The use of edible fungi to digest oily waste

Eamonn Browning

Barker College

Purpose: This paper aims to investigate and determine the capacity of edible Oyster mushrooms (Pleurotus spp.) to remove oil contamination from a growing substrate of coffee grounds and to examine the effect of this production method on the yield of the mushrooms. Design/methodology/approach: Sterile coffee grounds were divided into two containers and mixed with different amounts of canola oil. A control group of coffee grounds did not receive any oil. The substrate mixture was inoculated with commercially prepared Oyster mushroom spawn. The amount of oil present in the substrate was measured before and after myceliation. Findings: Substrates with no oil produced more mycelial growth than spawn bags inoculated with a low oil load. Spawn bags with low oil loads removed on average 73% of oil from the sample proving that mushrooms can effectively remediate the oil “contamination”. High oil load samples removed 57% of the oil. Research limitations/implications: Due to environmental conditions, the mushrooms did not properly fruit meaning that mushroom yield could not be calculated. Given the success of the oil removal, it would be very worthwhile to repeat this experiment and calculate mushroom yield. Practical implications: Edible fungi are clearly able to remove oil from their growing substrate. This could be applied to the remediation of cooking oils before they are disposed of via waste. If this could be shown to also produce edible fungi, this would be an important step toward sustainable food production. Social implications: This could be implemented in small scale food production systems and be used to help process waste and grow edible fungi. Originality/value: The paper investigates mycoremediation of a vegetable oil and examines the extent to which it is effective against the amount of contaminant that is present. Keywords: Mycoremediation, Oil, Fungi. Paper Type: Research paper.

Literature Review

Fungi are responsible for converting dead matter into nutrients for the soil, and thus contribute to the environmental ecosystem (Stamets, 2010). Fungi can be very large organisms with the largest recorded Fungi measuring to 10 square kilometres, located in Eastern Oregon. It is the largest known organism and has an estimated age of 2,400 years (Casselman, 2007).

Fungi can grow in diverse environmental conditions. They are being studied in diverse applications within food production, packaging, and waste remediations (Pullano, 2020). Their ability to decompose organic materials has meant that mushrooms are increasingly being investigated for their capacity to breakdown environmental toxins and manage waste through a process of bioremediation, or more specifically, mycoremediation (Nikola, 2020).

At the same time, one of the most significant food waste problems is the disposal of used and excess cooking oils. Oils cannot be safely disposed of in water waste as they are toxic to marine and freshwater ecosystems. Canola oil contains Polycyclic Aromatic Hydrocarbons (PAHs) which inhibit standard plant growth. Oil can be detrimental in landfill and soil/compost waste because PAHs alter soil grain size, blocking of pores preventing ventilation and water holding capacity of soil (Filho et al., 2017).

Edible Fungi

Oyster mushrooms can be grown in a laboratory setting using spawn bags and a substrate as a growing medium, mixed in with grain spawn. Coffee grounds are a suitable substrate and another form of cooking waste that can be utilised and remediated (Sayner, 2018). Through inoculating the coffee ground substrate with mushroom spawn, the mixture is then sealed in a spawn bag as seen in Figure 1., and placed in a dark environment as it allows the spores to reproduce (JayLea, 2022). After Mycelia networks have grown, slits are made in the bag, exposing the Primordial formations to the air, prompting mushroom fruiting, and the spawn bags are placed in a lit humid environment to encourage mushroom fruiting (MycoHaus, 2020).

Figure 1: Process of Inoculation of Spawn grain into a substrate. (After: Stamets, 2000, pg 82)

Mycoremediation

Fungi species, including oyster mushrooms, (Pleurotus spp.) remove toxins from their environment through a process called mycoremediation (Greenberg, 2019; Mahan, 2015). Fungi use three main methods in decontaminating their environment; biodegradation, biosorption and bioconversion (Kulshreshtha et al., 2014). Biodegradation is a process which the fungi use to break down and recycle a complex molecule into its mineral constituents thus removing their toxicity to the surrounding ecosystem (Mahan, 2015; Ali et al., 2020). Enzymes including ligninase and cellulase excreted from the mycelial, breakdown chemicals into minerals that are less damaging to the soil substrate, thus stimulating more mushroom and plant growth (Akhtar & Mannan 2020). Secondly, biosorption is a process of active transportation and storage of minerals and compounds into the cell (Daccò et al., 2020). They can then be further broken down and recycled for the plant (Kapahi & Sachdeva, 2017). Lastly, the process of bioconversion utilises harmful waste in the cultivation of fungi to enhance gross yield and mushroom growth (Kulshreshtha et al., 2014).

An experiment conducted by Horel and Schiewer (2020) identified the extent to which fungi can bioremediate hydrocarbons over a 28-day period. The study utilised spawn bags contaminated with fish biodiesel. At the conclusion of the experiment, more than 48% of the fish biodiesel had been removed by the bioremediation processes. This investigation identified fungi's ability to remove hydrocarbons from substrates. However, it lacked in effective graphical representation of the data to aid in identifying the type of trend. Another investigation examined the effect oil has on oyster mushroom growth (Chukunda and Simbi-Wellington, 2019). Their research determined that with a 50% increase in oil contamination, there can be an expected 20% decrease in oyster growth. Whilst the experiment produced results that proved the hypothesis, no control was used therefore degrading the validity of the experiment. These investigations confirmed mycoremediation can be produced in a laboratory setting and with specific oil types. Practically, investigations into other cooking oils would be valuable as well as investigating bioremediation of high oil loads compared to low oil loads as well as its effect on mycelial growth.

Additionally, fungi can readily adapt to their environment, providing the ability to grow in harsh locations and under different stresses that other natural bioremediation agents cannot (Selbmann et al., 2013). When conditions become too harsh for plant growth, fungi focus on extremotolerance and change their biological processes to best adapt under environmental pressures (Gostincar et al., 2010). The application of this is by introducing an adaptive species with mycoremediation properties to a harsh and contaminated environment, the natural processes of the mushrooms will allow the soil to be alleviated from harmful toxins and theoretically produce a natural edible crop that yields nutrition at the same time (Selbmann et al., 2013). There is little found practical investigations into fungi extremotolerance that produces quantitative results that mushrooms can adapt to grow in environments with high oil concentrations, therefore there needs further research into the effect of high oil loads on the growth of fungi to determine their ability to grow in such conditions.

This research aims to overlay these two food production dilemmas and seeks to determine whether mushrooms can be used to bioremediate oily food waste. By using an edible mushroom species in the mycoremediation process, it may be possible to use the breakdown of food waste to produce another food source. All these effects combine to affect the diversity and population of beneficial microbes (Klamerus-Iwan et al., 2015).

Scientific Research Question

To what extent can oyster mushroom (Pleurotus ostreatus) cultivation decrease the concentration of cooking oil in the growing substrate?

Scientific Hypothesis

In determining the capacity of oyster mushrooms in removing oil contamination from a growing substrate of coffee grounds and examining the effect of the yield that different amounts of oil may have it is hypothesised:

1 That oyster mushrooms will remove greater concentrations of cooking oil from a soil substrate when the initial concentration of oil is lower.

2 In substrates with the greatest amount of oil, the mushroom yield will be lower than in a substrate with less oil contamination and the substrate with no oil will produce the greatest yield of oyster mushrooms.

Methodology

A greenhouse that could hold nine spawn bags was sterilized using a surface cleaner and constructed in the lab. From the clear plastic sides, a 30cmx30cm patch was cut out and replaced with screen material and duct taped in place to provide ventilation for mushroom growth (as seen in Figure 2). 6.75kg of used coffee grounds was collected over a two-day period from a local café and was stored in the freezer immediately after collection to prevent mould growth (contamination). The coffee grounds were sterilised by evenly dividing and spreading out in batches onto baking trays lined with fresh baking paper and was placed in the oven in 170 degrees Celsius for 40 minutes as shown in Figure 3. After sterilization, the coffee grounds were poured into large sterile zip lock bags, sealed, and returned to the freezer to prohibit growth of mould.

Nine large containers were washed using detergent and dried. An electronic scale that measured to 0.00 grams accuracy was used to divide the coffee grounds into nine 750g samples as shown in Figure 4. 100ml of unused supermarket brand canola oil was added to each of 3 of the substrates and mixed thoroughly. In another 3 separate containers, 300ml of canola oil was added to each of the substrates and thoroughly mixed, whilst another 3 containers were left without oil contamination. 100ml and 300ml were determined to be a large enough difference to determine a comparison between low and high oil load when combined with 750g of coffee grounds in a spawn bag, providing enough room for mycelium growth within. 720 grams of commercially grown Pleurotus oyster mushroom grain spawn was separated into 9x 80g portions (as seen in Figure 4.). The grain spawn

Figure 2: Greenhouse apparatus including mesh ventilation and humidifier.

Figure 3: Oven sterilization of coffee grounds.

portions were tipped into the substrates and thoroughly mixed by hand into each substrate ensuring that the grain was broken down and spread to the entire substrate mixture.

From each substrate mixture, one 6g sample of substrate was collected from the centre of the mixture and placed into a 15ml centrifuge tube. The tubes were labelled and placed into the freezer to inhibit mycelial growth. These nine samples would later be analysed as the “before” sample. The containers were emptied into mushroom spawn bags which were then labelled and sealed. Spawn bags were placed in a dry

dark environment to prompt myceliation for three weeks.

After three weeks, two bags were observed to have been contaminated by mould growth and were discarded from the experiment (see Figure 5). Unfortunately, these bags were both from the same group: the high contamination group. This meant that the high oil contamination group only had one bag in the treatment.

Figure 4: 750g coffee ground substrates; 80g Pleurotus sp oyster mushroom grain spawn; spawn bags.

Figure 5: Discarded contaminated high oil load spawn bags.

Spawn bags were removed from the dark environment and X-shaped slits were made with a sterile scalpel where the mycelial growth had pressed against the edge of the spawn bag to allow mushroom growth outside the spawn bag. The bags were placed in the greenhouse shown in Figure 6. Bags were numbered and placed randomly on different shelves to account for the proximity to the humidifier in the chamber. The greenhouse was sealed, and the humidifier placed on a low setting, placed on a 15 minute on/off timer. The intent of this was to maintain a stable environment to produce maximum results. Spawn bags were taken out of the greenhouse after 7 days as no growth was observed and placed in the fridge for 24 (cold shock) hours and returned to the greenhouse to shock the mushrooms into growing.

The spawn bags were removed after three weeks in the greenhouse. For each spawn bag, a test tube was used to collect a sample by plunging the open end through the middle of the substrate to the bottom of the bag, twisting and removing it. 6g of the collected sample was transferred using a sterile stirring rod into the 15ml centrifuge tube (see Figure 7). The foil was used to catch spilt substrate pieces and funnelled them into the centrifuge tube. The tube was then labelled with the amount of oil from the spawn bag the sample came from. This was repeated for all the spawn bag mixtures. Each of the centrifuge tube samples from before the mycelial growth and after the mycelial growth were filled with distilled water up to the top of the tube.

Figure 6: Greenhouse loaded with randomly assorted spawn bags.

Figure 7: Sample measurement of 6g apparatus.

Table 1: Oil layers from centrifuge tubes of 6g low oil load samples and high oil load samples of both before and after mycelial growth and average.

Oil layer in centrifuge tube Control Low oil load A Low oil load B Low oil load C Low oil load average High oil load

Oil before mycelial growth (mm) 0.0 4.0 5.0 6.0 5.0 7.0 Oil after mycelial growth (mm) 0.0 1.0 1.0 2.0 1.3 3.0 Oil loss (mm) 3.0 4.0 4.0 3.6 4.0

The tubes were loaded into the centrifuge and spun at 4000rpm for 60 minutes. The layer of oils in the centrifuge for the sample before mycelial growth was compared to the centrifuge sample of after the mycelial growth, both from the same spawn bag substrate by using callipers to measure the thickness of the layer in mm. Observations were made on the amount of mycelial growth in the control spawn bags with no oil, the bags with low oil load of 100ml of inoculated canola oil and bags with high oil load of 300ml of inoculated canola oil.

An online t-test calculator was used to compare the means of amount of oil from the sample before and after mycelial growth to test the hypothesis and determine if a significant difference exists between them. The amount of oil before and after mycelial growth between the samples from the low oil load and the high oil load were averaged and placed in a column graph on the horizontal axis and oil layer in centrifuge (mm) on vertical axis, completed in excel. Error bars were calculated and graphed using excel.

Results

The oil layer in centrifuge tube before mycelial growth and after mycelial growth in the 100ml contaminated spawn bags labelled as the low oil load, and the 300ml contaminated spawn bag labelled as high oil load is shown in Table 1. The average for the low oil load before and after mycelial growth was calculated whilst the absence of more than one set of data for high oil load meant that no average could be calculated. This was then plotted in a column graph showing standard error in Figure 8. Figure 8 shows a general trend of an oil loss after the mycelial growth.

There was a 73% oil loss between the oil layer in the sample from before the mycelial growth compared to after in the low oil load average and a 57% decrease in oil from the samples with high oil load.

Figure 8: Amount of oil (mm) in 6g sample before and after mycelial growth

A t-test was used to compare the mean of the low oil load before mycelial growth and the low oil load after mycelial growth to determine whether the two resulting samples are significantly different. The null hypothesis is that there is no significant difference between the means. The alternative hypothesis is that the means are significantly different. The alpha value was 0.05. The t-value is 5.5. The test was a one-tailed hypothesis. The P-value was 0.002664 and the result is significantly less than the alpha value of 0.05. As the P-value is greater and the alpha value, there is a significant difference in the mean amount of oil in each sample.

Observations were made on the amount of mycelial growth in each of the spawn bags and the ability to produce mushrooms. Control spawn bags with no oil contamination had more vigorous mycelial growth, producing primordial formations that protruded out from the bag. Spawn bags introduced with 100ml of oil contamination had mycelial growth however lacked substantial primordial growth. Spawn bags with 300ml of oil contamination showed far less mycelial growth showing splotches of exposed substrate where there was no growth and patches of mould growth as shown in Figure 9.

Figure 9: Image of spawn bags after mycelial growth. Control spawn bag; left, Low oil load; middle, high oil load; right.

Discussion

The first hypothesis tests whether oyster mushrooms can effectively bioremediate (remove) different amounts of canola oil from a growing substrate. The result from this experiment proves that there is a significant decrease in oil results from mycelial growth. Using a student t-test, the p-value was found to be less than the alpha value of 0.05 (p=0.02664). The null hypothesis is that there is no significant difference between the mean amounts of oil. The alternate hypothesis is that there is a significant difference between the mean values. As the p-value was found to be less than the alpha value, therefore, the null hypothesis can be rejected, and the alternate hypothesis is accepted. Thus, it can be reliably concluded that mycelial growth removed oil from the substrate, reducing the oil load. The second hypothesis could not be tested because the fungi did not fruit properly. As this crop failure happened across all test and control groups this can be assumed not to be a result of the oil treatment.

Figure 8 shows that a low oil load had a larger oil loss than the higher oil load, therefore, proves the hypothesis true and that mushrooms will remove cooking oil from a coffee ground substrate more efficiently when the initial concentration of oil is lower. Low oil load average had a 73% removal, and the high oil load had a 57% oil removal. 16% difference confirms that Oyster mushrooms were more effective at bioremediating oil concentrations when the oil contamination was lower than when there was a high oil concentration. Therefore, mycoremediation of oil by Oyster mushrooms would be more effective if there is a greater amount of mushroom spawn over contaminant amount. This is consistent with findings from other research. For example, research on the effects of crude oil on the growth of Oyster mushroom (P. ostreatus), found that high concentrations of oil resulted in a decrease in the fungi’s natural processes and functions, and thus inhibited its growth (Chukunda and SimbiWellington, 2019). Ultimately, they concluded that fungal ability to transform PAHs into reusable products decreased as the concentration of PAHs increases, due to inability to obtain other resources from the substrate and environment. This is consistent with the findings of this study and therefore likely to be true of vegetable oils as well as petroleum.

Observations of mycelial growth determined that in substrates with a greater amount of oil, the mycelial growth was lower than in substrates with little to no oil contamination. The spawn bags with no oil contamination developed primordial formations which would later develop into the mushrooms. The spawn bags with low oil loads did develop large amounts of mycelial growth; however, did not produce primordial formations. It is therefore concluded that the presence of oil delayed or inhibited mushroom growth. There is no research to indicate a direct explanation for this; however, it is plausible that the oil delays the mushroom development and that with more time, the mushrooms might develop in substrates with oil present. High oil load bags did not produce much visible mycelial growth. Repetition of the experiment over a longer time frame may be one possible direction for future research.

One key and unexpected outcome was that the experiment was unable to produce any fruiting bodies (i.e., mushroom yield). Despite an attempt to cold

shock the spawn bags, no fruiting occurred. This was mostly likely due to environmental factors rather than the experiment because the control bags did not fruit either. The timing of the experiment meant that it was conducted during late autumn. Unseasonably cold conditions and an error with the laboratory thermostat meant that the laboratory temperature was dropping too low during the overnight period. This meant that the initial intention to measure the yield of the mushrooms (through mass of the fruiting bodies) was unsuccessful and the control for the experiment that would have been used to compare the yield produced, was less effective in comparing the end results for the loss of oil. However, it was still possible to qualitatively observe the extent of myceliation through measuring the difference between substrate before and after (the difference was assumed to be mycelial growth). From this it was concluded that in substrate containing low concentrations of oil, mushroom growth was inhibited; however, mycelium was still able to develop, whilst in high oil loads, mycelial growth was inhibited.

In two of the high oil load spawn bags, unwanted mould growth developed and needed to be discarded. Whilst this contamination is strictly a non-result, it is highly suggestive that the Oyster mushroom was being inhibited, allowing other fungi to flourish. Out of the nine spawn bags produced, contamination only occurred in bags with the high oil load. Therefore, I hypothesise that high concentrations of oil made the environment more susceptible to fungal growth as the desirable mycelia is inhibited by the oil, however undesirable species were able to survive.

The experiment was limited in the amount of time to let the spawn grow in a dark environment and a limited time to allow them to grow in the greenhouse. Therefore, if the experiment was conducted over a longer period, there may have been primordial formation growth in spawn bags with low oil loads, and therefore would have concluded that presence of oil delays growth of those formations and not inhibit it.

Due to the lack of mushroom growth, measuring the yield of mushroom growth had to be adapted to observing the mycelial growth, thus turned finding quantitative data into recording qualitative data, and thus limited the capability of solidifying the relationship with a t-test.

Future directions of research would involve repeating the experiment and extending the growth time to test for mushroom yields. Furthermore, extending that research to testing the safety and edibility of the mushrooms, thus contributing to the application of the research and investigation, wherein bioremediation can also produce food for impacted communities.

Conclusion

In conclusion, this experiment showed that the amount of oil in a substrate is decreased through mycelial growth using the natural process of mycoremediation. It showed that as the amount of oil in a substrate increased, there is a decrease in the effectiveness for Oyster mushrooms to remove the contaminant from the substrate and furthermore, increases in oil meant a decrease in mycelial growth and a susceptibility to mould growth.

The method employed in this research task was an effective way to determine if the active process of mycoremediation was present, wherein it showed the relationship between the amount of oil before and after mycelial growth. However, the experimental period impacted the methodology so that it had to be changed to produce a result in yield to test the hypotheses. This could be adjusted in the future to accommodate more time to the mycelium to grow and thus, as future research, the yield of mushroom production would be invaluable towards practical uses of this research.

The application of this research conforms to addressing vegetable-based oil pollution, where environments impacted with similar oil contaminant can be bioremediated using Oyster mushrooms; however, to be successful would require a high proportion of mushroom grain compared to contaminant for it to be effective.

In conclusion, this research has produced promising results to provide a scientific basis for growing edible fungi on coffee grounds that have been spiked with oily food waste. This could be an inherently sustainable food production system where waste (coffee grounds and cooking oil) is converted to an edible product (mushrooms) and the remaining substrate is much more compostable and environmentally inert than the original materials.

Acknowledgements

Dr Alison Gates mentored and assisted in the design of the project, from the apparatus, practical work,

resources, report writing guidance and is acknowledged for her efforts and involvement in this investigation.

Mrs Lucy Pitkin assisted, aided, and supported the methodology, specifically through her efforts in showing and teaching how to care for the mushrooms. Furthermore, her best efforts in aiding in getting the mushrooms to produce fruit bodies.

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