microbiological issues -...

Microbiological issues

Successful mushroom productiondepends on many factors

• raw materials• heat treatment• inoculation rate• bag type• filling volume• incubation time• overall hygiene• growing conditions• climate• SPAWN QUALITY

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First rule: the spawnshall be beyond suspicion!Quality standards for spawn– Pure culture

• Free of imperfect molds• Free of other mushroom mycelium• Free of bacteria

– Even aspect• Loose grains with correct

moisture content• Evenly incubated

– Vigorous• In well breathing vessel• Young• Properly conserved

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Spawn producers are the top of the pyramid

These

are

nothing

but

steps

of

multiplication

Mother culture

Mother spawn A

Spawn productionMother spawn B

Spawn

Substrate/compost

Mushrooms

The effects of our actions increase top-downWe have an enormous responsability

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How to estimatethe quality of spawn?

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Visible problems in spawn

Mold infection Stroma Soft spawn

Bacterial infection Suffocated spawn Overaged spawn

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Invisible problems

How to find out if spawn is invisibly infectedwith imperfect molds or bacteria?

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Testing procedure

Turn the bag Push the content Push a few grains Seal the bag cornerupside down to one side to the upper corner below the grains

Flame scissors Cut the corner Drop a few grains Gently move the grainsabove the seal on a petridish around on the plate

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Evaluation after 5 daysof incubation:

Pure culture Mycelium Myceliumcontaminated with contaminated with

imperfect mold bacteria

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How can you be sure to produceaxenic spawn?

There is no magic involved

Mycelium production is nothing but applied microbiologywww.mycelia.be 10

Welcome to the world of micro-organisms: bacteria and fungi

• Where the enemy is invisible

• Where competitors arestronger and quicker

• Where only a good insight willlead to the correct measures

• Where strict rules areneeded to achieve asuccessful production

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Bacteria: characteristics• Kingdom Prokaryotae:

– Single-celled organisms– No organized nucleus

• Asexual reproduction through celldivision: a new cell wall grows throughthe center forming two daughter cells,each with the same genetic material asthe parent cell

Some groups of bacteria, such asBacillus, are able to transforminto thick- walled survival spores,which are resistant to extremecircumstances, like 120°C.

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Imperfect fungi: characteristics

• Kingdom Eukaryotae: cells with real nucleus

• Mycelium composed of long, branchingfilaments called hyphae

• Grow in/on a specific organic substrate

• Formation of sporangia andnumerous airborne asexual spores

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Imperfect fungi versus bacteriaFungal sporesand conidia:

have a diameter of 1 to 10 µ. are produced in huge amounts:

each spore can generate up to 1012

spores in a few days fungal spores may survive for over

20 years keep floating in the air for a long

period

Bacteriaand endospores:

may have a size of less than 1 µ their individual cells divide every

20 minutes under optimalconditions

endospores are highly resistant

to extreme circumstances remain in the air for a long period,

often attached to dust particles

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Fungi and bacteriahave one thing in common:

They love the substrateson which we want to cultivate our mycelium!

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Microorganismsand spawn production

in which production stepsdo micro-organisms play an important role?

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Mycelium production process:critical steps

Substrate Cooling

Pretreatment Inoculation Inoculum

Additives Sealing

Bag filling Mixing Packaging

Sterilisation Incubation Cold storage

Cooling Dispatching

workshop cleanroom area packing area

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Substrate Cooling


Additives Sealing



Cooling Dispatching


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Substrate Cooling


Additives Sealing



Cooling Dispatching


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Substrate Cooling


Additives Sealing



Cooling Dispatching


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Each of these risk factorsare related to microorganisms

Phase: Problem: Solution:• Sterilization • Endospores surviving 1. Correct sterilization

in the substrates procedure

• Cooling , • Microorganisms 2. Air filtration withinoculation connected to dust HEPA filters

• Inoculation, • Microorganisms 3. Strict rules ofsealing shed by people conduct

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1. CORRECT STERILIZATIONPROCEDURE

Goal: killing all microorganisms, also endospores

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Fungal versus bacterial infections

mold infections areeasily discovered

are often a result of insufficient

hygiene after heat treatment fungal spores die when

exposed to 60- 65°C

bacterial infections aredifficult to spot

are often a result of insufficient

sterilisation bacterial endospores survive

up to 120°C

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Correct sterilization procedure

• Bags loosely stacked inautoclave

• Evacuation of air fromautoclave and from bags

• leedi g duri g e tiresterilization

• Correct use of condenstationtrap

• Temperature in the centre ofeach bag shall be 121°C for30 minutes

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Example of sterilization cycle2 kg- bags in 560 liter- autoclave

In autoclave

Inside the bag

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2. AIR FILTRATIONBY MEANS OF HEPA FILTERS

Goal: creation of a dust- free working areawww.mycelia.be 26

Air is never empty!

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What is in the air?

• Gases: 77% N2, 21% O2, 1% Argon, 0,04% CO2, 1% of H2O vapor

• Dust:– mineral dust– soot– orga i dust ge erated y sa ills, agri ultural a ti ities …

• Airborne micro-organisms:– fungal spores– mycelium fragments– bacterial endospores– living bacteria, often attached to dust particles

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Examples of dust production

Organic dust from straw chopping Mineral dust

Organic dust from sawmill Soot from cheminee

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Airborne micro-organismsmade visible on agar media

• Advantage:gives a picture of realcontamination pressure

• Disadvantage:results are obtained onlyafter 3 days of incubation

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• Advantage:immediate results

• Disadvantage:gives no picture ofreal contaminationpressure

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Air quality: dust levels

Air quality class Number of particles ExamplesUS FED STD 209E of 0,5 µ and more

per cubic foot

Class 10 000 000 More than 1 million Dusty, e.g. wood chopping, areaClass 1 000 000 100 000 to 1 million Mixing and bagging areaClass 100 000 10 000 to 100 000 Food packing area

Class 10 000 1000 to 10 000 Cleanroom area(class 1 000)Class 100 Less than 100 LAF conditions

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Air quality: examples

Wood sawing area: Truck loading area: Bag filling area:Class 100 million Class 10 million Class 1 million

Spawn packing area: Cleanroom area: Laminar Air Flow:Class 100 000 Class 10 000 Class 100

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Methods for air purification

• UV: kills microorganisms up • low efficiencyto 1m distance from the UVsource

• Ozone: does not kill all • insufficientmicroorganisms

• Formaldehyde vapor • health risks

• Ultrafiltration of air through • highly efficient if rulesHEPA filters are followed!

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HEPA filtration of air

For the creation of ultra hygienicworking conditions

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HEPA filter - structure

HEPA = High Efficiency Particulate Air

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HEPA filter - filtration classes

HEPA class retention (total) retention (local)

E10 > 85 % ---

E11 > 95 % ---

E12 > 99.5 % ---

H13 > 99.95 % > 99.75 %H14 > 99.995 % > 99.975 %

U15 > 99.9995 % > 99.9975 %

U16 > 99.99995 % > 99.99975 %

U17 > 99.999995 % > 99.9999 %

HEPA filters remove at least 99.95% of airborne particlesof 0.3 micron (µm) and larger.

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LAF (Laminar Air Flow)Creation of a dust free working space

dust-free air dusty air

• Prefilter, turbine and HEPA filter• Outcoming air: speed 0,47 m/ sec, USFS class 100• Eventually recirculation of air

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Creation of USFS class 100 airImportant points:• Air speed 0,47 m/ sec• Ventilator type: turbine

• Number of revolutions shall be ajustable in accordance withfilter- obstruction

• Perfect seals• Working space same dimension as HEPA• Smooth working surface and lining• Prevent dead spaces• Regularly checked HEPA filter• Frequent change or cleaning of prefilter• Correct working method so as to avoid turbulence

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LAF types

Crossflow unit Downflow unitHorizontal flow Vertical flow

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LAF maintenance

Visual control Air speed controlon dirt and damage

Particle counting Change HEPA filterswhen necessary

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Common mistakes

HEPA filter blocked by dirt Damaged filter

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Rules regarding the use of LAF

Keep the working surface Keep hands and headas empty as possible out of the flow

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Use of LAF: common mistakes

Flow disturbed by objects: increased risk of infection

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Sterile overpressureCreation of a dust free working space

• Way: unit with prefilters, turbine and HEPA filter

• Overpressure reduction between rooms:minimum 10 Pascal (1 Pa = 0.00001 bar)

• Eventually recirculation of air• Air USFS class 10 000 HEPA filter bag filter coarse prefilter

seal ventilator seal

Clean air out Dusty air in

pressure heat exchanger heat exchangerchamber (hot water pipe) (cold water pipe)

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Overpressure to cleanroom area

Overpressure system and piping on top of cleanroom

Cleanroom: HEPA filtered air in and outwww.mycelia.be 46

Overpressure system:critical points

• Correctly calculated turbine for desired overpressure

• Number of air exchanges per hour sufficient forUSFS class 10000

• HEPA filters with adequate filtering capacity• HEPA filters checked on obstructions• Perfect seals• Frame checked on leaks• Frequent change or cleaning of prefilter• Regular cleaning and disinfection of tubing

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Cleanroom area:filter maintenance

First prefilter: Second prefilter: HEPA filter:weekly yearly when clogged

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Floors and walls well finished

Flaking paintLoose edges

Smooth walls and floor Sealed edgeswww.mycelia.be 49

Taps and drains are forbidden

Taps: Drains: create aerosolssource of infections

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3. RULES OF CONDUCT

Microorganims, shed by people

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The number of particles spread by men per minuteis proportional to their activity

100 000 P. 500.000 P. 1.000.000 P. 2.500.000 P.5.000.000 P. 10.000.000 P.15.000.000 P.

relative rest regular movements heavy work

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Dust particle measurement with peopleParticles of 0,5 µ and more

Jogging suit: Lab coat: Complete outfit:cotton densely woven densely woven

polypropyleen polypropyleen

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Measuring dust- production by people per minuteParticles of 0,5 µ and more

Motionless: 900.000 p./ min. 350.000 p./ min. 29.000 p./ min.

Moving: 35.000.000 p./ min. 6.000.000 p./ min. 106.000 p./ min.(0,3%!)

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People are important vectorsof infection

Hence rules for cleanrooms:

• Restricted staff• Sterilized coveralls• Strict rules of conduct• Focus on training

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Cleanroom environment

Transfer of people

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Protocol for people’s transfer

Remove outerPut rubber gloves Put hairnetclothes and shoes

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Put sterile overall Close zipper entirely Disinfect gloves

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Tie mouth mask Put cap Put shoes

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Disinfect gloves Rub hands Disinfect shoes

Enter cleanroomwww.mycelia.be 60

Cleanroom environment

Rules of correct behaviour: examples

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Examples of correct behaviour

Bare skin Complete protectionwww.mycelia.be 62


Torn glove Change glove

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Skin touched Disinfect hands

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Critical step: emptying autoclave

Trolleys are disinfected after use Charts unloaded in sterile outfit

Substrates cooling in separate area Sterile overpressure essential

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Critical steps: inoculation and sealing

Body and hands are always away from the flow,behind the spawn bags

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Examples of wrong behaviour

Hands between LAF and bag Hands over the bag opening

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Cleaning and disinfection

Cleaning tools Correct choice Correct dosageare kept in the sluice of disinfectantia of disinfectantia

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Surface disinfection: all the time

working surface shelves trolleys

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Surface disinfection: all the time

Before cleaning After cleaning

Before cleaning After cleaningwww.mycelia.be 70

Disinfection and cleaning after work

Remove production rests from corners and hidden spaces

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Disinfection and cleaning after work

Vacuum cleaning: only at the end of the day

Disinfecting floors: no drying!www.mycelia.be 72

Production waste

Waste is dropped in Bag is tightly closed garbage is removedclosed garbage can. after production daily.

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Result of bad cleaning

Organic matter on the floor Spiders and insects

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Examples of bad hygiene

Cooling room: Inoculation room:bad hygiene conditions dirty pedal of sealer

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Air quality control

the air quality of a number of important samplingareas shall be regularly tested

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Before starting a myceliumproduction unit

• Consult thrustworthy sources:– Books– Internet

• Follow a training with teachers:– With a scientific background– Having industrial experience

• Make a design• Make a cost-benefit analysis• Study the market opportunities

• Decide if it is worth the investmentwww.mycelia.be 77

Thank you for your attention!

Mycelium producer and training centerVeldeken 38a, 9850 Nevele, Belgium

www.mycelia.be

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microbiological issues -...

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