THOUGHTS ABOUT MICROBIOLOGICAL SUBSTRATE SELECTIVITY IN PICTURES

Of old I prepared a substrate by hydrothermal method in boiling water. Eventually I started to reduce the temperature of process. Afterwards I decided to blow the filtered air through a treated warm substrate and heat it at the temperature upper than 40°C for some time.

On the mushroom farm “Saturn” (Russia) there was no clean inoculating room and so I tried soft conditions for pasteurization of the wet substrate in the farm blenders and maintain the substrate at the temperature about 45°C. The technology was reliable when the qualitative waste materials were used.

The Russian company “VEK” already had a clean inoculating room and incubating room with air recirculation and filtration. There we also practiced the same method, the sterilized air was fanned to the farm blenders and then the substrate was maintained at the temperatures of about 45°C.

Hereby, step by step I arrived at a conclusion that a substrate should be fermented. After several experiments on different mushroom farms, I began to think that a good substrate of oyster mushroom is possible to prepare by fermentation in tunnels, but only by wholesale.

Then I was invited to the Ukrainian farm to arrange a substrate production. The wet wastes in BAGS were pasteurized in the room with capacity of only 2,5 tons of substrate! The decision was in soft pasteurization+ fermentation+ clean inoculating and incubating rooms. As it turned out, a selective substrate can be prepared in small!

The next pasteurization room was even smaller, only 1 ton of substrate! And the same method was applied.

So, a substrate for oyster mushroom can be prepared by fermentation in any amounts. The point is in an economical expedience.

As far as sterilization, it has been out of my capacity until 2005-2006 years. These years were filled with the remarkable events. They are:

- first publications about Chinese sterile technologies of substrate preparation for cultivated mushrooms;

- my correspondence with Wang Shigiang, a professional mushroom technologist from Shanghai;

- I was invited to the first Russian sterile mushroom farm to eliminate a reject. The reason of reject was in incorrect design, management and absence of actual clean zone. We succeeded.

As a result, I realized that both technologies, sterile and not sterile, can be used. The

point is in professionalism. But the last word is in manufacture stability and economy.

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SUBSTRATE MICROBIOLOGICAL SELECTIVITY. OPTIMISTIC CHAPTER

The processes required for obtaining microbiological selectivity for oyster mushroom

substrate should be solid state aerobic fermentation. It means that we want to receive sort of protective layer of thermophile aerobic microorganisms on the surface of the substrate which will discourage all competitors of oyster mushroom. These aerobic microorganisms should assimilate nutrients and thus don’t give nutrients to competitors.

Please, pay attention to Picture 1.

Picture 1

The grain of oyster mushroom mycelium (Chinese Black strain) is put in Point 1. In 4 days, when the oyster mushroom colony is 3 cm of radius, Trichoderma is

added to Point 2. The picture is taken in 4 more days of thermostation. (the diameter of the Petri dish is 98 mm on all pictures)

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In the Petri dish with the standard potato glucose agar medium we put the grain of oyster mushroom mycelium of Chinese Black strain (further this strain is on all pictures). In 4 days of thermostation at the indoor temperature, when oyster mushroom colony has grown approximately to 3 cm of radius (Point 1), Trichoderma culture was pricked on the opposite side of the dish (Point 2). The Picture was taken in 4 more days of thermostation. It is clearly seen that as Trichoderma was growing, the mycelium threw back first and then, after colonies had contacted, it stopped. Sort of protective layer was formed.

Let us change the experiment and put a pinch of aerobic fermented substrate in the Petri dish with the same medium. Let us thermostate it during 3-4 days at the indoor temperature to find out if there are bacterial colonies there. Picture 2 shows that our substrate contains a lot of bacterial microflora.

Picture 2

In the Petri dish with potato glucose agar medium a pinch of aerobic fermented substrate was put. The dish was thermostated during 4 days at the

indoor temperature.

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Now let us place Trichoderma (the same as on Picture 1) in this dish on the surface of medium. Let us again thermostate it within 4 days. And look at Picture 3. The star in the circle is the point where Trichoderma was added. There are no signs of Trichoderma growth. Only the color of the bacterial colonies has changed a little and the gaps between them have grown down.

Picture 3

The same Petri dish as on Picture 2. In the marked point Trichoderma is added. The dish was thermostated within 4 more days. There are no signs of

Trichoderma growth. Only the color of the bacterial colonies has changed and the gaps between them have grown down.

Probably, the current substrate lot wasn’t microbiologically selective. The experiment

was incorrect. We put the grain of substrate on the nutrient medium, and then within 4 days we maintained almost ideal conditions for microflora growth. And only after that we added Trichoderma culture. To my mind, the experiment showed only a potential microbiological selectivity of the substrate. Let us check it.

The next substrate lot is taken. Let us add at a time the grain of oyster mushroom

mycelium (Point 1) and Trichoderma culture on the opposite side (Point 2) and thermostate them within 5 days. Look at Picture 4. On the 3rd day the radius of the oyster mushroom colony is about 25 mm and, as it seems to me, its growth has stopped. In the morning of the 5th day Trichoderma touched the colony of oyster mushroom mycelium. The picture is taken in the evening of the 5th day. Trichoderma still “grabs” the colony of oyster mushroom mycelium.

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Picture 4

The grain of oyster mushroom mycelium is placed in the Petri dish with potato glucose agar medium (Point 1). At a time Trichoderma culture is put in Point 2.

The dish is thermostated within 5 days. In the Petri dish with potato glucose agar medium I added a pinch of substrate from

the tunnel. Then I shook it and threw out the substrate. I tried sort of dirty the surface of agar medium by the substrate microflora. Then in Point 1 (see Picture 5) I put the grain of oyster mushroom. And in Point 2 I pricked Trichoderma culture. The next step is thermostation within 7 days. The result of the experiment is on Picture 5. The growth of oyster mushroom colony has changed in comparison with its growth on the clean potato glucose agar medium. The mycelium is growing in sort of “plumage”. The radius of its colony on the 5th day is about 45 mm. After that, as it seems to me, the growth has stopped. Trichoderma mycelium is growing in the separate winding “branches”. In the morning of the 7th day Trichoderma and the mycelium of oyster mushroom contacted. The picture was taken in the evening of the 7th day.

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Picture 5

In the Petri dish with potato glucose agar medium the oyster mushroom substrate from the tunnel was added. The surface of the nutrient medium is sort

of dirtied by the substrate microflora. In Point 1 there is a grain of oyster mushroom substrate. In Point 2 Trichoderma culture is pricked. The picture was

taken in the evening of the 7th day of thermostation. The radius of oyster mushroom colony before it connected with Trichoderma mycelium had reached

45 mm. Compare the picture with Picture 4.

I wonder how the mycelium of oyster mushroom will grow in the same conditions but without Trichoderma. There is the same Petri dish with potato glucose agar medium, the same substrate and mycelium of oyster mushroom. Look at Picture 6. The picture was taken in the morning of the 7th day. The “plumage” growth of mycelium is clearly seen there (pay attention to the green circle I marked. Soon we will return to it).

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Picture 6

The surface of potato glucose agar medium is “dirtied” by the microflora of fermented substrate. The grain of oyster mushroom mycelium is put in Point 1. The dish is thermostated within 7 days. The picture was taken in the morning of

the 7th day. Compare it with Picture 7.

Compare the result once more with Picture 4 and 5. It inspires, isn’t it? I wonder how the mycelium of oyster mushroom keeps on growing in our

experimental dish. Look at Picture 7. This is the same dish as on Picture 6. Pay attention to the selected fragment. Another day has passed. It is the morning of the 8th day. Sure enough, the mycelium is growing in sort of “plumage”. But it tries to grow on the bacterial colonies and avoid the clean zones of potato glucose agar medium!

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Picture 7

The same dish as on Picture 6 (the selected fragment). Another day has passed. The mycelium of oyster mushroom tries to grow on the bacterial colonies and

avoid the clean zones of potato glucose agar medium!

A day after the mycelium covered almost all surface of the dish. So, we can assume that the fermented substrate of oyster mushroom shows

microbiological selectivity towards Trichoderma. And can we say about other mold contaminants?

I separated one fast-growing mold among the suspicious whitish spot on the

substrate block. The colony of clean white color quickly (within 2 days) seizes the whole surface of potato glucose agar medium in the Petri dish. And during a night it turns in the cottony dirty gray formation.

Let us try to add the culture of this mold in the Petri dish with potato glucose agar medium and fermented substrate of oyster mushroom immediately after the substrate was put. Look carefully at Picture 8. You can also see several olive green colonies of molds. It is no wonder as the sample was taken from the substrate block and was added to the Petri dish directly in the growing room. The picture was taken on the 5th day. The colonies of molds almost stopped in growth on the 3rd day. And after that they didn’t grow too. On Picture 9 you can see the fragment of the same dish on the 9th day. Even on the 30th day (after my return from the trip) they didn’t grow!

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Picture 8

The growth of molds stopped on potato glucose agar medium with fermented substrate of oyster mushroom. The picture was taken on the 5th day of

thermostation. On the control dish the gray cottony mold seized the whole surface in the morning of the 3rd day.

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Picture 9

The fragment of the same dish as on Picture 8. The picture was taken on the 9th day. The growth of colonies has stopped on the surface of medium.

So, the microflora of fermented substrate completely stopped the growth of these molds. And other molds?

In 2002 I collected pure cultures of mold contaminants from mushroom productions. The growth of the molds I had managed to check was retarded by the microflora of fermented substrate.

PESSIMISTIC CHAPTER. RATHER REALISTIC

So, we produced a fermented, selective substrate. But I wonder why there is a reject

of substrate. I suppose there are 2 groups of reasons:

MYCELIUM In fact, a reject of mycelium connected with the quality of mycelium doesn’t refer to

the topic of the article. Anyhow, let us describe them shortly. A MYCELIUM ISN’T SPREAD EVENLY I propose to look at Picture 10 where it is shown, and then at Picture 11

(consequences).

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Picture 10

Uneven spread of mycelium in the substrate.

Picture 11

The result of uneven spread of mycelium in the substrate. No chances to better it.

USE OF OUTSTANDING MYCELIUM If the outstanding mycelium you use was stored without violation of temperature

conditions, you might receive very slow process of growth. If there are no problems with sanitation on your farm, then the block will be white, without any visible defects. However, late fructification and poor yield are guaranteed.

USE OF MYCELIUM WITH STROMA Use of mycelium with stroma is fraught with stroma on a substrate block. The result

of it is unpredictable date of fructification and unpredictable fall of yield. Sometimes some mushroom growers try to remove stroma films manually when

chopping mycelium. Sometimes it really helps, sometimes it helps partly. Look at Picture 15.

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Picture 15

The mycelium with stroma was used during inoculation. The films were thoroughly removed manually. It almost helped. But the harvest of the first flush was below 35 kg of mushrooms per a ton of substrate in comparison with control

at the same lot of substrate. USE OF POOR MYCELIUM Please, read my article “Tactics of work with mycelium”.

INFECTION OF SUBSTRATE Let us look once more at Pictures 1-9. We receive a potentially selective substrate.

It is very important not to infect it before its full growth of microflora and its colonization by oyster mushroom mycelium.

It means that it is necessary to avoid: - infection of substrate by contaminants during the process of substrate

preparation; - infection of substrate during inoculation; - infection of substrate during transportation or transfer of inoculated substrate

blocks to an incubating room. - infection of substrate blocks in the first days of incubation. Such infection can be easily evaded. The following things are necessary: - overpressure inside a tunnel or any equipment for substrate preparation; - filters on air supply system in a tunnel or substrate machine or farm blenders; - clean inoculating and incubating rooms; - perforation of formed substrate blocks only after their transportation to an

incubating room and thorough disinfection;

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- drop in air humidity in incubating rooms (though I don’t support this step, but sometimes it is the simplest method to reduce a risk of substrate infection during incubation).

NOTE: Dear colleagues, if you are interested in the pictures illustrating infection of

fermented substrate during inoculation, incubation, etc., please feel free to write me. I will send them to you. My e:mails are viktor_yakush@mail.ru and viktor_yakush@yahoo.com.

But I know the farms where all these measures are more or less observed.

Nevertheless, they also have rejects of substrate. What are the reasons of it? SUBSTRATE COULD NOT REALIZE ITS POTENTIAL SELECTIVITY Actually, we have received a potentially selective substrate. With the help of our

experiments in the Petri dishes we made sure of its potential. However, in dishes we create almost ideal conditions for microflora growth: temperature, conditions for interchange of gases. But we don’t create the conditions necessary for microflora growth when we work with a real substrate block. As well as for the growth of oyster mushroom mycelium!

So: VIOLATION OF INCUBATION TEMPERATURE CONDITIONS There are optimal temperature conditions of substrate incubation. The optimal

temperature in the substrate during its incubation is from 26 to 28°C. The substrate is allowed to heat up to 30-32°C for 2 or 3 days or up to 35°C for a day.

Please, look at Pictures 12;13;14.

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Picture 12 The picture shows overheat of the substrate up to 32°C for 2 days. There is a

small area of the secondary growth in the center of the block. The fall of harvest might not happen. (It is the picture of the cross section of the block).

Picture 13

This block was overheated up to 37°С for a day. There is a zone of “burned” mycelium and a zone of the secondary growth in the center of the block. I can’t predict the forecast of harvest for this substrate (It is the picture of the cross

section of the block).

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Picture 14

The mycelium was growing during a month at the substrate temperature of 18°C. No comments.

TOO WET SUBSTRATE When we speak about optimal humidity of the substrate, as usual we refer to

humidity necessary to receive maximum harvest. Now we speak about high humidity of the substrate at which the normal conditions of gas exchange in the substrate block are violated in the first days after its inoculation. .

The attributes of too wet substrate are: - liquid formation in the bottom parts of block; - spots where the mycelium couldn’t grow; - even after mycelium growth the block remains slow and soft; - even after obtaining a white block we receive late fructification. Look at Picture 16.

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Picture 16

The attributes of the growth in too wet substrate are: - there is a liquid in the bottom part of the block; - there are spots where mycelium couldn’t grow; - the block is slow and soft; - even after a full growth and obtaining a white block there is a late

fructification.

I will make several examples. Russian company “Saturn” has optimal humidity in the range of 65-68% for various

substrate receipts and strains. Russian company “VEK” drew a conclusion that the optimal humidity of the substrate

that will give 2 maximum flushes is 70-72%. In Russian company “Kaskad” in 2002 the best results of the harvest were obtained

at the substrate humidity of about 72%. On the farms where composed substrates (hull and straw) were used, the optimal

substrate humidity was dropping if there was more part of hull in the composition. According to the words of business partner of Italian company “VIERRIBI” Lucho

Ranzato, the optimal humidity of the substrate that will give 2 quick flushes is 70-72% (for various receipts of substrate).

The optimal humidity of the substrate in Spanish company “FungiSem” is 70%. TOO PACKED SUBSTRATE Too high density of the substrate is another way to prevent a preparation of the

substrate with normal gas exchange, with aerobic conditions in the block during the period of its microflora growth and its colonization by oyster mushroom mycelium.

Look at Picture 17. The humidity of the straw substrate is about 73%. It is quite enough for the good growth of block. But the substrate wasn’t filled evenly and condensations are well felt by fingers. Normal gas exchange is violated exactly in these places and problem zones appear.

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Picture 17

The substrate block isn’t filled evenly. There are places with high density; they are easily filled by fingers. Conditions of gas exchange are violated exactly in

these places. The mycelium grows too slow there if at all.

What is the optimal density of the substrate block filling? According to my experience: - Russian company “Saturn” tried to receive the density of no more than 0,45 kg per

liter. But sometimes they didn’t manage as the substrate was much chopped in the farm blenders.

- in Russian company “VEK” in 2002-2004 the density of filling was about 0,45 kg per liter. The reason is the same.

- Russian company “Novoselki” grew oyster mushroom on shelves in the growing rooms for Agaricus. They had permanent problems with the harvest of the second flush when the density of the substrate was less than 0,32 kg per liter.

- Ukrainian company “Tavriya Agro Capital” in 2005-2006 had 0,4 -0,45 kg per liter. - in Russian company “BIRG” the density of substrate block filling was 0,4 kg per liter

according to the engineering manual.

According to the literature and communication: - the density of filling in Hungary is about 0,35 kg per liter; - technologist of French company “EuroSubstrate” recommends 0,35-0,4 kg per

liter; - Lucho Ranzato from Italian company “VIERRIBI” said the density of filling is 0,4-

0,42 kg per liter.

I would recommend you to run documentation thoroughly and choose optimal parameters exactly for your substrate, but to start from the specified values.

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Unfortunately, almost always there are several abnormities: too wet substrate, too high density of filling, bad mixing of substrate with mycelium. In this case a grower can only guess if it will grow or not.

Earlier I photographed the history of the problem blocks. Sometimes the mycelium in the spots had grown only by the end of incubation or directly in the growing room. If eventually the block was white I went into raptures over the endurance of oyster mushroom! Despite of the adverse conditions the substrate had a strong growth of mycelium!

But then I began sampling of that white that grew at last on the spots and was

shocked. The white mycelium in the spot doesn’t mean necessarily the spawn of oyster mushroom. Just then I discovered a mold which you could see on Pictures 8 and 9.

Almost all molds have white mycelium. Organs of sporification are usually colored. And Trichoderma mycelium is white in vegetative period (see Points 2 on Pictures 4 and 5). And the dirty cottony mold on Pictures 8 and 9 was of clean white color in the first two days of the growth in the dish.

So, there is no sense to cause difficulties for the growth of oyster mushroom. ABSENSE OF AEROBIC CONDITIONS FOR SUBSTRATE FERMENTATION IN A

TUNNEL It is one of the most difficult questions. Aerobic fermentation of the substrate means

certain oxygen concentration in the gas phase of substrate during its solid state fermentation. The question is how to measure the oxygen concentration?

The third year I work with the specialized meter of Mickey Foley design (Mushroom Climate USA). Besides, there are a lot of sites in the Internet which sell a great amount of devices for measuring oxygen concentration in the gas phase of the substrate. You should only prepare air properly, cool and drain it.

According to recommendations on building of tunnels in magazines, we can draw a conclusion that the recommended air consumption is in the range of 200 to 300 m3 per hour of a ton of loaded substrate. The part of the fresh air is from 5 to 10% during fermentation period. I guess these are good recommendations. I would also recommend maintaining the specific air consumption NOT LESS than 300 m3 per hour of a ton of the substrate with the part of the fresh air not less than 5% during pasteurization and NOT LESS than 15% during fermentation period if your tunnel is properly designed.

Unfortunately, there is a small amount of such tunnels. Sometimes only oxygen meter allows adjusting a tunnel. At the end of 2006 I had to

adjust such a “strange” tunnel in Ukraine. And we succeeded! So, if you only design a tunnel, invite a specialist. Do you need a meter? Yes, it

would be desirable during the start and adjustment. I would also recommend big companies to purchase it. It costs approximately $500-600.

To conclude, any technology can be used. You should only know well the processes

and be a serious and scrupulous specialist. Viktor Yakushenko Oyster mushroom consultant Russia Ukraine

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