Optimization of Gmelina tree (Gmelina arborea) and Eggshells in mushroom culture

medium to maximize mycelial growth rate and fruit body yield of Oyster Mushroom

(Pleurotus ostreatus)

Ken Lorenz Anielo M. Alonzo

Khristian Marion D. Gonzales

Jan Marnelli G. Sayson

Eunice Bianca E. Somera

STEM 12- Graham Bell

In partial fulfillment of the requirements for the subject Capstone Project

San Jose City National High School-Senior High School

 INTRODUCTION

Applying mushrooms’ health benefits has a long tradition and is very common in

Asian countries. Mushrooms have been used extensively, owing to their nutritional and

medicinal value, for thousands of years. Cool, damp places like mountains and forests

mushrooms mostly grow but the ability to cultivate different mushrooms has improved

greatly over the past decades. This results to large numbers of scientific studies on over

270 medicinal mushrooms over the past three decades, especially in Japan, China and

Korea; They have confirmed the traditional uses and also demonstrated new applications

for health benefits. The widely cultivated species of mushrooms in the Philippines are

White Button (Agaricus bisporus), Shiitake (Lentinula edodes), and Oyster (Pleurotus

ostreatus).

Oyster mushroom (Pleurotus ostreatus) grows at moderate temperatures ranging

from 20°C to 30°C. It is perfect to grow in tropical countries such as the Philippines. The

fruit bodies of this mushroom are distinctly shell of spatula shaped with different shades

of white cream, white, or light gray. Cultivating this type of mushroom does not require

complicated substrate preparation technique and even grows on non-fermented, almost

fresh plant residues and uncontrolled environmental conditions. This type of mushroom is

known to have low amount of calories, fat, gluten, cholesterol, and sodium at the same time

they are high in vitamins, B1, B3, B5 and B12, C, and D (Hobbs, n.d).

Gmelina tree (Gmelina arborea) is suitable for general utility purposes, especially

light construction general carpentry, and utility furniture, with excellent woodworking

properties. It is native in Asia especially in Pakistan, Sri Lanka, Thailand, Vietnam, China,

and Philippines. It is widely used as furniture in the Philippines that the waste shreds left
after the production of one product are left to mix with air that helps grow fungi and be

inhaled by human beings; Gmelina tree shreds, though biodegradable can cause serious

health problems such as lung diseases.

Eggshells are high in Calcium in a form of Calcium Carbonate (CaCO3) which is

known to give strength to things such as rocks, seashells and of course, eggshells. A good

quality eggshells will contain, on average, 2.2 grams of Calcium. Actually, amounts as low

as 78% of an eggshell contains Calcium Carbonate and the remaining mass is composed

largely of Phosphorus, Magnesium, and trace amounts of Sodium, Potassium, Zinc,

Manganese, Iron, and Copper (Experiment of Calcium Carbonate Composition of

Eggshells, 2018). According to AM King’Ori (2011) discrarded eggshells are often used

as plan fertilizer and are effective liming sources. Eggs are widely used for cooking in

homes, and most of the times eggshells are thrown away by the trashcan.

Statement of The Problem

Mushroom cultivation is a rapidly propagating industry and commercially available

culture media which contain essential such as thiamine (Vitamin B1) and biotin (Vitamin

H or vitamin B7) components are expensive.

The main problem of the study is to determine the potential use of Melina (Gmelina

arborea) wood and Eggshelss as a culture media for oyster mushroom.

Specifically, it sought to answer the following

a) Is there a significant difference on the ramification period of oyster mushroom

subjected to different culture media?

 b) Is there a significant difference on the growth increment of oyster mushroom

subjected to different culture media?

c) Is there a significant difference on the mycelial weight of oyster mushroom

subjected to different culture media?

d) Is there a significant difference on the number and weight of mushroom produced

subjected to different culture media?

Objectives of The Study

This study aims to:

a) Determine if there is a significant difference on the ramification period of oyster

mushroom subjected to different culture media

b) Determine if there is a significant difference on the growth increment of oyster

mushroom subjected to different culture media?

c) Determine if there is a significant difference on the mycelial weight of oyster

mushroom subjected to different culture media

d) Determine if there is a significant difference on the number and weight of

mushroom produced subjected to different culture media

Hypotheses of the Study

H0:

a) There is no significant difference in the ramification period of the oyster

mushroom that subjected into different culture media

 b) There is no significant difference in the growth increment of the oyster mushroom

that subjected to different culture media

c) There is no significant difference in the mycelial weight of the oyster mushroom

that subjected into different culture media

d) There is no significant difference on the number and weight of mushroom

produced subjected to different culture media

Significance of the Study

The results gathered from the study will add to the body of knowledge about

mushroom cultivation. Mushroom cultivator and Gmelina tree-growers will benefit in this

study because Gmelina tree shreds and Eggshells were considered waste product and have

no general use, so if the study will be successful, Shreds coming from Gmelina tree and

residue coming from Eggshells will not be considered a wasted product anymore.

Scope and Limitation of the Study

This study will evaluate the effectiveness of Gmelina tree shreds and Eggshells as

culture mediums for mushroom growth. The rate of mushroom growth will not be

examined. The ramification period, growth increment, mycelial weight and number of

oyster mushroom produced will be examined in 14 days. The study will be conducted in

Central Luzon State University.

 REVIEW OF RELATED LITERATURE AND STUDIES

Review of Related Literature

Eggshells

Eggshells are waste materials from hatcheries, homes and fast food industries (Phil

and Zhihong, 2009; Amu et al., 2005) and can be readily collected in plenty. Eggshell waste

disposal contributes to environmental pollution. Challenges associated with disposal of

eggshells include cost, availability of disposal sites, odor, flies and abrasiveness (Phil and

Zhihong, 2009). However, they can be processed into saleable products like fertilizer, used

in artwork, human and animal nutrition and building materials and to produce collagen

from the membranes (Phil and Zhihong 2009; Amu et. al. 2005). Shell membranes consist

of collagen as component. The collagen is extracted and has diverse uses in medicine,

biochemical, pharmaceutical, food and cosmetics industries. These uses minimize their

effect on environmental pollution.

Eggshells contain and trace amounts of other micro elements, i.e. magnesium,

boron, copper, iron, manganese, molybdenum, sulfur, silicon and zinc (Bee, 2011).

Eggshell calcium is probably the best natural source of calcium and it is about 90%

absorbable (Bee, 2011). It is much better source of calcium than limestone or coral sources.

The composition of the eggshell is approximately 98.2% Calcium Carbonate, making it a

good soil amendment material. Amu et. al. (2005) reported that eggshells can be alternative

to lime as a soil stabilizing agent while Amu and Salami (2010) in the search for alternative

stabilizing agents, reported that pulverized eggshells improved the plastic indices of

lateritic soil samples.

Gmelina tree (Gmelina arborea)

Gmelina tree (Gmelina arborea) is suitable for general utility purposes, especially

light construction general carpentry, and utility furniture, with excellent woodworking

properties. It is native in Asia especially in Pakistan, Sri Lanka, Thailand, Vietnam, China,

and Cambodia. Since the 1960s it showed a fast growth in Brazil, Gambia, Malaysia, and

Philippines (Vikaspedia.in, 2010). Gmelina fruit and leaves have good nutritional value

with protein content of 21.50% and 13.20%, respectively. However, the fruit and leaves

were observed to contain some toxic component whose concentration in fruit include

tannins (3.20g/100g), flavonoids (5.30g/100g), phytates (5.80g/100g) and saponins

(4.60g/100g), respectively while the leaves showed concentrations of 1.20, 1.80, 3.10, 0.85

and 0.72g/100g of tannins, oxalates, flavonoids, phytates, and saponins, respectively

(Augustine, et. al., 2018).

Oyster Mushroom Cultivation

Very few mushroom have been successfully artificially cultivated although there

are a great number of mushroom species that are edible. The most popular are European or

White button mushroom (Agaricus bisporus), oyster mushroom (Pleurotus spp.), paddy

straw mushroom (Volvariella spp.) and Shiitake (Lenitus edodes). European mushroom is

more specific in its temperature requirement which ranges between 14-20º C (Optimum 16

± 2ºC) during cropping. Paddy straw mushroom can be successfully grown at places where

temperature range is 28-30ºC. Oyster mushroom can be grown in temperature range pf 20-

33ºC. Due to its easy way of cultivation in a wide range of temperature and high yielding

performance, it is becoming more popular in many parts of the country. There are several

Pleurotus spp., these are as follows: P. ostreatus, P. flabellatus, P. sajor caju, P. florida, etc.
These species are well known for their flavor and delicacy and they can grow wild in the

forests which can be cultivated in thatched, polythene, brick or stone houses. Oyster

mushroom can be grown on various substrates like paddy straw, corn stalk/cobs and

vegetable plant residues. (Cultivation technology for Oyster mushroom, 2005)

Making Mushroom Compost

The important thing to remember when making own compost for mushroom is that

need both a carbohydrate source and nitrogen source. These sources must be enough or

adequate to feed the microbes that will break down the mushroom compost. The microbes,

just like people, needed energy which is the carbohydrates (for mushrooms), and protein

which is the nitrogen (for mushrooms) to live. They in turn reward the people fresh

pH level on Mushroom Growth

On mycelium growth, the pH level of the compost has a major influence. The pH

level of compost should not be underestimated of importance because high quality

mycelium is the foundation for high quality mushrooms (Buth J., 2007)
 Review of Related Studies

Cultivation of (Pleustorus sajor-caju(Fr.)) Using Different Plant Wastes

Oyster mushroom was cultivated on different treatments with different combination

of plant wastes as substrates. These plants wastes are rice bran, sawdust, coconut waste and

ipil-ipil leaves. Had significant higher yield than in any other treatments (Durante, E.S.,

1982)

Adaptability of Two Imported Strains of Oyster Mushroom (Pleurotus sajor-caju)

Under CLSU Condition

This study compared the adaptability of two strains oyster mushroom on two

formulated substrates with the combination of rice bran. The result showed that the two

strains have no significant difference on the number of days from opening to harvesting

(Villaceran Jr., 2005)

Cultural Growth Evaluation of Pleurotus cystidiosus to Indigenous Culture Media

Pleurotus cystidiosus was subjected to different indigenous culture media. The

treatments are potato sucrose gulaman, sweet potato sucrose gulaman, corn cob sucrose

gulaman and sayote sucrose gulaman. The result showed that the corn cob is significantly

lobger in ramification period compared to the control medium, however, it was also the

excellent alternative indigenous material used.

 SUMMARY OF REVIEW OF RELATED LITERATURE AND STUDIES

Several culture media are made by different mushroom experts in the cultivation of

mushrooms. Some of these media may be affordable for Filipino farmers because of

unreasonable prices.

Substrates used in conducting the study have common composition that made up to

be alternative substrates to potatoes. They all have starch, mixture of amylose and

amylopectin and glucose as well that are needed in making a mushroom culture media.

The researchers included the studies because the materials used as substrates are

plant parts/materials just like the researchers used in the present study. For example,

Gmelina wood and Eggshells has also high protein content like the saw dust. The previous

studies also used the Oyster mushroom (Pleurotus sajor-caju(Fr.)) as the subjected of the

study.

Since there is no study on Gmelina and Eggshells as substrate for mushroom

cultivation yet, the researchers had a hard time finding related studies. However, the

reviewed studies are still relevant in the sense that the topic is about the cultivation of oyster

mushroom.

On the reviewed literature, different conditions for mushroom growth were stated.

The nutrient contents of Gmelina and Eggshells were stated and the nutrients needed by

mushrooms are nitrogen and carbohydrates and Gmelina and Eggshells satisfies the second

condition, being a fruit thates. is rich in carbohydrates.

 RESEARCH METHODOLOGY

Conceptual Framework

The independent variable will be the different culture media for oyster mushroom,

and it will be evaluated through measurement of the mycelial weight, ramification period

and daily growth of oyster mushroom.

Independent Variable
Different Culture Media: Dependent Variable
 Potato sucrose gulaman  Ramification period
(+control)  Growth increment
 Gmelina tree shreds  Mycelial weight
sucrose gulaman  Number and Weight of
 Gmelina tree shreds with mushroom produced
eggshells sucrose
gulaman

Fig. 1 Conceptual framework of the study

Operational Definition of Terms

Oyster mushroom – This is one of a variable in the study which is one of the most widely

eaten mushrooms. This is influenced by the different culture media.

Melina wood - fast-growing "white wood" that is used as substrate which is independent

variable.

Culture Media – special medium where the oyster mushroom was cultured.
Growth increment - the change of size of the cultured mushroom in quantitative data

Mycelium – vegetative part of a fungi that a thread like hyphae which consist of a mass of

branching and produces mushroom.

Mycelial weight – the mushroom’s heaviness in broth culture in terms of grams.

Ramification – the particular minute or hour that the Petri dish was covered with mycelia.

Substrate – where a mushroom lives, grows, and obtains its nourishment.

Research Locale

The study will be conducted in Philippine Carabao Center, Central Luzon State

University, Science City of Munoz, Nueva Ecija, Philippines. The melina wood will come

from a local furniture manufacturer. The study will be conducted within November 2019

Research Design

The research will use Complete Randomized Design. The study will focus on the

independent variable which was the culture media with three treatments. T1(potato sucrose

gulaman), T2(Gmelina tree shreds sucrose gulaman), T3(Gmelina wood shreds with

eggshells sucrose gulaman). There were three replicates for each treatment.

Subjects of the Study

Since there are many ways to cultivate mushrooms like using tiessa fruits which is

also a waste and doesn’t have any much of a use and considered as junk. The researchers

came up with an idea to use Gmelina tree shreds and Eggshells, which are also considered
useless and a waste product, to be an effective culture medium for mushroom growth. The

researcher chose this subject because there are many waste products being produced and

the researchers want to make use of this so-called waste product in a way that helps the

environment to grow and also to lessen the waste being produce. With proper procedures

and test to be conduct, the researcher will study if using Gmelina tree shreds and Eggshells

are an effective culture medium for mushroom growth.

Research Instrument

For the research instrument, the researchers decided to do the observations.

Observation technique has two types: structured and unstructured. The researchers did not

use the unstructured type wherein the researchers listed data based on what’s happening or

occurring. For this study, the structured type will be used as the researchers use tables and

guidelines right after the observations. As the days passed, the researchers observed day

by day the development and growth of the mycelia with the proper care and environment

perfect for the specimens. As the observations will proceed, the researchers will study how

long it will it take for the mycelia to cover up the petri dish, the change in diameter of the

cultured mushroom in terms of mm, the weight of mycelia and the number and weight of

mushrooms on each substrate. Gathering and collecting data about the mass production of

strawberries will be accomplished through observation. Since oyster mushroom is not the

only variable or subject of the study, the researchers will observe the effectiveness of

gmelina tree shreds as a medium of growth and also the effectiveness of eggshells that will

act as a fertilizer in gmelina tree shreds substrate. Will it make the mycelia produce more

mushrooms than usual sucrose agar? Will the eggshells help the medium of growth to be

healthy? There were so many questions but through observation, those questions can be
answer. Lastly, the researchers will produce 3 media of mycelia and after that it will be

transferred to the different media, the researchers then will observe which medium will

produce oyster mushroom faster and more among the media. Since the researchers will use

structured type of observations, the collection of data and the observations will take place

as the growth and development of the oyster mushroom and the procedures of the study

will happen then and be listed on the checklists or tables.

Data Gathering

The dry weight of mycelia will be determined to quantify the mycelial production

of strains. a 10-mm agar plug from the 7-day old culture of Plearotus ostreatus will be

aseptically transferred to 50 ml broth cultures. The mycelia mat will be harvested after 7

days of incubation under 28-30°C. After another 14 days, the samples will be blotted on a

filter paper to remove excess moisture and will be air dried on an air conditioned room for

48 hours and then oven dried for 2 hours at 50°C. Lastly, the mycelia will be placed in a

dessicator for 1 hour. The growth increment will be measured in mm using an electronic

caliper and the ramification period when mycelium has fully covered the petri dishes will

be observed. The weight of samples will be determined using analytical balance.

Research Procedure

Materials

T1 T2 T3

Potato – 250g Gmelina tree shreds- Gmelina tree shreds-

250g 125g
Sucrose -10g
Sucrose- 10g Eggshells- 125g
Gulaman - 20g
Gulaman- 20g Sucrose-10g
Water- 1L
Water- 1L Gulaman-20g

Water- 1L

Preparation of the culture media

Known weights of potato, Gmelina shreds and Eggshells will be chopped and

boiled in water until softened. sugar and gulaman will be added to the strained swcoction

and the mixture will be boiled again until homogenous. the process will be done for each

treatment.

Sterilization

The boiled media will be transferred into the erlenmeyer flask.

Inoculation

40-50 ml of culture media will be poured aseptically intro petri dishes then, it will

be allowed to solidify. to ensure uniformity of age, a 7-day old revived culture of pleurotus

sajor-caju will be used. mycelia block from revived culture will be transferred to petri

dishes. the treatments will be incubated for 14 days and will be kept at room temperature
Methods of Data Analysis

After growth increment was measured in mm using an electronic caliper, the

ramification period when mycelium has fully covered the petri dishes was observed and

the weight and quantity of samples was determined using analytical it will be then analyzed

The results will be analyzed using ANOVA (analysis of variance) and the treatment

means will be compared using duncan's multiple range test at 5% level of significance.