The importance of fungi investigation in the effect of cullulose enzymes on the growth rate and fungal spore density with Pleurotus ostreatus

The importance of fungi investigation in the effect of cellulase enzymes on the growth rate and fungal spore density with Pleurotus ostreatus

Sam Young

Barker College

Purpose: The purpose of this paper is to provide a new growth accelerant in fungal growth, specifically oyster mushrooms as to find the effect of the enzyme cellulase on the growth and sporification density and production of oyster mushrooms. This in which is to find new and faster ways to grow oyster mushrooms and fungi for both agricultural and industrial use. Design/methodology/approach: The experiment used a substrate mixture of hydrated lime (250 ml) mixed with 5kg of sugarcane mulch inoculated with Pleurotus ostreaus oyster mushroom breed of spawn, distributed in 10 mushroom bags. Once the pinhead stage occurred the mushrooms bags were cut to allow for growth of oyster mushrooms outside of the bag, and 4 were chosen to be sprayed with diluted cellulase (diluted in distilled water) (9 sprays per bag) and 4 were chosen to be controls through size and length of mushroom being paired with similar pairs. The weight of the bags was measured every 5 days and after the 15 days mark the 5 mushroom caps or caps from the test were cut and put on a piece of paper to produce a spore print. (figure 2). Findings: The research found that the synthetic introduction of the cellulase enzyme caused an increase in both growth rates (seen through larger weight percentage reduction in the mushroom bags). This partnered with the higher density and yield of the spore prints with the introduced cellulase. Research limitations/implications: The paper was limited to one specific breed of mushroom as well as only having a sample size of one enzyme dilution for the cellulase (5 ml of cellulase per 200ml of distilled water). In future tests more trials can be conducted, as well as the tests with different oyster mushroom breads rather than just PO and using different dilutions of cellulase. Practical implications: The results showed a clear indication for the use of cellulase to help stimulate and increase both the growth rates, spore density, and spore yield. In which provides a new stimulant for oyster mushroom growth. Social implications: The results of the findings can be used to understand and develop new growing techniques in both fungal spores and growing of edible oyster mushrooms. Originality/value: To the knowledge of the researcher, the use of synthetic introduction of any enzyme to see the effects of anything on mushrooms, nor has there been any recording of the effect of the introduced cellulase enzyme on growth rate of oyster mushrooms and fungal spore yield. Keywords: Growth rates, Fungal spores, Spore density.

Literature Review

Importance of mushrooms to humans

Mushrooms have been consumed by humans for as long as human recorded history, its first recorded use hundreds of years BC, as shown in the roman times to be perceived as the “food of the gods” (Valverde et al, 2015). They have also been shown to have a large health benefit showing attributes of anticancerous, anti-cholesterol, anti-allergenic properties as well as showing aspects of rich in carbohydrates like, chitin, hemicellulose, beta, and alpha glucans, mannans, xylans, and glycans for most farmed edible mushrooms (Jayachandran et al, 2017). Due to their dietary ability, mushrooms can provide a clear substitute for products harder to grow or have a longer turnover rate such as beef or cattle for the food shortage crisis leaving 800 million hungry (DeRose et al, 1998). The importance of mushrooms and their turnover rate of most edible mushrooms like oyster mushrooms being 21-28 days (Sharma et al, 2013). The use and importance of mushrooms can be seen in the recent mushroom cultivation and farming as one of the largest agricultural areas, however, is shown to have to combat supply increase of 30-fold worldwide from 1978 to August the 14th 2017, this in which has mainly increased in the 21st century due to the investigation of medicinal benefits and a renewable substitute for products that use more resources (meat) (Royse et al, 2017).

K. Piska al (2017) furthers these ideas of nutritional value and ability of mushrooms specifically Pleurotus Ostreatus (PO), as being the most soled oyster mushrooms across all continents except Antarctica. Investigating the possible abilities and nutritional values it may hold for further use, demonstrating the richness in primary and secondary metabolites. showing that only 100 grams of the fresh fruiting bodies can give 15% of the recommended daily intake of vitamin C, 40% of niacin, riboflavin, and thiamin, and 0.5 mg of vitamin B12. This species is also characterized by a high content of oleic acid (40%), linolenic acid (55%). Emphasising its ability and use in areas of medicine, agriculture, and food showing a clear indication of use for healing and restoring civilisation diseases, such as diabetes. He in which explains how the use of PO can be shown in many areas, demonstrating its ability to grow in harsher climates and use less resources for the same product when comparing to meat or other fruits. He shows the ability for PO to be developed in uses beneficial to humanity such as the ability to grow in harsh environments and the ability to develop amounts of vitamin c in a shorter growth period. Hence the ability mushrooms provide to humanity can be seen in the abilities PO is able to give, thereby showing its importance through its abilities that benefit humanity indicating further research in cultivation, production, and abilities demonstrating the benefit of edible mushrooms to humanity and thereby its importance.

Mushrooms (PO) physiology and effects of environmental and chemical changes on it

Mushroom physiology mainly refers to the nutrition, metabolism, growth, reproduction and death of fungal cells (GM Walker, et al). Fungal cells like any generally will rely and depend upon the biotic and abiotic factors that are in their necessary environment. When specifying with the tested mushroom (oyster mushroom (PO), it is found that the mushroom physiology is more structured and is formed to be much largely collected to the environment,showing that when the mushroom is put in a different temperature and container it was shown to have significant effects on the respiratory rate solubility and water content (Rajarathnam et al., 1983). The further effectors of the PO’s physiology are further shown chemically through chemicals such as oxygen and nitrogen synthetically added within the actual system showing a significant change in growth rate with synthetic introduction (Liu, Z et al., 2010). Therefore, showing that both chemical and physical environmental factors will affect the overall health and quality of the product it is clear to see that the effect of mushroom size and growth is affected both chemically and environmentally. The second factor is also the negative indication of certain chemical changes (seen in fig 1) both temperature and moisture level can affect the growing as well as the decay of the oyster mushroom. Oyster mushrooms and fungi are both effected ab y environment and chemical factors there by the testing should be further based on the effects of the changes of environment or chemical concentration change.

Figure 1: The effect of environmental water change and its dehydration with oyster mushrooms.

Identification of a research gap

There is limited research in a large factor of the biotechnology of enzyme production with relation to production rates (such as phosphorylase and cellulase). Indicating that the point of research gap, indicated through the lack of enzyme experimentation on both the qualitative growth and consumption rate of the substrate as well as the spore yield and density. Hence the purpose of this experiment is to investigate this, in which it hopes to see if there is an effect in the growth rate and spore density when it is synthetically introduced with cellulase, providing information in whether this could be used as a viable biotechnology in growing mushrooms more efficiently.

Figure 2: Mushroom life cycle.

Figure 3: A mushroom spore print

Figure 4: mushroom spore print example

Scientific Research Question

What is the effect of synthetic introduction of the cellulase enzyme on the fungal spore density, observable characteristics, and overall growth rate of oyster mushrooms?

Scientific Hypothesis

Primary: The introduced cellulase enzyme will increase the fungal spore density, indicated by the higher density and colouration of the spore print.

Secondary: The growth rate of the oyster mushrooms will increase with the introduced cellulase, indicated by the higher percentage of weight loss in the mushroom bags containing cellulase.

Methodology

Experimental design

The experiments were conducted in the science extension lab at Barker College in the winter of 2022 (May to June). The experiments were arranged in a randomised complete design with 4 replications per test. Before starting the experiment, safety equipment was worn, such as gloves and eye protection as well as a mask (prevention of contamination control), as well as priming two large jugs of 10L capacity heating the water over pasteurisation temperature (80C), this also included the buying of cellulase solution from sigma Store.

Pasteurisation of substrate

The sugar cane mulch was put in a 50-litre capacity esky in which it was wet down with water (approximately 8 litres). In which afterward 250 millilitres hydrated lime was carefully added on the top of the mixture. The hydrated lime was mixed into the substrate until completely dissolved (cannot see hydrated lime powder). In which the substrate hydrated lime mixture was then put evenly in two separate pillowcases to strain the water. In which place both pillowcases in a suitably sized esky that can fit the two pillowcases comfortably (pillowcases can fit without any added pushing or force rather than closing the lid). In which once the water reached 80C by the jug, the water was then poured in the esky with the pillow cases until the pillow cases where fully submerged. Once the pillowcases were fully submerged the esky was then closed. After 2 hours the esky was then opened in which each pillowcase was then moved to a sink to then drain. One drained the pillowcases of substrate where then moved to an unstainable tabletop in which the substrate was then taken out of the bag and onto the table and spread out around the table to cool to at around 35 degrees celsius. The substrate was then spread out along a sterilised bench top (about 3 inches of substrate depth).

Setting up for the growing of mushrooms

The growing of the mushrooms consisted of the materials said above. 5 kg of sugar cane mulch was sterilised in a hydrated lime solution in which was placed in the solution for 2 hours. After which the mulch was put in 10 separate sterile spawn bags (evenly) in which the mulch was then settled in the bag. That in which the oyster mushroom fungal spores were then placed on a separate equal container. This in which was then weighed to make sure each container contained the same number of fungal spores (controlled). Each container of spores was then put into the mulch filled spore bags and then mixed through evenly (mixed with gloves on to prevent contamination through oils of hands).

Inoculation of the substrate

Taking the bag of the broken-up spawn, it was then sprinkled along the length of the cooled substrate until spawn is used and evenly distributed in the substrate the substrate was then mixed well (to ensure even distribution of spawn in substrate control). In which the inoculated substrate was then put into mushroom bags (5 handfuls per bag), then put under the sink with constant temperature.

Setting up greenroom environment

The green room was sterilised and cleaned using alcohol wipes. Ensuring that the temperature was approximately 25-28 degrees (as is the optimum temperature), an automatic humidity sensor dispensing water vapor every 4 minutes was introduced. In which once the spawn showed the visual development of the hyphal knot stage, the mushroom bags were then put in the greenhouse environment.

Cutting the mushroom bags

Once there was visible indication of the next phase of the fungal growth and development (pinhead stage seen in fig 5). The bags were cut with 4cm length crossing directly above the pin head areas. lines to allow for growth outside of the mushroom bags, in which after 5 cuts were made on each bag, they were then put back in the green house until about a 2 cm of full body mushroom growth was found in each bag.

Mixing cellulase solution

Gloves were worn throughout dealing to prevent contamination with oils as well as breather directly over it was prevented to also prevent contamination of the cellulase and the substrate. The cellulase solution was removed from a refrigerator unit whilst still in the bag and was then put in a ratio 0f 5ml of cellulase per 200ml of distilled water. This in which the mixed solution was put in a spray bottle and then refrigerated until use.

Post oyster mushroom fruiting

Once each mushroom bag had oyster mushrooms over 2 cm in length, the mushroom bags were matched with similar size and shape of the developed mushroom outside. This in which each paired bag was separated to a control group and a test group either test a or b was random choice as the container could only fit two and there were four containers). Everyday afterwards, the diluted cellulase was then sprayed on the test mushroom bags (TMB).

Spore printing

After a three-week period of the mushrooms being sprayed with the enzyme solution, the oyster mushroom was then cut 0.5cm at the stalk from the pileus in which was then placed gills down in the center of a black piece of paper this in which each piece of paper with the mushroom was then covered with a sterilized container (sterilised using pasteurization).

Data analysis

Mushrooms were cut (same amount cut from each bag 1). Each mushroom was put on a piece of paper (the paper was marked as either control or test, test was the mushrooms with the introduced cellulase, and control was the mushrooms without introduced cellulase). Each mushroom was given a number to identify both the mushroom and their spore print (seen through figure 4 as an example of a spore print). Spore prints were measured qualitatively out of ten, in which the mushroom with the largest spore prints were given a score of 10 (10 being the largest spore print and 1 being the smallest). This similarly to the spores developed on the mushrooms itself and the way they were measured (10 being the largest number of developed spores on the mushroom, and 1 being the lowest developed spores on the mushrooms). However, the bag weight was measured quantitatively over a 15-day period and weight was recorded every 5 days (measured in grams).

Discussion and Results

Table 1: Bag weight loss over 15-day period C= control, T= test, N= not used in experiment. (1 or 2 is group)

Bag number Group (TCN) Weight (g) Day 0 Weight (g) Day 5 Weight (g) Day 10 Weight (g) Day 15 Weight Loss (g) Day 15

Percentage Weight Loss (%)

1 C1 1071.63 993.6 983.5 976.52 95.11 8.8 3 T2 910.41 870.26 820.66 790.25 120.16 13.2 5 N 875.02 820.94 806.92 782.13 92.87 10.6 6 C2 779.2 753.45 732.93 718.61 60.59 7.8 7 C1 959.41 923.46 903.83 873.73 85.68 8.9 8 C2 841.58 809.93 788.5 765.13 76.45 9.1 9 T2 1002.19 930.67 907.98 866.67 135.52 12.5 10 N 941.51 905.63 880.57 829.91 111.6 11.9

Mushrooms require basic food and other nutrients to grow. Oyster mushrooms are no exception. To cultivate and grow oyster mushrooms in a laboratory, the compounds and nutrients must be provided for their growth and other life processes. In the current study a specific strain of oyster mushrooms (PO) was successfully grown to whole body mushrooms in the laboratory scenario.

The experimental design was based on the idea of both the many testable features of mushrooms in the experiment such as weight spore density. This partnered with the large range and use of both the qualitative and quantitative data that will be retrieved i.e., the spore print and spore density as well as the bag weight (quantitative). This in which caused the creation of the secondary hypothesis of bag weight to then provide other forms of data showing the benefit of the use of cellulase. Another influence in the experimental design

Due to the mushrooms only being able to take nutrients through the mushroom bags, the mushroom growth rates were recorded with the weight loss from the mushroom bags. This is due to the mushrooms structure, being their use of aerobiotic respiration meaning that it releases waste in air and ground. This is due to the fungi’s myselial structure in which all fungi and mushrooms share in which is used in the mushroom anatomy to then make the nutrients into a nutrient rich liquid which then can be more efficiently absorbed this in which is then secreted from. In which the then waste of the liquid is both evaporated and secreted outside of the mushrooms as well as inside PO’s environment to then be re absorbed. Their process of aerobic respiration as well as require nutrients for other chemical and cellular reproduction (e.g., mitosis) and release waste and other products throughout the growth cycle due to the chemical processes, the bag's overall weight will be reduced as no new substrate is being added. The mycelium absorbs the sugar cane mulch for nutrients for their chemical processes. The mycelium/mushrooms produce waste with their chemical processes. Products from the waste can only originate from the substrate (due to the only nutrients available for the mushrooms in the mushroom bags being the sugarcane mulch substrate). It was consuming the nutrients from the sugar cane mulch and releasing waste products outside the bags. During the investigation, half the mushroom bags were sprayed with diluted cellulase enzymes (5mL of cellulase per 200mL of distilled water sprayed nine times) to identify its effects on the mushroom's growth rate and sporification yield.

Data from Table 1 and Figure 4 showed a weight loss difference between the oyster mushrooms sprayed with cellulase and without (an average 30-gram weight difference between the two). The average weight reduction between the control mushroom bags (CMB) and the test mushroom bags (TMB) ranged between 60.59 and 135.52 at 15 days after the spraying of cellulase was initiated. The mushrooms sprayed with cellulase showed an apparent higher weight reduction, CMB showed an average of 8% weight loss compared to a TMB showing an average of 12%. (table 1 fig 4) This is due to the mushrooms with introduced cellulase having a significantly higher ability to break down sugars faster as they have a higher count of cellulase enzymes in the mushroom body. As indicated similarly by B. Adney and J. Baker (2008) [6], cellulase concentrations were able to break down glucose concentrations. Indicating that higher amounts of the enzyme (cellulase) were able to break down the quantities of glucose faster. Showing cellulase breaks down the glucose. As well as showing similar results to Meidute, S., et al (2008) showing results of increase in respiration and absorption of complex carbon sources like glucose or enzymes, showing the effect of it through respiration being tenfold for the

mushroom with introduced complex carbon sources to the control.

Effect of introduced cellulase on the spore density with both the spore print and spores on mushrooms.

Fungal spores are shown to be a vitally important aspect of growing any mushrooms. It is like a seed of a plant. The research investigated the effect of the introduced cellulase on the oyster mushrooms' spore production, density. This in which was measured using the same CMB and TMB mushrooms. The mushroom caps were cut on day 15 from both CMB’s and TMB’s. The cut mushroom caps left on spore paper for three days. In which the TMB caps showed a clear indication of more densely packed with spores (table 2 and fig 5), as shown with the scoring system (described in the method). The TMB oyster mushrooms showed a clear indication of higher spore density on the mushroom and on the spore paper (table 3 and table 2). The score was shown to be an average of 3 for the CMB caps section and an 8 for the TMB caps for the spores on the mushrooms. They also showed that the TMB caps had a much faster and higher density of spores produced on the spore paper on the mushrooms than CMB over the three days showing visible spore prints on the first day, except for one case. This in which the qualitative measurement showed a similar difference being on average for the CMB to be 5/10 and the TMB caps spore score to be an 8/10. Initially, this was considered the result for the question. As cellulase was demonstrated as one of the leading products in mushroom spores’ pre-germination (1x10-3 units of cellulase per spore) (Hagerman et al 1985).

The growth rate in both cases can be seen with the large differences in the weight loss percentage averages with the CMB and TMB, showing a 4% difference (30 grams difference as an average) and the spore scores tests, which indicates higher density and yield (spore paper - test score 8 whilst control score 3). This directly supports the hypothesis as it indicates that the use of cellulase can be used to increase both the growth rate (seen in Figure 9) of the mushrooms and the spore density.

Figure 5: column graph weight loss per bag over the 15 days as well as the test section and control average

Figure 6: Column graph of all the percentage weight losses averages.

Figure 7: Spore prints of mushrooms

Table 1: Spore print ranking test and control data

Spore print number Score out of ten (1 smallest spore print 10 largest)

Test 1 4

Test 2 10

Test 3 8

Test 4 9

Test 5 6 Control 1 1 Control 2 2 Control 3 3 Control 4 7 Control 5 5

Figure 8: The spores developed on the mushrooms as shown. The number indicating the specific mushroom cap.

Figure 9: On the left is the control and on the right is the test as shown much larger amounts of stems and larger in size for the test group

Limitations

The experiment's limitation is mainly introduced by the lower amounts of repetition, only having four for each case (being the control and the tests). This means the issue of repetition may challenge. This partnered with the reduced repetition of only using 5 for both tests and controls causes an issue with reliability as the five may have been outliers.

Table 2: Spores developed on the mushrooms measured qualitatively and given scores for each

Mushroom number Scores

Test 1 6 Test 2 9 Test 3 8 Test 4 10 Test 5 7 Control 1 4 Control 2 5 Control 3 1 Control 4 3 Control 5 2

Further research

More research should be conducted on the complete mushroom life cycle to show more in-depth findings on the effect of cellulase on the spore yield, density, and growth rate. In addition, a greater variety of mushroom breeds and amounts of cellulase concentrate to distilled water would deepen the research. Combined with more accurate equipment, such as a constant stable temperature environment in a controlled greenhouse and a more accurate humidifier to have a more constant humidity percentage should be considered for further research.

Conclusion

The results supported the primary and secondary hypotheses. The primary hypothesis stated that the introduced cellulase enzyme would increase the fungal spore yield, indicated by the higher density and colouration of the spore print on paper as well as the spore growth of the mushroom itself. This is seen clearly through the qualitative results of both tests showing the test score average to be higher than the control score average. The secondary hypothesis stated that the growth rate of the oyster mushrooms would increase with the introduced cellulase, indicated by the higher percentage of weight loss in the mushroom bags sprayed with cellulase. This is demonstrated to be supported with the results of the bag weight loss over a period, showing the percentage weight reduction average to be 4% higher in the TMB’s than the CMB’s average. As the data supports the hypotheses, it is clear to indicate that the enzyme cellulase does in fact, positively affect both the growth rate and the sporification yield and density of oyster mushrooms. This in which could show a new way of farming that decreases the growth cycle of mushrooms, as well as increases yields per growth cycle.

Acknowledgements

Thank you to Dr Alison Gates as supervisor of the project. Thank you for help with the experiment and countless hours of feedback and encouragement.

Thank you to Dr Matthew Hill for help in both statistics and format and structure of the report.

Thank you to Mrs Virginia Ellis for assisting me with issues of heating and experimentation.

Thank you for Dr Terena Holdaway-Clarke for facilitating open hours in the lab for me to conduct the research and helping me with the practical side of the assessment in moving the mushroom bags around and helping weigh the mushroom bags.

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