Pa ge 1 Am. J. Environ. Clim. 1(1), 2022 The American Journal of Environment and Climate (AJEC) is a blind peer-reviewed journal, that aims to publish the article(s) immediately after submission of the corrected version by the author. The journal publishes articles that include those containing substantially supported theories, innovative works, substantial experimental results, and/or containing useful and constructive discussions or reviews standardized to regional or international acceptance. The AJEC reviews manuscript within approximately one month of submission and publishes accepted articles on the internet immediately upon receiving the final versions. Frequency: Three issues per year Area of publication: Environmental Science & Technology, Environment Pollution, Policy on Environment and Climate, Climate Change, Sustainable Development. Editorial Team Dr. Muhammad Farhad Howladar Department of Petroleum and Mining Engineering Shahjalal University of Science and Technology Sylhet, Bangladesh Professor Anand V. Shivapur Department of Civil Engineering Visvesvaraya Technological University Belagavi, Karnataka state, India Dr. Sofia Binte Ehsan Institute of Climate Change, Universiti Kebangsaan Malaysia, Malaysia Dr. Velichko Dimitri Vladimirobitch Russian State Geological Prospecting University, Russia Dr. K. J. Sreekanth Research Scientist, Energy Efficiency Technologies Program (EET) Energy and Building Research Center (EBRC) Kuwait Institute for Scientific Research (KISR) Safat, Kuwait Dr. Mohammad Tofayal Ahmed Department of Petroleum and Mining Engineering, Jessore University of Science and Technology, Bangladesh Dr. Asif Rayhan National University of Malaysia, Malaysia Dr. Md. Mahfuzul Islam Institute for Environment and Development Univeriti Kebangsaan Malaysia, Malaysia Ar. Sajal Chowdhury Department of Architecture Chittagong University of Engineering & Technology (CUET), Bangladesh Dr. Parnuwat Usapein Acting Director of Rattanakosin College for Sustainable Energy and Environment Rajamangala University of Technology Rattanakosin (RMUTR), Thailand Dr. Vikrant Katekar Research Scholar, Indian Institute of Technology, Mumbai, India Dr. Sheikh Tawhidul Islam Professor & Director, Institute of Remote Sensing Jahangirnagar University, Bangladesh Contact: American Journal of Environment and Climate (AJEC) E-Palli LLC Address: 2055 Limestone Rd Ste 200C, Zip Code: 19808 Wilmington, Delaware, USA Email: ajec@e-palli.com Phone: +1 3024070506 URL: https://journals.e-palli.com/home/index.php/ajec https://journals.e-palli.com/home/index.php/ajec/about/submissions mailto:mailto:ajec%40e-palli.com?subject= https://journals.e-palli.com/home/index.php/ajec Pa ge 1 American Journal of Environment and Climate (AJEC) Growth and Yield Performance of Oyster Mushroom Cultivated in Combined Cassava Peels, Coconut Residue and Coffee Waste Substrates Irish B. Elsisura1*, Mary Amor G. Figueroa2 Volume 1 Issue 1, Year 2022 ISSN: 2832-403X (Online) DOI: https://doi.org/10.54536/ajec.v1i1.206 https://journals.e-palli.com/home/index.php/ajec Article Information ABSTRACT Received: March 22, 2022 Accepted: April 1, 2022 Published: April 8, 2022 The oyster mushroom (Pleurotus ostreatus) is an edible mushroom that belongs to the class of Basidiomycetes. It has reached sufficient market maturity because of its flavor, shelf-life durability, and protein and fiber content. Besides their nutritional, medicinal, and economic value, they may help the country’s agricultural waste management, bridge environmental issues, and contribute to climate change resolution advancements. A study on different varieties of agricultural substrates derived from waste materials such as cassava peels, coconut residue, and coffee waste was investigated and compared to sawdust, the common substrate for oyster mushrooms. The effects of different substrates on the morphological characteristics of P. ostreatus, percent contamination, and yield parameters were recorded and analyzed using the Analysis of Variance in Completely Randomized Design, and their significant results were compared using Tukey’s HSD. Results showed that different substrate mixtures did not significantly influence the morphological characteristics of P. ostreatus. Moreover, sawdust, the common substrate for oyster mushrooms, showed the lowest percent contamination as compared to other substrate mixtures. Contaminants found in cassava substrates include Trichoderma spp., Aspergillus spp., Fusarium spp., Neurospora spp., and Penicillium spp. 80% of cassava peels combined with 10% coconut residue and 10% coffee waste significantly increased the number of fruiting bodies and produced the heaviest fresh weights of oyster mushrooms. Stipe length and pileus diameter were also significantly influenced by this substrate mixture, which is comparable to the common substrate. However, further research on the varying proportions of these substrate mixtures on the performance of oyster mushrooms is recommended. Keywords Cassava peel, Coconut Residue, Coffee waste, Oyster mushroom, Growing media 1 Student Researcher of Surigao State College of Technology, Mainit Campus, Magpayang, Mainit, Surigao del Norte, Philippines 2 Faculty Adviser of Surigao State College of Technology, Mainit Campus, Magpayang, Mainit, Surigao del Norte, Philippines * Corresponding author’s e-mail: ielsisura.tc@ssct.edu.ph INTRODUCTION Oyster mushrooms have reached sufficient market maturity because of their delightful flavor, durability in shelf life, extraordinary protein and fiber content, and nutritional and medicinal features. These kinds of mushrooms can establish and degrade a variety of lignocellulosic substrates and other wastes, which are produced primarily through the activities of the agricultural, forest, and food-processing industries. Because of the more favorable climate conditions in Southeast Asian countries such as the Philippines, growing one’s food poses few to no challenges. The country’s main crops are rice, corn, coconut, sugarcane, bananas, pineapple, coffee, mangoes, tobacco, and abaca. Secondary crops include peanuts, cassava, sweet potatoes, garlic, onion, cabbage, eggplant, lime, lemon, rubber, and cotton. These are all export-quality products that the country is most proud of. But with the assortment of waste materials it produces, it piles it up to the extent of harming the environment itself, which supplies all of it, ironically. But the ballooning effect of our population reflects the agricultural and waste management of our country. Today, only a part of this waste is used, mainly as provisions for goats and other pastured animals. In many regions where cassava is cultivated and processed, volumes of cassava peels are dumped along the roads, especially in wet seasons, and can generate offensive odors and unsanitary conditions. A valorization of cassava waste would not only resolve this environmental predicament but also contribute to the local economy. The coconut residue generated after milk extraction is a food waste by-product, but only a fraction of it is used as fertilizer or cow feed. As a result of these activities, the by-products can contribute to the additional trash in the community. By-products from coffee beans are useful substrates for the cultivation of P. ostreatus in coffee-producing countries like the Philippines because this species is fast-growing, substrates are easily accessible, and the mushrooms (or fruiting bodies) are a beneficial source of nutrition and revenue. The researcher recognized the fact that there was not enough identified data that involved the previously indicated substrate combinations in developing P. ostreatus, although individual studies have shown that these substrates are effective in growing oyster mushrooms as previously mentioned. P. ostreatus is uncommon in our area, and the researcher was strongly convinced that the need to discover and innovate ways to cultivate it was significant. It was anticipated in this research to compare and contrast the yield performance of oyster mushroom (P. ostreatus) cultivation in a rural setting using different agricultural waste materials such as cassava wastes, coconut residue, and used coffee grounds, along with a constant percentage of supplementations from rice bran, limestone, and molasses and sawdust as common substrate. The researcher compared five https://doi.org/10.54536/ajec.v1i1.206 https://journals.e-palli.com/home/index.php/ajec Pa ge 2 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 substrate mixtures based on the yield performance in the production of oyster mushrooms. Subsequently, the study determined which of these substrates produced the most viable yield. Through this study, Philippine households and farmers, particularly those in Mainit, Surigao del Norte, can have the opportunity and means to learn more about cultivating edible mushrooms such as oyster mushroom. Making these concepts a reality on a micro-to-commercial scale can suit the people’s nutritional demands, not only as a supplement but also with profitable future engagements. It can also provide an opportunity for everyone with the resources to address and contribute to the country’s current understanding of agricultural waste management not only ecologically but also economically. LITERATURE REVIEW For numbers of years, oyster mushroom (P. ostreatus) has displayed as one of the most cultivated mushrooms in the world, largely in Brazil (Lee et al., 2002; Sánchez, 2010; Royse, 2013). Pleurotus species’ nutritional value, comprises high protein of 25-50 %, nine essential amino acids, and very low 2-5 % fat content, which makes it fitting as a diet food for health-conscious individuals. The sugar content is reasonable and ranges 17-47%, including minerals such as calcium, potassium, sodium with vitamins such as niacin, riboflavin, vitamin B1, B5, B6, C, and D (Caglarirmak, 2007; Syed et al., 2009). In addition, oyster mushrooms are good source of extraordinary stamina and vigor, and they are utilized in the manufacturing of many continental recipes. They also have medical properties such as anti-cancer, anti-cholesterol, and anti- tumorous. Mushrooms can help with diabetes, ulcers, and lung illness (Quimio, 1976). Mushroom protein is a combination of animal and vegetable protein (Kurtzman, 1976). Mushrooms are also potent in Niacin, Pantothenic acid, and Biotin (Subramanian, 1986). The good thing is, we can easily grow it on agricultural and industrial waste. More than half of the overall yield from the land is wasted as straws, leaves, stalks, roots, and so on (Zadrazil, 1978). These pollutants can be repurposed into food, and the environment may be less polluted as a result (Hayes, 1978). The population of the Philippines is climbing to 110,623,413 (as of Sunday, March 21, 2021; based on web). With this increase in population is the increase in the demand for food and agricultural products. As a result, it had been estimated that only about 50% of the city household’s solid wastes are pulled by garbage collectors, while 38% of the households incinerate their garbage, and a further 12% of it is dumped in vacant lots (Holmer et al., 1999). The bigger portions of the solid waste that are being dumped at the city landfill site are from private households (54%) and commerce and institutions (28%). About 40 to 50% of the city wastes are biodegradable. However, if organic waste is properly processed to form compost, it can be put to good use in urban agriculture and horticulture as a fertilizer and soil improver. Farmers and gardeners can benefit from it diligently. The use of organic waste provides a lasting increase in the waste control situation in cities. However, this assumes that urban waste management is integrated consistently with urban horticulture and agriculture (Guanzon & Holmer, 2015). In Thailand, they can easily grow oyster mushrooms (P. ostreatus) in local conditions if the appropriate specifications of food and moisture for growth are available (Shah et al., 2004). Pleurotus ostreatus demands few environmental controls, and their fruiting bodies are not often attacked by diseases and pests, and they can be cultivated simply and cheaply. All this makes P. ostreatus cultivation an excellent alternative for the production of mushrooms when compared to other mushrooms (Sánchez, 2009). Cassava (Manihot esculenta) belongs to the higher classification Manihot; and in order Malpighialesis, which is a dominant food crop for approximately 700 million people, especially in African countries. A substantial quantity of waste is usually produced throughout the processing, mainly consisting of tuber peels (Sonenberg et al., 2015). Previous reports on the utilization of cassava waste for the cultivation of P. ostreatus and P. pulmonarius have shown that yields were reasonable (Adebayo et al., 2009; Onuoha et al., 2009; Obodai et al., 2014). Cassava by-products as substrates can give yields of up to 100 percent BE of oyster mushrooms which compare well to traditional substrates such as sawdust (Frimpong-Manso et al., 2011). Cassava-based composites thus have the potential to be economically profitable for the production of oyster mushrooms (Sonnenberg et al., 2015). Coconut palm (Cocos nucifera L.) is a tropical plant that belongs to the Kingdom Plantae, division Magnoliophyta, class Liliopsida, order Arecales, and family Arecaceae. The adult de-husked coconut generates 50% wet meat or core, 33% shell, and 17% water. Raw coconut meat has a chemical composition that contains about 3.33 percent protein, 33.49 % total fat, 15.23 % starch, 3.23 % total fiber, and a range of minerals and vitamins. The meat of the coconut kernel is used to make coconut milk in mostly Asian counties like the Philippines. Fresh coconut kernel is finely shredded and hand-squeezed or expeller pressed to produce coconut milk. As a result, a significant volume of coconut waste by-product after squeezing out milk is razed to the ground as waste (Sopit, 2007). Coffee grounds have been investigated as a potential remnant for mushroom growth. Coffee grounds’ chemical and structural properties enable for the reuse of lignin (23.90 g/100g dry product), nitrogen (12.79 g/100g dry product), and dietary fiber (60.40 g/100g dry product). The storage capacity of water is influenced by the particulate matter produced by coffee pressing (Ropciuc et al.,2016). Oyster mushroom cultivation on SCG for human consumption is totally possible, and there is significant potential for this extensive form of waste to be utilized and the caffeine content reduced. (Cabrera, 2018). Lime (CaCO3) is a key component in mushroom development; commercial mushroom production is dependent on correct substrate pH adjustment. (Khan https://journals.e-palli.com/home/index.php/ajec Pa ge 3 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 et al., 2013). Furthermore, molasses has been shown to promote the growth of several bacteria. Molasses contains sugar, nitrogen, and other nutrients that aid in cell development. As a result, it produced good results in the production of oyster mushrooms. (Erkel, 2009). Rice bran is clearly the most widely used and widely available organic component in substrates for the production of a wide range of edible mushrooms (Peng et al., 2000) MATERIALS AND METHODS Table 1. Research material, equipment, and raw materials used. Materials Equipment Raw materials Metal strainer Improvised mushroom bag sterilizer Cassava peels Cheesecloth Shovel Coconut residue 6x12 inches Polypropylene cellophane Shredder Coffee waste Empty bottle Pressurized sprayer Sawdust PVP pipe (2.0 cm thick) Triple burner gas tank Limestone Cotton wool Water drum Molasses Rubber bands Rice bran Soap Water Bleach Small-eyed net Basin Tarpaulin Black cloth Pail Spoon 70% Alcohol Denatured alcohol Denatured alcohol lamp Sacks LED light bulb The following materials, equipment, and raw materials were used in the study (Table 1). Acquisition of Pleurotus ostreatus Grain Spawn and Substrate’s Raw Materials The oyster mushroom spawn was procured from a commercial laboratory located in Taguibo, Butuan City. Different raw materials needed for preparing substrates for the study were gathered from the local area in Mainit, Surigao del Norte. Specifically, cassava wastes and coconut residue were from the farmers and neighborhood within Mainit, Surigao del Norte. The coffee-pressed grounds were collected from the nearest coffee shop within Surigao City, Surigao del Norte. Preparation of Mushroom Substrates Cassava wastes. Fresh cassava wastes were carefully cleaned with water, packed into nylon sacks and were submerged in water for three days to mimic natural fermentation. After three days, the fermented cassava wastes were drained for a day, and sun-dried for two days using a net or a table. The sun-dried cassava wastes were shredded using a shredder. When the desired texture and sizes were attained, it was then packed in a clean sack and stored in a dry place. Coconut residue. Finely shredded fresh coconut meats were soaked in warm water. The soaked shredded coconut meats were hand-squeezed and pressed through a metal strainer, and cheesecloth. In order to get lighter coconut milk, the process was repeated once or twice. After the milk extraction, the coconut leftovers were sun-dried for at least two days. The dried coconut residues were stored in a cool and dry place. Coffee grounds. Coffee-spent grounds that were gathered from a coffeehouse were dumped in hot water to eliminate chemicals that might limit fungal development and trigger contamination. To eliminate the extra moisture, cheesecloth was used to softly pressed it to allow any leftover moisture to travel through. The coffee waste was left to cool off in a room temperature for about a day or two. It was placed in a dry container and stored in a cool and airy corner. Building and Maintenance of Mushroom House The mushroom house was four meters from floor to top part, and with an area of four-square meters. It only has one door, no window, and was made of bamboo matting with a nipa roof. The mushroom house was cleaned thoroughly in its entirety by using pressurized water hose with soap, and bleach to ensure the removal of almost every unwanted bacterium or foreign biological substance that could alter the growing process and was regularly sanitized. Preparation and Bagging of Mushroom Substrates Recently prepared substrates were packaged in a 6x12 https://journals.e-palli.com/home/index.php/ajec Pa ge 4 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 inch polypropylene cellophane bags containing 500 grams each based on the different substrate mixtures (Table 2). The bottom ends of the bags were folded to allow them to stand on their own. As additional compost was added to the ultimate weight required, an empty bottle was used to compress it. To act as a bottle neck, a 2.0 cm thick and 2.5 cm long PVC pipe was put at the neck of each bag. A piece of cotton wool was inserted into the bags’ necks then covered with paper, and was secured with a rubber band. Then, it was labelled carefully and accordingly before proceeding to the next step. Sterilization of Bagged Substrates Bagged substrates were sterilized for six hours using the improvised mushroom bag sterilizer, fired with a triple burner stove. The sterilized bags were then removed from the improvised mushroom bag sterilizer to allow the second batch to be accommodated. After sterilization, the substrates were allowed to totally cool-off for six to ten hours before spawn inoculation. Table 2. Experimental treatments. Treatment Numbers Oyster Mushroom Substrate Mixtures Total Major Substrate Ingredients (78%) Supplemental Ingredients Sawdust (Control) Cassava peels Coconut residue Coffee waste Rice bran Molasses Lime- stone 1 100% - - - 20% 1% 1% 100% 2 - 100% - - 20% 1% 1% 100% 3 - 80% 10% 10% 20% 1% 1% 100% 4 - 70% 20% 10% 20% 1% 1% 100% 5 - 50% 40% 10% 20% 1% 1% 100% Grain Spawn Inoculation to Sterile Bagged Substrates The oyster mushroom grain spawn was inoculated to the sterile bagged substrates in a biosafety cabinet. The grain spawn was loosened first before inoculation by shaking it. Bags were opened carefully, grain spawn were aseptically inoculated to the substrates at one tablespoon per bag, and then was covered again for incubation. Incubation of Inoculated Fruiting Bags The inoculated fruiting bags were arranged properly by treatments and replication, then they were placed in a well-ventilated dark area, covered in black cloth, and layered by tarpaulin or any plastic material to prohibit the direct light to our fruiting bags, until fully colonized. Fruiting bags were regularly monitored for colonization period. Hanging of Fully-colonized Fruiting Bags After colonization, the bags were sorted and hung using a rope and a wire as a lock to the thread. To depict each replication, fifteen mushroom fruiting bags were arranged in a row of every treatment piled at one foot apart. These were then regularly monitored until the appearance of pinheads’ formation. Management of Fruiting Bags Spraying water at the back portion of the fruiting bags were done twice a day, at nine o’clock in the morning and three o’clock in the afternoon. The fruiting bags were enclosed in a net to avoid insect and pests’ infestations. The floor was completely covered with a damp cloth to keep the mushroom house’s humidity level stable. The time pinheads were observed, the cotton and paper cap of the fruiting bags were removed to allow the fruiting body to develop. During the stage of fruiting body formation, the room was illuminated with diffused light. Bags were checked for contamination on a daily basis. To prevent the spread of contaminants, defective bags were disposed or eradicated as soon as they were observed. Contaminations were identified based on their morphological characteristics and were compared to published and identified common contaminants of oyster mushrooms. Harvesting of Oyster Mushroom Fruits Harvesting was done when the cap of the oyster mushroom reached its maturity with its maximum diameter and in convex shape, by hand picking. Fruiting bodies were twisted and pulled carefully from the base of the stem to avoid contamination. Data Gathered During the course of the study, the following parameters were recorded. 1. Morphological characteristics. This parameter includes period of colonization or spawn run, pinhead formation, fruiting body formation and full maturity of oyster mushroom. a) Days from Spawning to Complete Spawn Run (S-CSR). This refers to the number of days the substrates were fully colonized by the fungus from the day of inoculation. b) Days from Spawning to Pinhead Formation (S-PF). This pertains to the number of days the fungus produces pinheads’ formation from the day of inoculation c) Days from Pinhead Formation to fruiting body formation (PF- FBF). This refers to the number of days the fruiting body was observed after the pinhead formation, and d) Days from Fruiting Body Formation to Full Maturity (FBF- FM). This represents the ideal number of days to harvest after fruiting body formation. 2. Percentage Contamination. It refers to the percentage of discarded fruiting bags due to contamination in every treatment. This was computed using the formula: https://journals.e-palli.com/home/index.php/ajec Pa ge 5 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 3. Harvest Data. This parameter includes number of fruiting bodies per treatment, average mycelia height, average mycelia diameter, and weight of fresh fruiting bodies of oyster mushroom on different substrate mixtures. a) Number of fruiting bodies. Immediately every after harvest, fruiting body was counted by determining the caps of the fruits. b) Average stipe height. It was gathered by measuring the tip of the stem or the stipe of the oyster mushroom to the base of the cap. A ruler was used and was expressed in centimeters. c) Average pileus diameter. The pileus or the cap of the oyster mushroom was measured across using a ruler and was expressed in centimeters. d) Weight of fresh fruiting bodies of oyster mushroom. It was gathered every after harvest. Electronic balance was used to gather the data and was expressed in grams. Statistical Analysis The study was analyzed using Analysis of Variance (ANOVA) in Completely Randomized Design and the differences among treatment means were compared using Tukey’s Honest Significant Difference (THSD) as Post- Hoc analysis. RESULTS AND DISCUSSION Morphological Characteristics of P. ostreatus Grown in Different Substrate Mixtures Days from Spawning to Complete Spawn Run (S-CSR). The colonization period of oyster mushroom using different substrate mixture is presented in Table 3. Results of the Analysis of Variance (ANOVA) showed no significant differences. Results of the study showed that 100% cassava peel has the shortest colonization period (33.33 days from inoculation), which was followed by 80% cassava peels + 10% coconut residue + 10% coffee waste with 34.67 days of colonization period, and 70% cassava peels + 20% coconut residue + 10% coffee waste with 35 days of inoculation. The longest period of colonization was recorded in substrate mixtures of 100% sawdust (Control) and 50% cassava peels + 40% coconut residue + 10% coffee waste with both 35.33 days. These results suggest that different substrate mixtures did not influence the period of colonization of P. ostreatus. Days from Spawning to Pinhead Formation (S-PF). The pinhead’s formation period of oyster mushroom using different substrate mixtures is also presented in Table 3. Same results of the ANOVA were recorded showing no significant differences among treatment means. Shortest pinhead formation was recorded in substrate mixtures with 100% cassava peels with 37.33 days. Eighty percent cassava peels + 10% coconut residue + 10% coffee waste and 70% cassava peels + 20% coconut residue +10% coffee waste have shorter period of pinhead formation with both took 38.00 days from inoculation. These were followed by the control (100% sawdust) with 38.67 days of pinhead formation. Substrate mixture with 50% cassava peels + 40% coconut residue + 10% coffee waste registered the longest number of days of pinhead formation with 40.00 days from inoculation. However, general results of this parameter suggest that these substrate mixtures did not significantly influence the pinhead formation of P. ostreatus. Days from Pinhead Formation to Fruiting Body Formation (PF- FBF). The period of fruiting body formation of oyster mushroom using different substrate mixtures is presented in Table 3 as well. ANOVA results showed no significant differences among treatment means. Data showed that whatever substrate mixtures used, there is no differences in period of fruiting body formation which only took three days from pinhead formation in all substrate mixtures. Days from Fruiting Body Formation to Full Maturity (FBF- FM). The time taken from fruiting body formation to pin maturation is also presented in Table 3. Results of the ANOVA revealed no significant differences among treatment means. Results of the study showed that all substrate mixtures used had no significant influenced on the period of full maturation of P. ostreatus which only took three days from fruiting body formation in all substrate mixtures. Generally, results imply that different substrate mixtures used in the experiment do not influence the colonization period, pinhead formation, fruiting body formation, and full maturation period of P. ostreatus. These findings could be attributed to the cellulose and lignin properties of both cassava peels and sawdust (Frimpong-Manso et al., 2011) (Horisawa et al., 1999). The results also confirmed Stanley & Nyenke’s (2011) findings that cassava stimulated luxuriant mycelial growth rate and extension Table 3. Morphological characteristics of oyster mushroom in different treatments. Substrate Mixtures S-CSR 1,ns S-PHF 2,ns PHF-FBF 3,ns FBF-MP 4,ns 100% Sawdust (Control) 35.33 38.67 3 3 100% cassava peels 33.33 37.33 3 3 80% cassava peels + 10% coconut residue + 10% coffee waste 34.67 38.00 3 3 70% cassava peels + 20% coconut residue + 10% coffee waste 35.00 38.00 3 3 50% cassava peels + 40% coconut residue + 10% coffee waste 35.33 40.00 3 3 CV (%) = 2.68 2.52 0.00 0.00 https://journals.e-palli.com/home/index.php/ajec Pa ge 6 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 can be comparable to that of a commonly used substrate such as sawdust. ns=not significant 1 =S-CSR (Days from Spawning to Complete Spawn Run) 2 =S-PHF (Days from Spawning to Pinhead Formation) 3 =PHF-FBF (Days from Pinhead Formation to Fruiting Body Formation) 4 =FBF-MP (Days from Fruiting Body Formation to Full Maturity) Days from Spawning to Pinhead Formation (S-PF). The pinhead’s formation period of oyster mushroom using different substrate mixtures is also presented in Table 3. Same results of the ANOVA were recorded showing no significant differences among treatment means. Shortest pinhead formation was recorded in substrate mixtures with 100% cassava peels with 37.33 days. Eighty percent cassava peels + 10% coconut residue + 10% coffee waste and 70% cassava peels + 20% coconut residue +10% coffee waste have shorter period of pinhead formation with both took 38.00 days from inoculation. These were followed by the control (100% sawdust) with 38.67 days of pinhead formation. Substrate mixture with 50% cassava peels + 40% coconut residue + 10% coffee waste registered the longest number of days of pinhead formation with 40.00 days from inoculation. However, general results of this parameter suggest that these substrate mixtures did not significantly influence the pinhead formation of P. ostreatus. Days from Pinhead Formation to Fruiting Body Formation (PF- FBF). The period of fruiting body formation of oyster mushroom using different substrate mixtures is presented in Table 3 as well. ANOVA results showed no significant differences among treatment means. Data showed that whatever substrate mixtures used, there is no differences in period of fruiting body formation which only took three days from pinhead formation in all substrate mixtures. Days from Fruiting Body Formation to Full Maturity (FBF- FM). The time taken from fruiting body formation to pin maturation is also presented in Table 3. Results of the ANOVA revealed no significant differences among treatment means. Results of the study showed that all substrate mixtures used had no significant influenced on the period of full maturation of P. ostreatus which only took three days from fruiting body formation in all substrate mixtures. Generally, results imply that different substrate mixtures used in the experiment do not influence the colonization period, pinhead formation, fruiting body formation, and full maturation period of P. ostreatus. These findings could be attributed to the cellulose and lignin properties of both cassava peels and sawdust (Frimpong-Manso et al., 2011) (Horisawa et al., 1999). The results also confirmed Stanley & Nyenke’s (2011) findings that cassava stimulated luxuriant mycelial growth rate and extension can be comparable to that of a commonly used substrate such as sawdust. Percent Contamination of Oyster Mushroom Bags Prepared With Different Substrate Mixtures Percent of contamination. The percent contamination of oyster mushroom bags prepared with different substrate mixtures is shown in Table 4. Results of the ANOVA revealed significant differences among treatment means (p > 0.01). The least percentage of contamination was recorded in the control bags with 100% sawdust (11.11%), followed by the substrate mixtures of 80% cassava peels + 10% coconut residue + 10% coffee waste and 50% cassava peels + 40% coconut residue + 10% coffee waste comparable percent contamination of 37.78% and 35.56%, respectively. Higher contamination was observed in substrate with 100% cassava peels with 71.11% contaminated bags. While the highest contamination was recorded in substrate mixtures with 70% cassava peels + 20% coconut residue + 10% coffee waste with 84.44% contamination. Common contaminants found in the contaminated fruiting bags were presented in Table 5. The fungal contaminants observed were as follows: Trichoderma spp., Aspergillus spp., Fusarium spp., Neurospora spp., and Penicillium spp. These results suggest that 100% sawdust as substrate for P. ostreatus had the lowest percent of contamination. Hence, it can be the ideal substrate for oyster mushroom to lower contaminant population. Moreover, cassava mixed with coconut residue and coffee waste can enhance contaminant population. These may be due to the fungal contaminants usually associated with higher percentage of cassava-based substrate like Aspergillus spp. (Obadina, 2006), and coconut-based substrate Fusarium spp. (Manimekalai, 2010). Number of Table 4. Percent of contaminated oyster mushroom bags in different substrate mixtures. Substrate Mixtures Replication Total Mean** I II III 100% Sawdust (Control) 6.67 13.33 13.33 33.33 11.11a 100% cassava peels 66.67 73.33 73.33 213.33 71.11c 80% cassava peels + 10% coconut residue + 10% coffee waste 33.33 40.00 40.00 113.33 37.78b 70% cassava peels + 20% coconut residue + 10% coffee waste 86.67 86.67 80.00 253.33 84.44d 50% cassava peels + 40% coconut residue + 10% coffee waste 33.33 33.33 40.00 106.67 35.56b CV (%) = 8.02; **=significant at 1% level, THSD. https://journals.e-palli.com/home/index.php/ajec Pa ge 7 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 Table 5. Contaminants found in contaminated fruiting bags Substrate Mixtures Image of Contaminations Morphological Characteristics Fungal Contaminants 100% Sawdust (Control) Green-mold. Color yellow to green, gradually creeping into the black substrate (Druzhinina et al., 2011) Trichoderma spp. 100% cassava peels Produced white to yellowish or crusty on the substrate, foul-rotten smell. (Lopez - Arevalo et al. 1996)) Aspergillus spp 80% cassava peels + 10% coconut residue + 10% coffee waste Fusarium mold, can appear pale or brightly colored, with a cottony surface. (Manimekalai, 2010) Fusarium spp. 70% cassava peels + 20% coconut residue + 10% coffee waste Produced bright neon orange to reddish substrate. (Suada et al.2015) Neurospora spp. 50% cassava peels + 40% coconut residue + 10% coffee waste Produced initially white and become blue-green, gray- green, olive-gray, yellow or pinkish with time substrates. (Lopez - Arevalo et al. 1996)) Penicillium spp. Fruiting Bodies of Oyster Mushroom Grown in Different Substrate Mixtures Number of fruiting bodies produced in different substrate mixtures is presented in Table 6. Results of ANOVA revealed significant effects of these substrates to fruiting bodies of oyster mushroom on its first, second, and total flushes. Effects of the number of fruiting bodies is shown in Fig. 1. First Flush. Results of the study showed that fruiting bodies (43.15) were significantly higher in substrates with 70% cassava peel + 20% coconut residue + 10% coffee waste which was comparably higher as compared to substrate with 80% cassava peel + 10% coconut residue + 10% coffee waste with 37.25 fruiting bodies formed, and was followed by the substrate with 100% cassava peels with 34.30 fruiting bodies. Lower number of fruiting body formation was observed in substrate with 50% cassava peel + 40% coconut residue + 10% coffee waste with 30.70 fruits formed. Lowest number of fruiting bodies on the other hand, was recorded in control (100% sawdust) with only 23.21 fruits formed. Second Flush. Results showed that 50% cassava peel + 40% coconut residue + 10% coffee waste had the highest number of fruiting bodies with 50.09 fruits formed, which is comparably higher as compared to substrate with 80% cassava peel + 10% coconut residue + 10% coffee waste and 100% cassava peels with 46.56 and 35.00 fruits formed. Seventy percent of cassava peels + 20% coconut residue + 10% coffee waste and the control (100% sawdust) had the lowest number of fruiting bodies (19.75 and 21.49 fruits, respectively). Total Flush. Results showed that 80% cassava peel + 10% coconut residue + 10% coffee waste and 50% cassava peel + 40% coconut residue + 10% coffee waste had the highest number of fruiting bodies with 83.81 and 80.78 fruits, respectively. These were followed by the substrate with 100% cassava peels and 70% cassava peel + 20% coconut residue + 10% coffee waste having corresponding fruit formations of 69.30 and 62.90. Lowest number of fruiting bodies was recorded in the control (100% sawdust) with only 44. 70 fruits. Generally, the above results suggest that the substrate mixtures containing 80% cassava peel + 10% coconut residue + 10% coffee waste and 50% cassava peel + 40% coconut residue + 10% coffee waste can increase the production of fruiting bodies of oyster mushroom for about 36.08 - 39.11. These corresponds to the outcomes of the study of Sonnenberg et al. (2015) that demonstrated the potential of cassava peel as a substrate ingredient for the production of oyster mushrooms, with a yield comparable to that of sawdust. These results were further explained by Youri (2003), who showed that https://journals.e-palli.com/home/index.php/ajec Pa ge 8 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 sawdust and cassava peel are lignocellulosic material which consist of three main components, namely: cellulose, hemicellulose, and lignin that serve as a suitable substrate for mushrooms as it degrades lignocellulosic substrates through lignocellulosic enzyme production and utilize the degraded products to produce their fruiting bodies, and were also backed up by the studies of Sa´nchez (2009), and Grim & Wosten (2018). Furthermore, Ginterova and Janotkova (1998) discovered that plant oil with a high total fat content, such as that found in coconut residue, stimulates the formation of biomass in P. ostreatus. As a result, adding the appropriate amount of coconut residue can be used as supplemental substrate, increasing the fruiting bodies and yield of oyster mushroom (Sopit, 2007). Besides, Ropciuc et al. (2016), also observed that mushrooms grown on coffee waste had a respectable percentage of biological efficiency. As a result, combining these substrates can contribute to achieving the highest yield in weight parameters of oyster mushroom. Table 6. Yield of oyster mushroom in different substrate mixtures. Substrate Mixture No. of Fruiting Body (Per flush) Stipe Height (cm) ** Pileus Diameter (cm) * Fresh Weight (g. per flush) 1st * 2nd ** Total** 1st** 2nd** 100% Sawdust (Control) 23.21d 21.49c 44.70c 19.45a 21.15a 37.99bc 38.10b 100% cassava peels 34.30b 35.00b 69.30b 11.35bc 13.90ab 50.00ab 51.00a 80% cassava peels + 10% coconut residue + 10% coffee waste 37.25ab 46.56ab 83.81a 12.17b 14.34ab 63.63a 53.50a 70% cassava peels + 20% coconut residue + 10% coffee waste 43.15a 19.75c 62.90b 11.00c 13.40ab 52.10ab 32.25b 50% cassava peels + 40% coconut residue + 10% coffee waste 30.70c 50.09a 80.78a 9.44d 10.78b 32.18c 34.25b CV (%) = 19.84 14.34 12.31 2.91 21.88 13.97 7.69 *=significant at 5% level, THSD; **=significant at 1% level, THSD. Figure 1. Number of fruiting bodies of oyster mushroom grown in different substrate mixtures. Stipe Height (cm) and Pileus Diameter (cm) of Oyster Mushroom Grown in Different Substrate Mixtures Stipe height (cm) and pileus diameter (cm) of oyster mushroom grown in different substrate mixtures is also presented in Table 6. Results of ANOVA revealed significant differences on both parameters. Stipe height (cm). Results showed that the control (100% sawdust) has the longest stipe height with 19.45 cm. This was followed by the substrate with 80% cassava peel + 10% coconut residue + 10% coffee waste with 12.17 cm. height which is comparable with 100% cassava peels with 11.35 cm. stipe height. Shorter stipe height (11.00 cm.) was observed in 70% cassava peel + 20% coconut residue + 10% coffee waste which as followed by the substrate with 50% cassava peel + 40% coconut residue + 10% coffee waste with 9.44 cm. Pileus Diameter (cm). Results of the study showed control (100% sawdust) still showed the widest pileus with 21.15 cm. Comparably, wider pileus were observed in substrate mixtures such as 100% cassava peels, 80% cassava peel + 10% coconut residue + 10% coffee waste and 70% cassava peel + 20% coconut residue + 10% cof-fee waste with corresponding pileus diameter of 13.90 cm., 14. 34 cm., and 13.40 cm. Smallest pileus was recorded in substrate containing 50% cassava peel + 40% coconut residue + 10% coffee waste with 10.78 cm. wide. The above results imply that 100% sawdust has the longest stipe and widest pileus with 19.45cm. and 21.15 cm., respectively. Comparable pileus size was also observed in substrates such as 100% cassava peels, 80% cassava peel + 10% coconut residue + 10% coffee waste, and 70% cassava peel + 20% coconut residue + 10% coffee waste. These results inclined to the study of Sanjel et al. (2021), that discovered that the size of the https://journals.e-palli.com/home/index.php/ajec Pa ge 9 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 mushroom is dependent on substrates, and that poor in cellulose, hemicelluloses, and lignin materials can constitute physical barriers that are difficult to be broken down without the presence of lignin-degrading enzymes. In addition, major ecological factors that affects the stipe height, and pileus diameter in oyster mushroom includes the compactibility or the compressibility of material used as substrate. Because of the particle size, it increases the void ratio as moisture plays an important role in bonding because of sawdust and cassava peel’s cohesive or lignin characteristics (Azhar et al., 2015). It has been supported by several studies that substrate type such as its lignin characteristic contributes significant differences in stipe height, and pileus diameter of oyster mushroom (Besufekad et al., 2020; Nkwonta, 2013; Tsegaye & Tefera, 2017; Onyeka et al., 2018; Dubey et al., 2019). Figure 2. Pileus diameter (left), and stipe height (right) of oyster mushrooms harvested from different substrate mixtures. Fresh Weights (g) of Oyster Mushroom as Influenced by Different Substrate Mixtures Fresh weights (g) of oyster mushroom grown in different substrate mixtures is presented in Table 6. Results of the ANOVA revealed significant effects in the fresh weight of oyster mushrooms in first and second flushes. First Flush. Heaviest fresh weight (63.63 g) was recorded in oyster mushroom grown from substrate with 80% cassava peel + 10% coconut residue + 10% coffee waste. Comparable effects were observed in substrates containing 100% cassava peels, and 70% cassava peel + 20% coconut residue + 10% coffee waste with 50.00 grams and 52.10 grams, respectively. Lighter fresh weights on the other hand, were recorded in substrates with 100% sawdust (37.99 g.) and 50% cassava peel + 40% coconut residue + 10% coffee waste (32.18 g.). Second Flush. Heaviest fresh weight of oyster mushroom on the second flush were observed in 100% cassava peels and 80% cassava peel + 10% coconut residue + 10% coffee waste with weights of 51.00 grams and 53.50 grams, respectively. Lighter fresh weights were recorded in the other substrate mixtures such as 100% sawdust, 70% cassava peel + 20% coconut residue + 10% coffee waste, and 50% cassava peel + 40% coconut residue + 10% coffee waste with weights that ranged from 32.25 grams – 38.10 grams. Based on these results, it suggests that 80% cassava peel + 10% coconut residue + 10% coffee waste can produce a heavy fresh weights of oyster mushroom. Moreover, comparable results can be obtained using 100% cassava peels as substrate. These results confirmed the study of Sonnenberg et al. (2015) that cassava peels showed to be a potential ingredient in mushroom substrate for it produced more than 100% BE which is comparable to that the usual sawdust substrate. Moreover, several reports showed that the adding of coconut residue (Sopit, 2007), and coffee waste (Ropciuc et al., 2016) increases yield and gained a respectable percentage of biological efficiency in oyster mushrooms. Therefore, combination of these substrates can contribute to gaining the significant yield in weight parameters of oyster mushroom. CONCLUS ION Based on the above results, it can be concluded that various substrate mixtures do not significantly influence the oyster mushroom’s colonization, pinhead formation, fruiting body formation, and the period of maturation. Among other substrate mixtures, the common substrate (100% sawdust) can produce the least number of contaminated fruiting bags, as well as the widest mushroom pileus and longest stipe. Moreover, the substrate mixture with 80% cassava peels + 10% coconut residue + 10% coffee waste can produce the greatest number of fruiting bodies while also being the most viable in terms of weight yield. Thus, different mixtures of cassava peels, coconut residue, and coffee waste were an effective substrate mixture to produce viable yields and, therefore, can be used as a successful substrate for oyster mushroom cultivation. The findings of this study proved that cassava peels and/ or a combination of coconut residue, and coffee wastes can be use as substrate option for mushroom growers due to its high yield potential. Aside from their nutritional, medicinal, and economic value, oyster mushrooms (P. ostreatus) may aid in agricultural waste management in the country. These can help bridge environmental issues and advance climate change response. However, further research on the effects of these substrate mixtures to oyster mushrooms as well as its varying proportions, are recommended. REFERENCES Adebayo, G.J., Omolara, B.B., and Toyin, A.E. (2009). Evaluation of yield of oyster mushroom (Pleurotus pulmonarius) grown on cotton waste and cassava https://journals.e-palli.com/home/index.php/ajec Pa ge 10 https://journals.e-palli.com/home/index.php/ajec Am. J. Environ. Clim. 1(1) 1-11, 2022 peel. 8(2):215–218. https://doi.org/10.5897/ AJB2009.000-9039. 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