent wastewater media. 100 NTU Wastewater-Large Date of Experiment Sample Number 1 hr sedimentation 6/24 hr sedimentation 2/18/ % 47.68% 2/18/ % 35.15% 2/18/ % 38.01% 2/18/ % 27.11% 2/25/ % 29.92%

69 2/25/ % 35.27% 2/25/ % 37.25% 2/25/ % 38.17% 2/25/ % 48.16% 2/25/ % 38.14% 2/25/ % 40.06% 2/25/ % Average 29.85% 37.44% Standard Deviation 15.24% 6.11% 95% Confidence Interval 10.24% 3.88% This is all the collected micro-scale jar test turbidity removal data for medium Chiang Mai seeds at 1 hour and 24 hours of sedimentation in 100 NTU kaolin media. 100 NTU Kaolin-Medium Date of Experiment Sample Number 1 hr sedimentation 24 hr sedimentation 2/28/ % 75.35% 2/28/ % 72.01% 2/28/ % 76.83% 2/28/ % 77.54% 3/14/ % 82.73% 3/14/ % 82.99% 3/14/ % 81.72% 3/14/ % 80.00% 3/21/ % 75.82% 3/21/ % 75.56% 3/21/ % 75.62% 3/21/ % 82.56% 3/21/ % 84.69% 3/21/ % 85.57% 3/21/ % 86.40% 3/21/ % 82.99% Average % 79.90% Standard Deviation 13.97% 4.37% 95% Confidence Interval 7.45% 2.33%

70 Seed Mass Distribution Data Large Seeds Below, is collected data of the mass distribution of the large Chiang Mai seeds. Below is a table summarizing the collected mass data for the large seeds. Average Mass (g) Standard Deviation % Confidence Interval From this mass distribution data, the average mass of a large Chiang Mai MO seed with the shell on was 0.259±0.061grams.

71 Medium Seeds The medium Chiang Mai seeds were picked from the same trees as the large were picked from except the medium seeds were picked at a younger maturity. The figure below shows the mass distribution for the medium Chiang Mai MO seeds. The table below includes a summary of the mass data for the medium seeds. Below is a table summarizing the collected mass data for the medium seeds. Average Mass (g) Standard Deviation % Confidence Interval As seen in the table, the average mass of the medium Chiang Mai seeds with the shell was 0.107±0.036 grams.