Link to Video Summary: https://www.youtube.com/watch?v=UjjMqYULI_Y&t=8s
As many parts of the world are suffering from water scarcity, there is a growing interest in conserving water and one of the best ways to do this is to reuse or recycle grey water from the laundry. However, many detergents have harmful chemicals that can be detrimental to soil, plants and aquatic life. Therefore, I needed to find an environmentally friendly solution. I tested if greywater from soap nuts, a natural berry shell that produces soap when in contact with water, could be used for irrigation without harming the environment.
I tested the effects of soap nut and other grey waters on aquatic life, soil and plants (tall fescue grass and vegetables) and on the soil microbiome. Additionally, I saw if watering with greywater would lead to pathogenic contamination in the soil or plant.
I found that soapnut greywater was not detrimental and had a trend for higher plant growth and biomass than other greywaters. For all of the soil and plant nutrients tested, soapnut greywater did as well as regular water. However, the non-organic detergent led to Boron toxicity and salinity issues. I also found that for all treatments, there were no E. Coli and very low levels of Fecal coliforms. In addition, Soap nuts are cheaper than organic laundry detergents which makes soap nuts and its greywater not only a solution for California's historic drought, but also to the global water crisis. After conducting my research, I started my non-profit organization ‘The Grey Water Project’ in order to promote water recycling.
As many parts of the world are experiencing water scarcity, there is a growing interest in innovative approaches to sustain water resources.
I became interested in water conservation four years ago when I visited the county of Tulare where I saw first hand the crippling effects of California’s historic drought.
I was interested in reusing greywater (lightly used water) especially from the laundry. However, many commercial laundry detergents have harmful chemicals, affecting plants, soil, and aquatic life. Therefore, the goal of my project was to find a laundry detergent that was environmentally friendly, cheap, and whose grey water can be used for irrigation. So, I started investigating soapnuts, the berry shell of the Indian soapberry that naturally releases soap and tested it against other laundry detergents and regular water.
Question: Will the grey water from soap nuts harm the environment espeically soil and plants causing pathogenic presence or affect nutrient levels in the soil and plants when compared to regular water and the grey water from an organic and non-organic detergent.
Hypothesis: There will be no significant differences in E.Coli and Fecal coliform levels(indicator organisms), levels of Soil macro and micro-nutrients, pH, soil CEC, leachate quality, plant height, plant biomass, plant nutrients and in the soil microbiome between the grey water from soap nuts and regular tap water.
I broke down my project into 4 phases: Testing soapnut grey water on:
Four years ago when I visited the county of Tulare for an Archery competition, I saw first-hand the effects of a crippling drought. I talked to people whose wells ran dry, leaving them waterless, and farmers who have lost their year's crops and I was touched by their stories. I went to India soon after this, and I found that California is not alone in dealing with drought when I learned about the impact of failed monsoons. People's lands had become unfarmable and whole villages had been abandoned. Many people moved to the cities taking whatever work they could find. It was then that I understood how water scarcity is a global issue. Droughts and water scarcities affect about 2 billion people worldwide. My home state, California, is experiencing the longest duration of drought in its history, which as of November 27th 2018, has lasted 362 weeks and affected about 93% of the state’s population according to the US Drought Monitor.
I became interested in conserving what water we already have and I started learning about reusing greywater (used water from the laundry, sinks and showers). According to the EPA, Americans use more than 7 billion gallons of water a day for landscape irrigation, half of which goes to waste. A family of four generates about 243 gallons of reusable water daily. I was most interested in recycling grey water from the laundry as it the simplest type to reuse. However, many commercial laundry detergents have harmful chemicals that can be detrimental to soil, plants and aquatic life. Therefore, I needed to find an environmentally friendly alternative that would also be cheap and whose grey water can be recycled for irrigation.
I heard about soap nuts from my grandmother and found that they have been used as a shampoo in India for hundreds of years. Soap Nuts are the shell of the Indian soap berry and they naturally contain soap. The chemical constituents of the nut have been found to be antiviral, fungicidal and molluscicidal(B.N.Suhagia, 2011). Unlike commercial soaps, neither animal fat nor chemical processing is used, and they are biodegradable. Current studies on grey water irrigation reveal a wide array of chemicals in greywater (WERF, 2011) highlighting the need for using environmentally friendly detergents. Since no research had been done on the soap nut greywater, I decided to test its effects on the environment and see if it can be used for irrigation.
In my first phase of research, I tested to see if grey water irrigation would affect aquatic life, and in the second phase I tested on soil and plant tissue nutrient levels in Tall Fescue grass to see if soapnut grey water would be a good candidate for irrigating lawns. Encouraged by the positve results from soapnuts, I researched on vegetables focusing on pathogenic contamination and I am currently investigating the soil microbiome.
Although my research is still ongoing, in order to create awareness in the community about grey water reuse, I started my non-profit “The Grey Water Project” through which I conduct workshops and presentations
Independent Variable: type of grey water (Soap nut(SN), Organic detergent(OD), non-organic detergent(ND), regular tap water(RW))
I grew a total of 154 replicates of vegetables (current year): a fruit, a leafy green and a root vegetable, namely tomato, spinach and radish as well as 48 replicates of tall fescue grass (previous year) with 3 replicates each for four types of grey water( Soapnut, reg.water, organic and non-organic detergents). The experiment was repeated for two types of soil (sandy and sandy loam) for 6 weeks in a controlled environment. Grey water irrigated soil without plants was used for the soil microbiome analysis.
Soil and Plant tissue testing
Both soil and plants were first dried.
All foreign particles were removed with a sieve and crushed with a mortar and pestle.
They were then tested for soil quality(pH, EC, sodium), Macronutrients(Nitrogen,Phosphorus, Potassium,Calcium,Magnesium,Sulfur), MicroNutrients(Zinc,Copper,Iron,Aluminum,Lead,Boron, Manganese), CEC,TOC and Nitrogen. The plant tissue was evaluated for nutrient content (Nitrogen,Phosphorous,Potassium,Boron,Calcium,Magnesium, Zinc,Copper,Iron and Manganese).
Pathogen Testing on Vegetables
I tested to see if watering with grey water would lead to contamination with harmful pathogens like E.Coli or Fecal Coliforms (FDA produce safety markers)on the outside or inside of all my vegetables and their soil (1400+ samples) at the end of four weeks.
Further overtime analysis was performed on spinach only in autoclaved, sandy soil by inoculating with 106 E.coli K-12 at the beginning of the experiment. Bacterial counts were taken everyday for a week followed by weekly measurements for 4 weeks.
Pathogen testing method:
The soil was tested for E. Coli and Fecal Coliforms by taking 5 grams of soil from each sample, and diluting to the desired concentration with Phospate Buffered Saline(PBS). Plant leaves were sampled and washed thoroughly with an amount of PBS proportional to its weight in order to move any coliforms from the plant to the buffer solution.The soil and the plant solution was then plated on a chromogenic media, incubated at 37C and counted after either 24/48 hours.
Plant Quality & Biomass:
I measured the plant height and looked for turf quality, crown density, shoot/blade color and disease presence every week. At the end of the experiment, the plant tissue was separated from the soil and weighed for biomass.
Leachate and Water Analysis
I also collected leachate from my plants every week. These were tested for pH, EC and TDS along with grey water samples using a Hannah Combo pen.
Soil Microbiome Analysis:
To achieve this, DNA was extracted from the soil samples using a Mobio Power Soil DNA extraction kit and Library prep was completed on Illumina Platforms. 16S Metagenomic sequencing was performed and the resulting data was analyzed using QIIME software.
Water, Soil & Plant Nutrient Analysis:
I analyzed all of my data using a one-way ANOVA (p<0.05) for all of the soil, plant and water characteristics tested (600 ANOVA’s) followed by post-hoc comparisons where necessary. I then classified them under four major headings Toxicity & Salinity (Boron,Sodium,TDS,EC and pH), Metals (Copper,Lead,Manganese,Zinc), NPK & C (Nitrogen,Phosphorus,Potassium,Carbon), and 'Others' (CEC,Sulfur,Calcium,Magnesium,Iron and Aluminum).
The soil constituents of interest with respect to greywater are Boron, pH, and salinity. I am showing my results from this year along my results from Tall Fescue grass last year as they have similar trends. The soapnut greywater treatment led to similar levels of soil and plant nutrients when compared to the regular-water treatment across the board. However, the non-organic detergent led to Boron toxicity issues, illustrated with very high levels of Boron both in soils and in the plant tissue(See FIGURE 1). These levels were significantly different from the other treatments, reaching 24.8 ppm in Tall Fescue grass - 5 times higher than the safe range for Boron which is < 3ppm and 9.47 for Tomatoes and 6.43 for Spinach. The plant tissue also saw high level of Boron, 3082.3 ppm where as the normal level is 25-60 ppm. This led to plant death in most cases(See PICTURE1)
Additonally, the non-organic detergent leachate EC showed high level of salts(See Figure 2). There was no significant difference between regular water and the treatments for the rest of the categories.
E.Coli and Fecal Coliform: E.Coli and Fecal Coliform levels were measured at the end of the plant cycle (Spinach,Chard&Radish:4 weeks, Tomato:12 weeks). There were no E.Coli found on any of the samples tested and all the grey water treatments resulted in low levels of Fecal Coliforms in the soil and outside of plants(FIGURE 3)
In order to further investigate, I autoclaved one type of soil(Sandy) and grew Spinach alone. The soil was inoculated with 106 E.Coli K-12 cells. As you can see from Figure 4 & 5 below, the E.Coli levels in the soil started out high and 105 Ecoli cells were recovered for the first four days in all the different grey water treatments. However,by the end of the first week, the levels of E.Coli dropped to undetectable for all the treatments and stayed the same for the remainder of the experiment. The same trend can be observed for the soil and outside of the plant fecal coliforms and total abcteria as well, regardless of the type of grey water.
Plant Height & Plant Biomass : Plants watered with soap nut grey water did as well as regular water. Soapnut grey water was not detrimental to plant growth with a trend for higher growth and plant biomass (See Figure 6).
Treatments with grey waters with different detergents showed a significant shift in bacterial populations in sandy soil. Cyanobacteria,Proteobacteria,Verrucomicrobia,Acidobacteria,Gemmatimonadetes, Crenarchaeota and Actinobacteria dominated in the sandy soil, but the distribution was more even in the general landscape soil which is more resistant to changes in microbial levels due to higher organic matter levels(Figure:6 & 7).
Soapnuts are cheaper than organic detergents making them a global solution (Figure:8)
After concluding my experimentation, I accepted my hypothesis. Soapnut greywater was not detrimental to plant growth with a trend for higher growth and plant biomass than regular water and other grey waters.
For all of the soil and plant nutrients tested, soapnut greywater did as well as regular water. However, the non-organic detergent led to Boron toxicity issues. High levels of Boron were found in both soils and in the plant tissue for the non-organic detergent.These levels were significantly different from the other treatments.
The non-organic detergent also had significantly higher EC levels in the leachate and in grey water when compared to regular-water. Though the leachate EC starts out within the “good” parameter(<0.8 ds/m), as the weeks progressed, the non-organic detergent saw levels of 2.65ds/m, which indicated salinity buildup.
Soap nut grey water was not significantly different from regular water and did not affect soil nutrient availability and plant nutrient uptake. This is significant because it shows that soapnut greywater can be used for irrigation without any negative effects.
With regards to E.coli and Fecal Coliforms, all grey waters follow the same trend where the levels approached zero after the second week of grey water irrigation regardless of soil type. Grey water irrigation of matured vegetables will see no effect on bacterial colonization.
Even when 106 cfu/g of E. coli K-12 was added to the soil through inoculation, bacterial counts declined and became undetected after 2-3 weeks, both in the soil and the plant resulting in no E.coli and very low levels of fecal coliforms at the end of the experiment across all grey waters. This indicates that grey water type does not have an effect on the bacterial content of plants tested
For the aquatic life testing, an LC50(acute toxicity test) and a daphnia heart rate test revealed that even in the lowest concentration, 50% of the population died with the non-organic detergent while all of them survived with soap nut grey water. Also, their heart rate did not change greatly when in soap nut grey water.
After the four year study, I now conclude that soapnut greywater does not significantly affect the environment, and can be used for irrigating landscapes and vegetables. In addition, soap nuts are affordable and cheaper than organic laundry detergents and are readily available globally which makes them an ideal solution for conserving water in drought stricken areas around the world.
I am currently continuing my project and looking at the effects of plant soil microbiome by growing Arabdiopsis Thaliana. Preliminary results reveal that the microbiome is affected by grey water irrigation in the sandy soil. In the outreach front, last year I was invited to partner with the United Nations Global Waste Water Initiative and I will continue to work with them to spread the awareness on grey water reuse. I am also working with the City of Fremont to promote environmental sustainability and my organization is a resource for the Fremont Green Challenge. I hope that my research and outreach to the community can help conserve water one drop at a time.
My name is Shreya Ramachandran, and I am a bookworm, a dedicated lover of learning and a citizen scientist. Science Fairs have been a huge influence on my life and they are what got me interested in biology. Through competing in science fairs, I was able to meet peers who shared my interests as well as professionals working in fields I was interested in. I was able to see a future for myself in the sciences.
I am inspired by scientists such as Hope Jahren whose memoir Lab Girl is a fascinating story of how dedication and passion can take you far. I am also inspired by reading about the groundbreaking science that is being done today. I hope that through my research and work in the future, I will be able to push forward the boundary of human knowledge and make an impact.
While I am not sure what college I will go to, I know that I will continue doing research and working on new projects. I am also very active in environmental efforts through my nonprofit “The Grey Water Project” to promote water conservation and grey water reuse. I will continue to work on water-related environmental issues.
Winning the Google Science Fair would mean the world to me as it would give me an opportunity to share my ideas with a larger audience through this prestigious global platform. By reaching more people, I can help more people in need in drought stricken areas.
I conducted my experiment partially at home and at a community lab. I followed all lab safety protocols. For my 1st phase of my project, Dr. Celine Pallud went over my project plan answered my questions. For the second phase of my project, Dr. Eric Espinosa answered my questions and motivated me towards a science career.
The lab is a BSL1 facility with all personal protective equipment provided. They have biohazard disposal and an EPA license upon request. Gloves and lab coat were provided and I was required to wash and sterilize my hands before leaving the lab.
Contact Person: Dr. Eric Espinosa
I would like to thank my grandpa Mr. T.M.Rajagopalan and grandma Mrs. Sudha Rajagopalan for their unconditional love and support. I also would like to thank my grandma for introducing me to soap nuts. Thank you to my parents, especially my mom for supporting me while I did this project. She bought me my materials for my grey water testing and leachate analysis.
I would like to thank Mrs. Pallud at UC Berkeley for allowing me to use the incubator in her lab for experimentation. I was able to go to her for help with difficult questions that I could not find answers to. I also would like to thank Dr. Eric Espinosa for advising me during the last phase of my project and oversee my work at the lab.
Thank you to the 3M Corporation for supplying me with a box of 3M petrifilm and DRG International Inc., for providing me with Chromagar ECC. I would also like to thank Tricity Ecology center for providing me a $600 grant to buy additional supplies.