CALVIN KARTHIK

Age 14 | Peterborough, ON

CWSF Best Project Award | Discovery, CWSF Challenge Award | Energy, CWSF Excellence Award | CWSF Renewable Energy Award Gold Medal | CWSF Honors Finalist

Edited by Harini Aiyer

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While visiting my grandparents in Kerala, India in 2018, my curiosity was piqued by their small backyard biogas plant that produced biofuel to run one cook stove for a few hours. Kitchen scraps were added daily into their digestor along with cow manure every six months. As these wastes degraded, methane was produced. This got me wondering. Steaming heaps of manure dot rural landscapes around the globe. Why are we allowing this valuable methane to be wasted? Through anaerobic digestion, animal manure can be converted into useable fuel, biogas, which is clean, renewable, and sustainable. The volume of kitchen scraps is limited; the challenge is to find abundant organic waste that can be combined with manure to increase the efficiency of biogas production. My research identified the agricultural byproduct spent mushroom substrate (SMS) as a potential candidate. SMS is a waste product of mushroom cultivation. To test my hypothesis that two wastes would be better than one, I built 33 biogas digestors and filled them will different combinations of manure slurry and SMS. Four different types of manure (dairy cow, sheep, horse and pig) were tested, with and without SMS. In every case, SMS increased biogas yield when added to the slurries. My new goal is to connect mushroom farmers and livestock farmers to introduce them to a new, sustainable method of producing fuel by combining their waste products, while simultaneously cleaning their farms, and reducing greenhouse gas emissions.

INTRODUCTION

Fossil fuels contribute to climate change through the process of combustion, releasing greenhouse gas emissions (GHG) into the atmosphere. (National Geographic Society, 2012). A recent study by the University of Rochester suggests that anthropogenic methane sources (e.g., landfills, livestock, and burning fossil fuels) in the atmosphere are 25-40% higher than initially thought; whereas natural methane sources (e.g., wetlands or geological seeps) are ten times less than originally thought (Shirkavand, Baroutian, Gapes, & Young, 2016). This is concerning, but also presents an opportunity to focus on technology to capture this methane. According to the Food and Agriculture organization of the United Nations, of all the gases produced by the degradation of animal waste, 55-65% is methane, which upon release in the atmosphere affects global warming 21 times more than CO₂ (Gupta, Aneja, & Rana, 2016). Methane is a powerful greenhouse gas that is many times more effective at trapping heat in the atmosphere than CO₂. As ruminants, such as, mainly dairy and beef cattle, contribute the largest proportion (61%) to livestock related GHG emissions, there is increased pressure to reduce their carbon footprint (Gupta, Aneja, & Rana, 2016).

Biogas is a clean and renewable source of energy that is generated by anaerobic digestion (AD) of organic matter, which can help us reduce our reliance on fossil fuels. Using biogas generators and the process of AD, methane can be captured and converted into usable fuel, reducing GHG emissions and combating climate change. Maximum bacterial biogas production occurs at the mesophilic range (35-40 ℃) and a pH range of 6.5- 7.5. Spent mushroom substrate (SMS) is a waste product of mushroom cultivation comprising raw materials, all of which are lignocellulosic biomass (plant material). For every 1 kg of mushrooms produced, 5 kg of SMS is created (Grimm & Wösten, 2018). Considering that the Netherlands alone produces about 50 million kg of fresh mushrooms and nearly 220 million kg of processed mushrooms every year, the resulting SMS globally is staggering (Heavenly Holland, 2018)! Some of this SMS is recycled by farmers, but much of it ends up in landfills, where it can lead to surface water contamination (Muchena, Pisa, Mutetwa, Govera, & Ngezimana, 2021). Plant cell walls are composed of cellulose, hemicellulose, and lignin. Lignin acts as a glue and bonds cellulose and hemicellulose giving strength to the cell walls. To access the sugars during AD that will eventually be converted into biogas, lignin must first be broken down through a process called delignification. During the growth of white rot fungi, especially oyster mushroom (Pleurotus Ostreatus) delignification occurs naturally in the substrate. White rot fungi possess the ability to grow on woody materials rich in lignocellulose due to their unique ability to degrade lignin (Pérez-Chávez, Mayer, & Albertó, 2019). When SMS is added to the manure slurry, bacteria in the manure will be ready to feed on the resulting simple sugar compounds in the medium. This biological pretreatment of lignocellulosic waste, using white-rot fungi, is eco-friendly, low cost, and accelerates the process of AD and biogas production, with less energy input required. The outlined environmental and economic benefits of biogas production, along with personal experience in seeing biogas generators used in India, provided the motivation for this project.

This project was designed to determine which of the 2 animal manure types, monogastric (MG) or ruminant (R) produces the most biogas in an anaerobic digester, and whether the addition of SMS increases the amount of biogas compared to controls. Four types of manure from 2 different animal digestive systems were tested individually or in combination with other manures or SMS. It was hypothesized that SMS would increase the amount of biogas produced in all manure types, compared to controls, due to its ability to pre-treat lignocellulosic material (Hypothesis 1). It was also hypothesized that MG manure would produce more biogas than R manure (Hypothesis 2). R waste has proven to be helpful in starting the fermentation process as it contains methanogenic bacteria; however, ruminants extract a more significant proportion of nutrients from food, and remaining lignin in R manure could be more resistant to anaerobic digestion. Over time, it was predicted that MG gas production would be more significant (Andrade, Xavier, Coca, Arruda, & Santos, 2016).

MATERIALS & METHODS

SMS from Oyster mushrooms and manures were sourced from local farmers. Two phases of experimentation were performed, using two collection methods referred to as the Balloon Method and Water Displacement Method. Phase 1 tested the production of biogas from cow dung (CD) and sheep manure (SM). Phase 2 tested the biogas production form CD, SM, horse manure (HM) and pig manure (PM). CD and SM are from ruminant animals and HM and PM are from monogastric animals.

PHASE 1: EXPERIMENTAL SET-UP

Phase 1 consisted of 3 trials and two collection methods (Balloon Method and Water Displacement Method) to observe and quantify biogas production. One litre plastic bottles were used as anaerobic digesters. The combinations of manure and SMS included CD, SM, SMS, CD+SMS, SM+SMS and CD+SM+SMS. Digesters were kept in a closed room with a space heater set at 30 – 35 ℃ in Phase 1.

**Table 1**: The manure to SMS ratio and the manure/manure combinations and their corresponding digestive systems.
Manure from two types of digestive systems were assessed including, R: Ruminant; MG: Monogastric.

Trial 1 - Balloon Method
Fresh manures and SMS were weighed and distributed into separate bottles, as per the ratios described in Table 1.

Bottles were filled up to 2.5 cm from the top with distilled water, slurries were shaken, a balloon was stretched over each bottle mouth and taped in place. Three repetitions of each bottle and 1 control bottle of distilled water made a total of 19 digesters. The balloons were classified as ‘Standing Straight Up’, ‘Half Up’, ‘Flopped Over’, and ‘Sucked In’ from greatest to least biogas production, respectively. Visual qualitative observations were taken daily for 21 days.

Trial 2 - Balloon Method
To verify results, the same 6 slurries listed in Table 1 were tested again, with only two repetitions of each bottle. The bottles were set up using the same parameters as trial 1 except, this time, SMS and manures were homogenized prior to adding. Balloon circumference was measured daily over 15 days.

Trial 3 - Water Displacement Method
Based on results in Trial 2, it was found that slurries with SMS added produced the most gas. One repetition of each slurry listed was tested using water displacement method (Figure 1). SMS and manures were ground prior to adding and then the bottles were filled with distilled water to 6 cm from the top: CD + SMS, SM + SMS, and CD + SM +SMS, along with SMS only, as the control. The trial was run for 10 days and the height of displaced water was measured daily.

Phase 2: Water Displacement Method

The experiment was expanded to include two more types of MG manure: Horse (HM) and Pig (PM). SMS was ground before adding, but manures were not. Three repetitions of each slurry were made using the same proportions as in previous trials (±0.01g) (Table 1). The trial was run for ten days, and the height of water displaced by the gas produced was measured daily.

**Table 2**: Summary of experimental design.
*Manure types included ruminant (R) or monogastric (MG). Manure treatments included cow dung (CD); sheep manure (SM); horse manure (HM); pig manure (PM). Experiments were conducted with or without the addition of spent mushroom substrate (SMS).

RESULTS

PHASE 1

Trial 1: From Day 1, the CD + SMS slurry had balloons ‘Standing Straight Up’, with maximum gas production in all 3 balloons on Day 3, followed by a reduction in gas from Day 7 onward (returning to “Flopped Over”). The SM + SMS slurry showed maximum biogas production on Day 1, as all 3 balloons were upright. In Trial 1, although SM + SMS showed the quickest gas production (Day 1), followed by CD +SM + SMS (Day 2), the slurry of CD + SMS showed the longest stretch of maximum gas production (Upright from Day 3 - Day 7).

Trial 2: The average balloon circumference was consistently highest for the CD + SM + SMS slurry. The largest circumference occurred on Day 2 (average 21 cm). This was consistent with Trial 1. By Day 15, the average circumference decreased to 17.5 cm. The next largest average circumference was seen in SM + SMS on Day 1 (consistent with Trail 1). By Day 15 it had reduced to the lowest average circumference of all 3 slurries (16 cm). Although CD + SMS did not get as big as the other two slurries, it remained most consistent, having the slimmest range between highest and lowest values (19.5 cm - 17.5 cm).

Trial 3: The height of water displaced into the water collector was measured in centimeters. On Day 1, SM + SMS had the highest value (4.4 cm), consistent with Trials 1 and 2. Day 3, CD + SM + SMS measured 6 cm, the highest value in the trial, supporting Trial 2, and stayed at that height. CD + SMS consistently had the lowest value, 4.9 cm.

PHASE 2

Phase 2 results supported Phase 1 as the slurry containing CD + SM + SMS produced the most biogas (Figure 2). Overall, biogas increased more in most slurries treated with SMS compared to manures on their own. Comparing individual manures, PM was highest, followed by CD, HM, and then SM. Overall, MG manure produced slightly more biogas than R manure without SMS. However, when mixed with SMS, PM and SM both outperformed CD. (Figure 3) PM showed a similar pattern of steady increase in biogas on its own (20.14 mL/day) and with SMS (26.42 mL/day); the addition of SMS widened the gap in total gas production (example day 10: PM = 202.72 mL; PM + SMS = 256.53 mL). (Figure 4) CD and CD+SMS also showed a similar pattern to each other, with marked increases. For example, on day 10: CD = 99.38 mL; CD+SMS = 119.66 mL, which have a rate of change of 20.14 mL/day and 17.55 mL/day, respectively. SM + SMS and HM +SMS also showed this marked increase (30 mL/day & 8.76 mL/day) at Day 4, as did CD + SM +SMS (28.74 mL/day). Even though SM alone produced the lowest value, when it was added to CD + SMS, more biogas was produced than CD + SMS.

DISCUSSION

According to my findings in this study, SMS makes a huge increase in biogas production when mixed with animal manures. Phase 1 revealed that SM with SMS was the most effective biogas producer. In fact, the CD + SM + SMS combination only increased biogas a small margin over SM + SMS. In Phase 2, using an average of all results from each slurry, it was determined that R manures produced more biogas than MG when treated with SMS (P < 0.03). Conversely, without SMS, MG manures produced more biogas than R, supporting the hypothesis that MG would make better biogas substrates than R and hypothesis 2 that SMS would increase the biogas yield when mixed with various manures. The null hypothesis for hypothesis 1 of this study that SMS has no effect on biogas production can be statistically rejected (Table 1) because in five cases, the addition of SMS increased the production of biogas over the same manure combinations without SMS (Table 1; T-test P< 0.05). The CD and SM produced a significant amount of biogas on the first three days when treated with SMS. Whereas HM and SM produced a significant amount of biogas until day 5 when treated with SMS. The marked increases in biogas in some of the slurries, noted in results, could be due to interactions among bacteria species in manure and SMS. The SMS used in Phase 2 was a lighter shade compared to Phase 1 and was obtained from a different farm. Future investigations to identify bacteria in manure samples should be done. Also, the temperature was slightly different between phases 1 and 2 which could have affected the activity of methanogenic bacteria.

Possible sources of error were explored and noted. Future work could include testing the composition of SMS prior to use. Also, the temperature was slightly different between phases 1 and 2 which, could have affected the activity of methanogenic bacteria. Other factors identified were moisture content of the manure, substrate particle size (manures were not ground the same way between phases), design of the digester - plastic bottles were used as the digesters, which are somewhat expandable and may affect measurements of biogas. Furthermore, the ratio of manure to SMS could be altered and tested. The current results could be further reinforced by autoclaving to sterilize manures to observe the effects of bacteria removal. Overall, this study is an opening to a world of renewable energy to help in the fight against global warming and climate change.

REFERENCES

Andrade, W., Xavier, C., Coca, F., Arruda, L., & Santos, T. (2016, September 15). Biogas production from ruminant and monogastric animal manure co-digested with manipueira. Retrieved from Google WebCache: https://webcache.googleusercontent.com/search?q=cache:9bDWs3ip7NUJ:https://dialnet.unirioja.es/descarga/articulo/6505137.pdf+&cd=2&hl=en&ct=clnk&gl=ca

Grimm, D., & Wösten, H. A. (2018, July 19). Mushroom cultivation in the circular economy. doi:https://doi.org/10.1007/s00253-018-9226-8

Gupta, K., Aneja, K., & Rana, D. (2016, June 1). Current status of cow dung as a bioresource for sustainable development. doi:https://doi.org/10.1186/s40643-016-0105-9

Heavenly Holland. (2018, January 30). Seven interesting facts about Dutch mushrooms. Retrieved from Heavenly Holland: https://heavenly-holland.com/mushrooms/

Muchena, F. B., Pisa, C., Mutetwa, M., Govera, C., & Ngezimana, W. (2021, March 28). Effect of Spent Button Mushroom Substrate on Yield and Quality of Baby Spinach (Spinacia oleracea). Retrieved from Hindawi: https://www.hindawi.com/journals/ija/2021/6671647/

National Geographic Society. (2012, October 9). Greenhouse Effect. Retrieved from National Geographic: https://www.nationalgeographic.org/encyclopedia/greenhouse-effect/

Pérez-Chávez, A. M., Mayer, L., & Albertó, E. (2019, March 21). Mushroom cultivation and biogas production: A sustainable reuse of organic resources. Retrieved from ResearchGate: https://www.researchgate.net/publication/332330326_Mushroom_cultivation_and_biogas_production_A_sustainable_reuse_of_organic_resources

Shirkavand, E., Baroutian, S., Gapes, D., & Young, B. (2016). Combination of fungal and physicochemical processes for lignocellulosic biomass pretreatment – A review. doi:http://dx.doi.org/10.1016/j.rser.2015.10.003

ACKNOWLEDGEMENTS

A very special thanks to Amanda McInnes and Dr. Neil Emery, my mentors who inspired and guided me through this project.

Many thanks to Danny Lee Rinker, PhD Retired Professor - Mushrooms - University of Guelph, Dr. David Meigs Beyer - Professor of Mushrooms - Penn State University, and Dr. Barry J Micallef Associate Professor - Plant agriculture - University of Guelph for their contributions to the project design and Mrs. Jill Emery for guiding me throughout.

Thanks to Farmers from Lindsey, Bailieboro, Millbrook and Keene who provided different manures, and Dave Kranenburg from Kendall Hills Farms who provided the spent mushroom substrate.

And one big thank you to my parents for their strong support and encouragement.