Final report for GW20-210
The past few years have seen the rise of controlled environment ‘vertical farms’ in almost every major city around the world. These precisely controlled plant chambers require a high level of carbon dioxide to maintain optimal photosynthetic levels. Currently, there are two main options to accomplish this – injecting pure C02 from cannisters or using a generator that is run on natural gas or propane. Injecting pure C02 is a cost-inefficient option while burning natural gas or propane increases the carbon footprint of the vertical farm. As cities trend towards a carbon-free future, alternative sources must be explored for C02 enrichment.
This problem can be addressed by linking a specialty mushroom cultivation chamber with a plant cultivation chamber via air exchange. Carbon dioxide respiration from mushrooms can be used to enrich leafy plants that photosynthesize. If enough carbon dioxide can be generated, this research will shift the paradigm for how growers enrich their plants with carbon dioxide. Furthermore, specialty mushroom cultivation can also play an important role in tackling the problems of local food insecurity, inefficient water use, and waste management by growing highly nutritious mushrooms on agricultural and industrial waste.
This research will focus on which specific species, substrates, and environmental conditions are ideal in the context of urban agriculture. The impacts will be measured by C02 levels, yield, and bio-efficiency. Outputs of this research will include a published scientific paper and an instructional guideline in order to empower a new generation of specialty mushroom cultivators.
- Measure which species of specialty mushrooms produce the highest amount of yield and usable carbon dioxide for plant chambers. The species of specialty mushrooms will be selected based on their market potential, nutritional profile, and demonstrated history of cultivation - Pleurotus ostreatus (Pearl Oyster), Hericium erinaceus (Lion’s Mane), and Lentinula edodes (Shiitake). The timeline of mycelial inoculation of the substrate bags to post-harvest is approximately six weeks – therefore, the project will be able to go through multiple rounds of testing within a one-year time period.
- Measure which substrate produces the highest amount of bio-efficiency and usable carbon dioxide for plant chambers. The substrate is a critical variable in terms of efficient mushroom production and may have a profound impact on the amount of carbon dioxide emitted by the mushroom for enrichment of plant chambers. The substrate can be divided into a carbon source and the nitrogen source. Whenever possible, this project aims to partner with local businesses for straw, coffee grounds, wood chippings, and other waste streams that can be used as substrate.
- Measure which controlled environment conditions are optimal for specialty mushroom production (humidity, temperature, ventilation, light, carbon dioxide level). It is important to know the precise conditions that the mushroom grow bags require during the different stages of cultivation. This project will test preexisting assumptions of ideal conditions for growth and see if any controlled changes in environmental conditions may result in higher yield or more carbon dioxide production.
8/1 – The project will begin by contacting producer cooperators and getting pre-project surveys on their current operations. The project will collect data on carbon dioxide enrichment, yield of specialty mushrooms per month, and amount of water used relative to biomass produced. (1 month)
8/1 – Environmental controls will be established at the vertical farm and series of grow tents to ensure that optimal conditions can be achieved during the fruiting phase. Furthermore, computer controls and sensors will be set up to continuously collect data. A carbon dioxide decay test will be completed to understand the leakage from these fruiting chambers. (1 month)
8/1 – Lab work will be undertaken to isolate clean cultures of mycelium and inoculate grain spawn bags of three different cultures. (1 month).
8/1 – Partnerships will be established with local businesses to collect post-consumer waste that can be used as a substrate for the mushrooms (1 month).
9/1 – The spawn bag will be used to inoculate substrate bags and placed in the fruiting chambers to collect data on carbon dioxide enrichment (2 months). Milestone for objective 1.
11/1 – Experiment will be repeated to ensure consistent results (2 months).
1/1 – New substrates will be inoculated to collect data on the optimal species and substrate combination using a two-factorial analysis. Test will be repeated (4 months). Milestone for Objective 2.
2/1 – A gas exchange conduit will be established at the vertical farm to measure carbon dioxide enrichment. Test environmental controls to find optimal conditions for specialty mushroom cultivation with the species/substrate combo. Test will be repeated. (4 months). Milestone for objective 3.
5/1 – Analyze results, establish educational materials, and set up workshop events. Disseminate information. (1 month)
6/1 – Work with producer cooperators and evaluate producer adoption. (2 months)
Commercial, dikaryotic strains of Pleurotus ostreatus, Ganoderma lucidum, and Trametes versicolor were obtained (Fungi Perfecti, LLC, Olympia, WA; Table 1). The cultures were maintained at water vultures at 4°C until being grown on glucose yeast extract agar (GYEA) consisting of 10 g L-1 glucose, 10 g L-1 brewer’s yeast, 10 g L-1 agar for 10 days in sterile conditions. After 10 days, the cultures were inoculated onto grain spawn composed of cracked corn. Cracked corn has been shown to have a shorter incubation period for mycelial cultures compared to other forms of grain spawn (Dulay et al., 2017).
Three substrate combinations were used for this study, each containing a principal carbon source and a principal nitrogen source (Table 1). The first substrate combination was 70% wheat straw dry weight (local sourced) and 30% whole, delinted cotton seed (local sourced) dry weight, which is commonly used at the University of Arizona’s Controlled Environment Agriculture Center. This combination is found in agricultural waste streams throughout the United States and provide reliable bio efficiencies and quick fruitification (Sánchez, 2009). Straw was the principal source of carbon source and cotton seed was the principal nitrogen source. The second substrate combination was 50% mesquite wood pellets (Bear Mountain BBQ Woods, Louisville, CO) and 50% alfalfa pellets (Haystack Farm and Feed, Culver, OR). Mesquite wood provides the principal carbon source and alfalfa provides the principal nitrogen source (Geesing et al, 200; Stamets, 2000). The third was a mix of 50% oak wood pellets (Bear Mountain BBQ Woods, Louisville, CO) and 50% soy hull pellets (Mushrooms Media Online, Hayward, WI). This substrate mix, also known commercially as Master’s Mix, is commonly used in industry for specialty mushroom cultivators throughout North America. Oak provides the principal carbon source while soy hull provides the principal nitrogen source (Stamets, 2000).
The substrate combination of straw/cotton was soaked overnight and drained for thirty minutes before packing them into the polypropylene bags with 0.3-micron filters for gas exchange (Fungi Perfecti LLC, Olympia, WA). Each bag was filled to a wet weight of 1360 grams excluding the weight of the polypropylene bag of 18 grams. For straw/cotton, the water content was 68% while for mesquite/alfalfa and oak/soy the water content was 60%. The water content ratio for the substrate combination of mesquite/alfalfa and oak/soy was selected in preliminary trials to maximize colonization without oversaturating the substrate.
Spawn was prepared in 5.5 kg (dry weight batches) per four bags, soaked for 24 hours with tap water, and drained for thirty minutes until water was not constantly draining. Subsequently, the spawn bags were autoclaved at 121°C and allowed to cool overnight. The substrate for straw/cotton was prepared in 2400 g (dry weight) batches with 1680 straw and 720 cotton seed. Substrate was soaked for 18 hours and drained for thirty minutes before being packed in batches of 1360 g in bags. The substrate for mesquite/alfalfa and oak/soy was prepared in 574 g (dry weight) batches with 816 grams of water to be packed in batches of 1360 g in bags. Each of these substrates were inoculated with grain spawn from cracked corn. The substrate was packed into polypropylene bags with a 0.3-micron filter patch that precludes the passage of contaminants but allows gas exchange. Polypropylene bags are often used in mycology laboratories due to the ability to be sterilized in an autoclave (Rutala et al., 1982).
The Experimental System
The system comprised of a total of ten 120-quart totes from Sterilite with an airtight gasket to safeguard both air and moisture. Each tote was outfitted with a CozIR-A C02 sensor (C02Meter, Ormond Beach, FL) with a measurement range of 0-10,000ppm and an accuracy of ±50 ppm+ 3% of reading (CozIR®-A 10,000 ppm CO2 Sensor). These were connected to the GaslabTM Software (C02Meter, Ormond Beach, FL) that allows for easy export of CSV files. The design schematic of the experiment includes two sets of five experimental units connected to a venting source and a computer using the Gaslab TM software to collect data (Fig. 1). The experiment was housed inside UAgFarm, a vertical farm facility located at the University of Arizona’s Controlled Environment Agriculture Center (Fig. 2). Furthermore, each tote was outfitted an air inlet and air outlet, each fitted with a one-way valve, positioned to allow fresh air to enter the system through negative air flow. The outlet was connected to a manifold system with a vacuum that periodically vented the totes for minutes every hour to keep C02 levels from rising above 10,000ppm after monitoring the rate at which mycelium bags produced C02 inside the experimental units. The venting system time interval (1 min duration) also allowed C02 to reach ambient conditions of the room at approximately 450 ppm.
For Pleurotus species, it is typical for mycelium to colonize the substrate for two weeks before placing them in fruiting conditions (Sánchez, 2009). Therefore, each experiment was conducted over a two-week period and monitored CO2 production of all three substrate combinations. There was a total of nine totes - three totes were dedicated to straw/cotton, three totes were dedicated to mesquite/alfalfa, three totes were dedicated to oak/soy, and one tote was used not filled with substrate but remained empty for establishing baseline C02 values throughout each experiment. The baseline C02 measurement was approximately 450 ppm. Each of the filled totes had five mycelium filled bags in it for a total of fifteen bags per substrate per species trial. Instantaneous measurements were taken in one-minute intervals and the raw data was extracted onto an Excel file. For each species, there was a repeated trial to collect additional data points.
Over the initial 24-hour period with Pleurotus ostreatus, the substrate combination of oak/soy had the highest CO2 output while the substrate combination of straw/cotton had the lowest CO2 output (Fig. 3). For the same time period, the total amount of carbon dioxide produced in grams was analyzed (Fig. 4). Furthermore, a trendline was added to show both the rate of carbon dioxide gained and the R2 value which shows how closely the trendline fits over the actual data. For this data set, five bags of Pleurotus ostreatus grown on a combination of oak and soy in a 120-quart tub generated .03 grams of carbon dioxide / minute with an R2 value of 0.99. The same technique of aggregating data and adding a linear trendline was used for analyzing data over the entire two-week period for all of the species.
For the entire two-week experiment, the data for Pleurotus ostreatus can be extrapolated for each of the substrate combinations (Fig 5). Five bags of Pleurotus ostreatus with a substrate combination of mesquite and alfalfa, in a 120-quart system, produced .019 grams of carbon dioxide per minute with an R2 value of 0.97. Five bags of Pleurotus ostreatus with a substrate combination of oak and soy, in a 120-quart system, produced .014 grams of carbon dioxide per minute with an R2 of 0.98. Five bags of Pleurotus ostreatus with a substrate combination of straw and cotton, in a 120-quart system, produced .0087 grams of carbon dioxide per minute with an R2 value of 0.98.
Using the constants shown in Table 2, the amount of carbon dioxide emitted was converted into moles of C02. The moles of C02 were used in the ideal gas law equation to calculate the grams of carbon dioxide generated per minute per bag (Table 3). The results can be shown in Table 4.
Furthermore, the rate of change was measured for the given time period in order to analyze when the most amount of carbon dioxide was generated (Figure 6). All three substrate combinations followed a similar trend with the highest rate of carbon dioxide output on day 6. The mesquite/alfalfa combination, which produced the most amount of carbon dioxide total over the entire two-week trial, produced on average a rate of 7.86 grams/day on day 6. The average rate on day 14 for all three substrate combinations was drastically closer than on previous days, with measurements between 2.59 grams / day and 3.32 grams / day.
For Ganoderma lucidum, the results from the two-week trial can be seen Figure 7. Five bags of Ganoderma lucidum with a substrate combination of mesquite and alfalfa, in a 120-quart system, produced .021 grams of carbon dioxide per minute with an R2 value of 0.98. Five bags of Ganoderma lucidum with a substrate combination of oak and soy, in a 120-quart system, produced .018 grams of carbon dioxide per minute with a R2 value of 0.98. Five bags of Ganoderma lucidum with a substrate combination of straw and cotton, in a 120-quart system, produced .016 grams of carbon dioxide per minute with an R2 value of 0.99. These values were higher than Pleurotus ostreatus across all three substrates.
Using the ideal gas law , the respiration rate of mycelium bags was calculated. Results are shown in Table 4. Furthermore, the rate of change for Ganoderma lucidum was measured for the given time period in order to analyze when the most amount of carbon dioxide was generated (Figure 8). All three substrate combinations followed a similar trend with the highest rate of carbon dioxide output on day 6. The mesquite/alfalfa combination, which produced the most amount of carbon dioxide total over the two-week trial, produced on average a rate of 11.7 grams/day on day 6.
Lastly, for Trametes versicolor, the results from the two-week trial can be seen in Figure 9. Five bags of Trametes versicolor with a substrate combination of mesquite and alfalfa, in a 120-quart system, produced .026 grams of carbon dioxide per minute with R2 value of 0.9942. Five bags of Trametes versicolor with a substrate combination of oak and soy, in a 120-quart system, produced .021 grams of carbon dioxide per minute with R2 value of 0.9935. Five bags of Trametes versicolor with a substrate combination of straw and cotton, in a 120-quart system, produced .020 grams of carbon dioxide per minute with a R2 value of 0.9922. These values were higher than both Pleurotus ostreatus and Ganoderma lucidum.
Using the ideal gas law , the respiration rate of mycelium bags was calculated. Results are shown in Table 4. Furthermore, the rate of change for Trametes versicolor was measured for the given time period in order to analyze when the most amount of carbon dioxide was generated (Fig. 10). The mesquite/alfalfa combination, which produced the most amount of carbon dioxide total over the entire two-week trial, produced a rate of 8.04 grams/day on day 5.
The data was presented on a box plot to show the mean, median, and quartiles for the various species/substrate combinations in Figure 11. The units for the responses are grams of carbon dioxide generated per minute. Trametes versicolor produced the most amount of carbon dioxide while Pleurotus ostreatus produced the least amount of carbon dioxide across all three substrates. The substrate combination of mesquite/alfalfa produced the most amount of carbon dioxide across all three species.
A two factor ANOVA was performed on both the species and substrate, as well as the interaction between the two factors. When looking at Type III Sum of Squares in Figure 12, the p-value for species was 0.0005, which indicates that species is a significant factor when measuring carbon dioxide output. The p-value for substrate was <0.0001, which indicates that substrate was a significant factor when measuring carbon dioxide output. Lastly, the species and substrate interaction had a p-value of 0.1178 which shows that the interaction is not significant when measuring carbon dioxide output at the alpha = 0.05 significance level, and it is appropriate to combine factors into a single analysis.
When looking at the distribution of response for the species in Figure 13, it is evident that Pleurotus ostreatus produces the least amount of carbon dioxide while Trametes versicolor produces the most amount of carbon dioxide. In Figure 14, Tukey’s Honest Significant Difference Test indicates that the Trametes versicolor produces significantly more carbon dioxide than both Ganoderma lucidum and Pleurotus ostreatus at the alpha = 0.05 significance level.
When looking at the distribution of response for substrate in Figure 15, the substrate combination of mesquite/alfalfa produces the most amount of carbon dioxide and the substrate combination of straw/cotton produces the least amount of carbon dioxide. This is further evidenced by Tukey’s test in Figure 16. The combination of mesquite / alfalfa produces significantly more carbon dioxide than the other two substrate combinations and the combination of oak/soy produces more carbon dioxide than combination of straw/cotton.
Lastly, Figure 17 shows the Q-Q plot for the various responses. Most of the data points lie along the trend line which indicates that the data set follows a normal distribution. This is further evidenced by Figure 18 showing the residual plot vs predicted value. The data points indicate normality of distribution.
The results from the chosen species and substrate combinations showed significant results in terms of carbon dioxide generation. However, there were many areas of improvements for this study. First, controlling the lighting may have an influence in mycelial colonization. The lighting conditions at the UAg vertical farm was arbitrary based on whether the person last in there forgot to turn the lights off. Next, an important future area of study is to use reusable jars with 0.3-micron filter caps instead of polypropylene bags with 0.3-micron filter patches. Polypropylene bags are single use which poses a finite resource when in environments where it is not easy to replenish supplies. Growing in jars has been a popular method of cultivating mushrooms in Asia because of its reusability, ability to be inoculated in masse, and ability to withstand sterilization.
Furthermore, although a trial period of two weeks was initially selected due to that being the standard colonization time for Pleurotus ostreatus on straw-based substrates, an area for improvement for this experiment is to extend the duration of trials from two weeks to several months. Although this method may not be ideal for trying to fruit mushrooms, it would give insight into how the rate of change of the various species and substrate combinations changes over time.
Perhaps one of the biggest applications for carbon dioxide respiration of mycelium is in the context of a Bioregenerative Life Support System (BLSS). Mycelium can not only convert inedible biomass from plants into mushrooms containing all nine essential amino acids, but also allow those same plants to be enriched with carbon dioxide from the mycelial colonization of the substrate (Gellenbeck et al., 2019). Although the atmosphere of Mars contains over 95% carbon dioxide, a bioregenerative system utilizing a controlled environment will contain mostly oxygen for the human crew to survive which highlights the need for a consistent source of carbon dioxide (Rothschild et al., 2018). It has also been suggested that a fungal outer wall combined with cyanobacteria will be used as the outer structure to produce oxygen for the habitat (Rothschild et al., 2018). Thus, it is necessary for more predictable methods of carbon dioxide generation that maximizes the synergies between the various food production systems. Figure 19 shows the various exchanges for a subsystem for human life support which includes using spent mushroom substrate to feed into a biodigester / bioreactor (Gellenbeck et al., 2019).
Furthermore, mycelium could play an important role in the future of amateur controlled environment agriculture. The cannabis market is industry is expected to grow at a compounded annual growth rate of 14.3% per year from 2021 to 2028 (Grand View Research, 2021). This has also led to the rise of amateurs growing cannabis within their private residence using grow tents and other controlled environment systems as more states legalize cannabis cultivation. Mycelium can offer a cost-effective solution for growers who do not want to invest in heavy infrastructure for carbon dioxide enrichment but would like to address the issue of carbon dioxide deficiency to produce higher yields.
There are already several companies in the market that already mycelium bags as a method for carbon dioxide enrichment. The most notable one is called “ExHale Homegrown CO2 Bag” who uses a proprietary blend of Trametes versicolor to market to amateur cannabis growers (Exhale). Future areas of study will be to measure different species and substrate combinations to optimize carbon dioxide generation during the incubation phase while minimizing fruiting.
Harnessing the power of mycelium for other applications are potential areas of study to further humanity. The efficiency for mycelium to convert waste from fish, insects, and plants in a bioregenerative system must be evaluated to support a human crew. It is essential for mycelium to be able to fully decompose and recycle the lignin, cellulose, and hemicellulose in order to maintain a closed-loop system. Furthermore, the application to use fungi in the context of astromycology to build the outer shell of habitats in harsh environments poses a great opportunity to expand the human consciousness.
Education and Outreach
On numerous occasions throughout the year, we provided consultations with growers at Rhiba Farms (Phoenix, AZ), Desert Pearl Mushrooms (Tucson, AZ), and Wild Gardens (Benson, AZ). On numerous occasions throughout the year, we provided on-site demonstrations to growers at Desert Pearl Mushrooms to help them get their business up and running.
On March 17, 2021, we delivered a one-hour webinar on specialty mushroom production and the integration of mushroom production into concurrent greenhouse production as part of a 3 day online conference on Controlled Environment Agriculture, hosted by the UA Controlled Environment Agriculture Center. Online registered participants were 82. https://ceac.arizona.edu/2021-online-greenhouse-crop-production-engineering-design-short-course
On May 12, 2021, we hosted a 2-hour online specialty mushroom production workshop, based at the University of Arizona. Participants (30) registered in advance and then picked up workshop supplies at the UA Controlled Environment Agriculture Center, 4 days in advance of the workshop. On the day of the workshop, we delivered lectures on the production of specialty mushrooms, including a discussion of CO2 production and the ongoing Western SARE project. The participants were then guided online in inoculating their pre-sterilized substrate bags that they picked up in advance. The workshop training modules are posted at http://www.azmushroomgrowers.org/presentation-slides.html.
During both the webinar and workshop activities, guidelines were provide on how to grow specific species of specialty mushrooms, details on the ongoing Western SARE supported research program and its potential for CO2 generation, and how the pairing of mushroom production and greenhouse-based plant production can reduce production costs and environmental impacts.