Unit 5: Mushroom Cultivation

• The 20th century began with a world population of

1.6 billion. The world’s population is expected to be

reached 9.3 billion in 2050 from the current worldpopulation of 7.8 billion as of February 2020 accordingto the most recent United Nations estimateselaborated by Worldometer.

• This over exploring human population creates huge

demand ------------------------

➢Nutritious health food to combat malnutrition

➢Industries for jobs and comforts

➢New cities for sheltering huge populations

➢More water for drinking as well as industries

• We need some alternative way for crop production like Aeroponics and Hydrophonics with limited land.

What is the way to feed 9.3 billion of peoples in 2050

with continuous reducing area of cultivated land ?

What is the way to feed 9.3 billion of peoples in 2050

with continuous reducing area of cultivated land ?

• Agro waste may be the boon of mankind .

• India generates about 350 million tones ofagricultural waste every year and the ministry ofnew and renewable energy estimates this wastecan generate more than 18,000 MW of powerevery year apart from generating green fertilizer forfarms. The country so far failed to find itsproductive use in the absence of enoughgovernment push and business model to work forfarmers.

• So, agro waste is the major problem of stubbleburning in India.

Stubble burning…………

• This agriculturalwaste that aregenerally set fireon field which iscalled as stubbleburning andproduce untoldamount of greenhouse gases andtoxic pollutants. Stubble burning at Uttar Dinajpur, West Bengal ,

India in the Year 2018

Recent scenario of stubble burning at Delhi• Much of the pollution is comingfrom farms in nearby states ofPunjab, Haryana, and WesternUttar Pradesh.

• Every year, farmers of neighbouringstates set fire to their own fields toclear them for the next season.Known as stubble burning, millionsof tons of crop residue are set fireand releasing untold amounts ofparticulate matter into theenvironment.

• This belt produces an estimated 34million tonnes (mt) of paddy strawevery season and burned within lessthan a month’s span between mid-October and around November 10.

on November 8, 2017

Air Quality Index was 1,010 . It was above the upper limit of the hazard

Delhi’s chief minister call his city a ---------------------------------------------------“gas chamber”

Mushroom can be the alternative source of food to feed over exploring population

If Earth Is Ever Hit By An Asteroid, Only Mushrooms Can Save Us From Going Extinct

• Asteroids may just be rocks thathave large orbits around the Sun.And they're fine when they staythere. But when they come crashingdown to Earth, they can cause a lotof damage.

• Earth surface will be covered withthousand of miles denseparticulate matters , sun light willnot be able to penetrate on earthsurface, photosynthesis in plantswould have ground to a halt, andherbivores starving to death, thecarnivores preying on themfollowing shortly after. .

Gwyn D'Mello | updated: Sep 16,

2019, 16:16 IST

• And it seems like the only thing that could stop us being wiped out is some fungus.

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WHAT ARE MUSHROOMS?• Mushrooms are fungal form of

life.

• The term mushroom is broadlydefined as follows: “a mushroom isa macrofungus with a distinctivefruiting body which can be eitherepigeous (above ground) orhypogeous (under ground) and largeenough to be seen with the naked eyeand to be picked by hand.”

•The most common type of mushroom is umbrella shaped with

pileus (cap) and stipe (stem), e.g., Lentinula edodes and some

species additionally have an annulus (ring), e.g., Agaricus

bisporus or a volva (cup), e.g., Volvariella volvacea ,or have both,

e.g., Amanita phalloides .

Mushroom Bodyi) Cap (pileus)- Colour (white, grey, yellow) and shape (umbrella,

kidney, cap) depend on species.

ii) Stype (stem)- Stype is stem like structure that supports the pileusand transports nutrients from the substrate to other parts ofmushroom.

iii) Gills -tissues that produce spores.

iv) Mycelia- absorbs nutrients from substrate

Mushrooms Varieties and their scope• There are more than 30,000 identified types of mushrooms

worldwide.

• 99% of these are safely edible and roughly 1% is poisonous.

• Yet there are still many undiscovered mushroom species andthe effects of some mushrooms on human health remainunknown.

• Nowadays, almost every country devotes more attention toresearch, experimentation, selection and development ofmushrooms cultivation technique.

• Some mushrooms have medicinal qualities and theirpopularity is rising too.

• They are rich in protein compared with other vegetables.

• Its production is one of the most promising and highlydesirable in developing countries to reduce proteinmalnutrition.

Objective of Mushroom cultivation

• It generate relatively cheap source of nutrition rich health food (totackle shortage of food and malnutrition).

• Use for bioconversion/ bioremediation of large scale easily availabledifferent lignocellulosic agro waste in term of large scale productionof food as well as the safe disposal of crop residues.

• Mushrooms with its flavour, texture, nutritional value and highproductivity per unit area are not only an excellent food source toalleviate malnutrition and ensuring food security, used mushroombed might be used as soil conditioner for good health of soil.

• Therefore, mushroom cultivation can be carried out to empower thewomen, unemployed young people and youth.

Objective of Mushroom cultivation

Why Do You Cultivate Mushroom ?

– Generate relatively cheap source of nutrition rich heath food i.e. tackle shortage of food and malnutrition.

– Use substrate as lignocellulose, agro wastes are easily available (to reduce emission of air pollutants) i.e. Integrated Waste Management

– Easy to cultivate (no hard work)

– Need very low capital investment

– Need no land or very small space

– No use of pesticides and manure of production i.e. Organic food and fiber

– High market demand

Commonly Cultivated Edible Fungi

Mushroom Cultivation• Mushroom cultivation has two main phases,

– spawn production and

– fruiting body production.

• The mushroom seed is generally referred to as spawn.

• Spawn is a medium that is impregnated with mycelium of apure culture of the chosen mushroom strain used asinoculums for initiating mushroom production.

• Its production is a precise laboratory procedure where

maintaining sanitation and purity of the spawn are critical.

• Cereal grains and sawdust are common materials used for

spawn making.

• The most commonly utilized grain types are rye, wheat,

sorghum and millet.

• Small sized grains couldn't be used for spawn making b/c of

sticking nature of the grain during soaking and sterilization.

• Grain spawn is made of cooked or soaked grains with small

amount of gypsum.

Mushroom Cultivation

METHOD OF SPAWN PREPARATION

There are four steps involved in spawn

production:

– Preparation of Fungal growth medium (PDA)

– Raising pure culture,

– Preparation of mother spawn and

– Multiplication of spawn.

Fungal growth medium

• Preparation of agar media is basic for

mushroom cultures.

• There are several types of agar media such as

potatoes, malt extract and corn meal.

• Potato dextrose agar and malt extract agar

are available in market and could be

prepared according to instruction given by

the manufacturer.

Potato Dextrose Agar Medium

Preparation

• We can prepare our own medium from fresh potato.

• Wash and slice about 40 g of potato and place in 100 ml of

boiling water in a flask and boil for 15 minutes.

• Filter the potato decoction using a piece of muslin cloth.

• Add 2 g of dextrose and 2 g of agar at boiling temperature

and adjust the volume to 100ml by adding water.

• Pour the liquid agar medium in culture tubes (5/10 ml each),

plug with non-absorbent cotton and wrap with news paper

and rubber band.

• Sterilize the medium and other accessory glassware in the

autoclave at 15lb pressure/inc2 for 15 minutes.

• Keep the culture tubes in a slant position so that the agar

slants are solidified.

• Next day use the agar slants for pure culture preparation or

store in refrigerator.

Potato Dextrose Agar Medium

Preparation

Pure culture preparation

There are two ways of raising pure culture

a. Tissue culture

b. Spore culture

Pure culture preparation

Tissue Culture technique

• Select young fresh fungal fruiting body(sporophore/basidiocarp).

• Clean the fruiting body from any debris with alcohol.• Split the fruiting body lengthwise with knife and avoid any

contact of the knife with the area we want to take for tissueculture.

• Sterilize the scalpel on aflame and take a small piece ofmushroom tissue using forceps.

• Inoculate the mushroom tissue to PDA or MEA media slantsaseptically.

• Incubate for hyphal growth at 25ºC ,• In a few day hyphea will grow out from the tissue and

covers the entire surface and the pure culture becomesready for further multiplication.

Spore culture

• Select a mature fungal fruiting body and cut the stalk .• Laid the gills down on a clean typing paper, glass or similar

surface.• After 12 hours most fruiting bodys have released thousands of

spores.• The spores are collected by spore map techniques.• Pick up the spores by the inoculating loop under microscope.• Inoculate the spore to the PDA or MEA slants as in tissue

culture under aseptic condition and incubate at roomtemperature.

• The spores germinate and will form mycelium in a few days.– The tissue culture method is the most reliable and recommended

for the fungal culture– In the process of spore inoculation contaminant bacteria and molds

may grow together making it difficult to get pure culture of themushroom.

Spore culture technique

Pure Culture

Spawn preparation

• Select clean wheat grain, wash the grains and soak

overnight.

• Next day remove the soaked grains and wash gently and

drain the water on a sieve

• Check the moisture content by hand; it should not be too dry

or too wet.

• Mix the grain with 10% wheat bran and 2% calcium sulfate

and calcium carbonate mixtures.

• About 350 g prepared supplemented grain substrate is filled

in glucose/milk bottles or polythene bag upto 2/3 volume

and plugged with non-absorbent cotton. The plugs are

covered with news paper and rubber band.

• These bottles are then autoclaved at 15 lb p.s.i. pressure at

126o C for 1.5 to 2 hr.

Preparation of mother spawn

Preparation of mother spawn• These autoclaved bottles are left in the room for 24 hours for

cooling and are kept on laminar flow under U.V. tube for 20-30

minutes before inoculation.

• A piece of mycelium (pure culture) grown in Petri plates is

aseptically transferred to these bottles and inoculated bottles

are incubated at 25oC. Inculated bottles are incubated at 22-

250C for Agaricus bisporus, Pleurotus spp. and Lentinula edodes

but at 30oC for Volvariella spp.

• Inoculated bottles are gently shaked on 5th and 10th day.

• This spawn prepared using pure culture mycelium on agar

medium in Petri plates as inoculant is referred as mother

spawn.

Preparation of mother spawn

Multiplication of mother spawn• Multiplication of spawn can be done in polypropylene bags

(heat resistant). Normally for half and one kg spawn the bags

should be of 35 x 17.5cm and 40 x 20cm size, respectively.

• Select well grown mother spawn.

• Open the mother spawn bottles on a flame and stir the spawn

using sterilized forceps to get the individual grains.

• Transfer few grains with the mycelium into sterilized

substrate (grains medium) under aseptic conditions and cover

the mouth.

• Mix the grains by shaking to uniformly distribute the

mycelium.

• Incubate the inoculated bottles at 25ºC till all the grains is

covered with the mycelium.

• Inspect the bottles regularly and discard contaminated ones.

• Within 10-15 days of inoculation mycelial growth covers the

entire substrate and the spawn is ready for use.

Multiplication of mother spawn

Definition of Spawn• Mushroom “spawn” is a medium that is impregnated with

mycelium of a pure culture of the chosen mushroom strain used as

inoculums for initiating mushroom production. Spawn

production is a fermentation process in which the mushroom

mycelium will be increased by growing through a solid organic

matrix under controlled environmental conditions. In almost

all cases the organic matrix will be sterilised grain, e.g. millet,

rye or wheat. The purpose of the grain spawn is to boost the

mycelium to a state of vigour such that it will rapidly colonise

the selected bulk growing substrate. The grain is an important

nutrient support as well as a vehicle for the eventual even

distribution into the growing medium of the mushroom

inoculant. Each individual grain becomes coated with the

mycelium and in fact becomes a mycelial capsule.

Steps in spawn production

Pleurotus Classification

Different species of Pleurotus

Mushroom bed preparation

Mushroom bed preparation• Lignocellulosic wastes as substrates were chopped (2-3 cm.

pieces) and soaked in water over night to moisten it and excess

water was drained off.

• The polythene bags (35 x 45 cm) were filled up with substrates

and multi layered technique was adopted for spawning. Each

bag was filled up with 1.5kg dry substrate and 250g spawn was

added to it.

• After spawning, the bags were kept in cultivation house where

the temperature and humidity were maintained around 25ºC

and 80 to 90% respectively.

• Some pores are made on each bed with needle for gaseous

exchange.

• The spawn run was completed within 20 -25 days.

• The polythene bags were tear-off following the spawnrun.

• The beds were water sprayed regularly to moisten it.

• Formation of fruit bodies as pinheads was evidentwithin 3-4 days after removal of poly bags.

• The beds were maintained up to the harvest of thethird flush, which was completed in 65 days afterspawning.

Mushroom bed preparation

Pleurotus spp. Spawn Run Parameters (Substrate Colonization)

• Relative Humidity: 90-100%.

• Substrate Temperature: Fastestgrowth 78-84°F. Thermal deathoccurs above 104°F /48 hr.

• Duration: 10-14 days forcolonization.

• CO2: 20,000 ppm or 20% byvolume. Growth is stimulated upto 28,000 ppm.

• Fresh Air Exchanges: None(0/hr).

• Light: Incubation in totaldarkness.

Pleurotus spp. Pinhead Initiation Parameters

• Relative Humidity: 95%. Air T: 55-60°F

• Duration: 7-14 days. CO2: less than600 ppm.

• Fresh Air Exchanges: 4/hr.

• Light: Phototropic, most responsiveto an exposure of 2,000 lux/hr for12 hr/day. Grow- lux typefluorescent lighting recommended.Diffuse natural light is sufficient.

• Watering: Regular misting once totwice daily until fruit bodies are 30-40% harvest size.

Pleurotus spp. Cropping Parameters

• Relative Humidity: 85-92%. Air T: 60-64°F

• Duration: 5-7 weeks. CO2: less than 600 ppm.

• Fresh Air Exchanges: 4-6/hr.

• Light: Same as for pinhead initiation.

• Harvest Stage: Directly before incurved margins elevates to plane.

• Flush Intervals: ~10 days.

• Watering: Regular misting to prevent caps from cracking and to keep resting pinheads viable.

Pleurotus spp. Harvest

• Matter Content: For every 100 g of freshweight of mushrooms, we get only 52.36 g ofdry matter after dehydration, loosing 47.64 gof water.

• Nutritional Content: Crude Protein has beenreported at 30.4% of dry weight.

• Yield Potential: Average commercial yield are1 kilogram fresh weight of mushrooms perkilogram of dry weight of straw substrate.

• Biological Efficiency: 100% or more.

Mushroom Food value

• It contains many vitamins and minerals but

very low on sugar and fat. It is easily

digestible. It is very popular in most of the

developed countries and is being accepted in

many developing countries.

• Mushrooms are a good source of vitamin B, C

and D, including niacin, riboflavin, thiamine,

and folate, and various minerals including

potassium, phosphorus, calcium, magnesium,

iron and copper.

•Good source of essential amino acids, non-essential

amino acids and amides, unsaturated fatty acids

essential for the diet and other dietary substances (e.g.,

enzymes, polysaccharides, peptides, ergosterol,

terpenoids and other metabolites) for use to supplement

the diet by increasing the total dietary intake.

•Shiitake are said to have antitumour and antiviral

properties and remove serum cholesterol from the blood

stream. Other species, such as Pleurotus (oyster),

Auricularia (mu-er), Flammulina (enokitake), Termella

(yin-er) and Grifola (maitake), all have varying degrees

of immune system boosting, lipidlowering, anti-tumour,

microbial and viral properties, blood pressure

regulating, and other therapeutic

• Treatment of Diabetes: Ganoderma lucidum thatreduce blood glucose are Ganoderma B and C.The principle is by enhancing utilization of bloodglucose by body tissues.

• Reishi helps to protect the heart from shortage ofblood supply, and it is ideal for both curing andpreventing heart diseases like nausea.

• Reishi can reduce the level of blood cholesterol,liporotein and triglycerides in hypertensivepatient All these effects contribute to preventingvarious kinds of stroke.

• Reishi can best regulate and activate the immunesystem and increase self defense capabilityagainst tumor.

Genetic Diversity of Mushroom Species

➢ Estimated total species 300,000

➢ Mushroom species known 15,000

➢ Edible mushrooms known 5,000

➢ Prime edible mushrooms 2,000

➢ Medicinal mushrooms known 1,800

➢ Edible ectomycorrhizal mushrooms known 900

➢ Edible & medicinal mushrooms commercially cultivated 100

➢ Medicinal mushrooms used as dietary supplements 50

Laboratory equipments and glassware

– Autoclave

– Laminar Air Flow

– Incubator

– Microscope

– Inoculation needle

– Culture tube

– Utensil

– Net

– Chopper

– Water

Consumable materials

– Potato Dextrose Agar (PDA)medium– Bottle/polythene bag (4cm x 6cm– Wheat grains– Calcium carbonate– Calcium sulphate– Non-Absorbent Cotton– Rubber band– News paper– Paddy straw/wheat straw– Formaldehyde– Mercuric Chloride– Alcohol