Welcome!

If you’re new to this site and topic, I suggest that you stop reading this article and recommend that you read first the following three articles. In these articles, I put all my effort to provide you the best and fast learning curve. More about the relationships of all the factors which make mushroom cultivation possible. I know its tempting but believe me, you will be confronted with many details which in the beginning are more confusing than helping.

I start with the basics (101) and then will dive deeper into the details with the next level (201). At level 301 we are really down to the nitty-gritty of mushroom cultivation and dissecting research papers to learn.

1. Things to Know Before Starting a Mushroom Farm
2. How the Substrate Influences your Mushroom Yield
3. How your Sterilization Method will Impact your Mushroom Yield

With that said, its inoculation time.

When it comes to inoculation, there are two methods

• Dry inoculation and
• Wet or liquid inoculation.

Dry inoculation means that for the transfer of the mycelium, typically certain types of grains are used. In the case of liquid inoculation, the mycelium is grown within a nutrient-rich solution.

Each one of them has its pros and cons, but for most mushroom growers out there dry inoculation is the way to go.

To understand the process of inoculation better, we will talk about

• the inoculation rate (spawn rate)
• if we should seal or not seal the bags
• if we should mix or not mix after inoculation
• how we should document what we are doing
• and about scheduling, so we don’t run out of new spawn.

In this article, I will only address the first three points. The other two can be found here and here.

INOCULATION RATE

How much Spawn and Supplement should I use?

The author investigated the influence of the spawn rate and commercial delayed release nutrient levels on Pleurotus cornucopiae (No. 608) yield, size, and time to production.

For the substrate, the author used cottonseed hull (75%), wheat straw (24%), and lime (1%). The wheat straw was chopped to a 5- to 15-cm length and watered to a moisture content of 68%.

The substrate was then steam pasteurized at 65°C for 45min, cooled down and then packed into 13.6 kg (crop I) or 9.1kg (crop II) bags of moist substrate (68%).

The substrate was then inoculated at various levels (1.25%, 2.5%, 3.75%, or 5% wet wt).

With that brief overview of the science paper, let’s find out what the author found. To do that, let’s look first for some seconds on the table xx. This table gives us an overview of all the results mentioned in the research paper for the crop I.

We can quickly identify the spawn rate and supplement level for each test run as well as the outcomes of them. But to understand these results better I out them into a surface diagram which helps us to spot the best results faster.

Table 1: Overview of all results from crop I[1]

The first diagram (Figure 1) gives us an inside into the influence of the spawn rate and supplement level on the days to production. Here we can clearly see that with lower spawn rate and lower supplement level, the number of days to production goes up. The fastest growth was achieved at a spawn rate of 5% and a supplement level of 6%.

Figure 1: Influence of spawn rate and supplement level on the days to production of crop I[2]

While a higher level of spawn rate and supplement level has a positive impact on the number of days to production, we see a clear negative correlation when it comes to the mushroom size (Figure 2).

By looking at the diagram, we see two primary impacts. The first is at the lower end of the spawn rate and the second is in the middle to the higher end of the spawn rate, but both impacts are dominant in the middle range of the supplement level. What do I mean? At a spawn rate of 1.25% and a supplement level of 3%, we see a spike (most significant size), and at a spawn rate of 3.75% and a supplement level of 3%, we see a valley (smallest size).

While the valley makes sense for me, the big jump at the 1.25%/3% mark looks more like an outlier to me.

If we excluded this point, we would get a beautiful even playing field when it comes to the mushroom size. Which would mean that both the spawn rate and the supplement level doesn’t affect the mushroom size.

Figure 2: Influence of spawn rate and supplement level on the size of crop I[3]

Next up on our list is the yield (Figure 3) and the biological efficiency (Figure 4). For both parameters, we see a similar result. Both diagrams are showing us a tilted rectangle with the highest point at the 5%/6% mark and the lowest score at the 1.25%/0% mark.

Figure 3: Influence of spawn rate and supplement level on the yield of crop I[4]

Both diagrams suggest, therefore, that the higher the spawn rate and the higher the supplement level, the higher the yield and biological efficiency.

But we also see that none of the achieved values were larger than 100%, which is as we already learned below the norm for the Pleurotus species (BE 100-200%). To understand this result better, we had to re-run this test, but this time with two or three different Pleurotus species in parallel and by using several other sterilization methods. As a last chance, we should take into consideration that the size of the wheat straw should be smaller.

If you want to understand the last part (influence of the particle size on the yield of Pleurotus) I go over a research paper that investigated this topic.

Figure 4: Influence of spawn rate and supplement level on the biological efficiency of crop I[5]

For the second crop, the author changed three variables. First, he used a smaller bag size (9.1 kg instead of 13.6 kg) and second, he increased the supplement level-up to 12%, and third, he cut back the spawn rate to a maximum of only 3,75%.

The results of these changes are shown in Table 2.

Table 2: Overview of all results from crop II[6]

The first diagram (Figure 5) gives us a similar result when it comes to the number of days to production. The best results were achieved at 2.5%/3% or higher values. This means that the higher the spawn rate and supplement level, the shorter the time it takes to get to production.

Figure 5: Influence of spawn rate and supplement level on the days to production of crop II[7]

If you look at the next diagram (Figure 6), you will see in the back a small green area that represents the largest mushroom size at the 1.25%/12% mark. On the opposite side in the front, you see a larger yellow area which represents the smaller mushroom sizes.

You may also notice that the range between the smallest and largest mushroom size is low. We are talking about only 3.6g. Which is similar to what we found for the crop I.

Figure 6: Influence of spawn rate and supplement level on the size of crop II[8]

The next diagram (Figure 7) is fascinating. It shows the impact of the spawn rate and the supplement level on the mushroom yield. First, we see that the higher both are, the higher the yield. With the maximum at the 3.75%/12% mark (dark blue area).

From this area, everything falls down. The range is with about 2.6 kg huge, especially if you consider that the lowest yield was 0.75kg. It could be an outlier like we saw with the crop I, but removing it from the diagram doesn’t change it that much.

Figure 7: Influence of spawn rate and supplement level on the yield of crop II[9]

When it comes to biological efficiency (Figure 8), we a similar behavior. The Rang is with 87% very high. This is due to the lowest BE of just 23%.

Figure 8: Influence of spawn rate and supplement level on the biological efficiency of crop II[10]

What is the best Spawn rate for Oyster Mushrooms

The author of the next science paper explored the impact of different spawn rate on the growth and yield of Pleurotus ostreatus.

He used only wheat straw as a substrate which was first pre-conditions by boiling it for 15-20 minutes and then dried. The goal of this treatment was to get rid of insects and other micro-organisms.

The substrate was then moistened until a moisture content to 70-90% and filled in bags, each weighing 500g. The bags then were sterilized in an autoclave at 15 Psi for an hour. I assume that the author used the standard temperature of 121°C for this treatment.

After the cool down the bags then were inoculated with sorghum grain spawn at different rates. (10-100g per kg substrate dry wt, 10g/step).

Okay, now let’s interpret the results (Table 3). By looking at this table, we can already recognize that with a higher spawn rate, we get better results. But let’s discuss each quality factor more in detail.

Table 3: Overview of all the results[11]

The first diagram (Figure 9) shows the duration until the pinhead formation. While in the beginning, we see only a small decrease in time, at the 6% mark, we see a sharp drop. But as we continue to increase the spawn rate, the time starts to rise again.

According to these findings, the optimum spawn rate would be 6-7%.

Figure 9: Influence of the spawn rate on the pinhead formation (days)[12]

With the next findings, the author studied the influence of the spawn rate on the rest phase between the flushes (Figure 10). Which is for me a new approach. While in the beginning, the rest phase varies a lot with the spawn rate, the time between two flushes almost doubled at the 5% mark. Interesting enough after this jump, the rest phase doesn’t change that much.

The optimum spawn rate regarding the time between two flushes (rest phase) would be 2-4%.

Figure 10: Influence of the spawn rate on the rest phase (days)[13]

The time from pinhead formation to maturation is short (Figure 11), and the variations are not that big either. But we still see a small decrease when increasing the spawn rate.

The optimum spawn rate would be 6% or higher.

Figure 11: Influence of the spawn rate on the maturation of fruiting bodies (days)[14]

The next diagram (Figure 12) gives us an inside into the number of flushes per bag. As clearly visible, the higher the spawn rate, the more flushes a bag generates. But if we overshoot (≥8%), we will experience a sharp drop.

Optimum spawn rate would be 6-7%.

Figure 12: Influence of the spawn rate on the number of flushes per bag[15]

Another interesting finding of the author is the number of bunches per bag (Figure 13). This number just grows each time we increase the spawn rate.

Optimum spawn rate: 10%

Figure 13: Influence of the spawn rate on the number of bunches per bag[16]

This is different when we look at the number of fruiting bodies we can get from each bag (Figure 14). Here the number starts increasing until we hit the 7% mark. After that, the number plunges down.

Optimum spawn rate: 7%

Figure 14: Influence of the spawn rate on the number of fruiting bodies per bag[17]

And finally, the influence of the spawn rate on the yield (Figure 15). Here again, we see an increase in the yield with a higher spawn rate. The maximum yield is achieved at the 7% mark.

Optimum spawn rate: 7%

Figure 15: Influence of the spawn rate on the fresh yield[18]

To put all our findings in perspective, I made a quick calculation of the correlation values for each quality factor (Figure 16). As we already know, a higher spawn rate is negatively correlated with the number of days for pinhead formation and maturation, but positively correlated with all the other factors.

Figure 16: Correlation between spawn rate and different quality factors[19]

The optimum spawn rate according to this research paper would between 6% and 7%.

What is the best inoculation rate for Liquid Cultures

The next research paper we will analyze explored the development of an alternative technology for the oyster mushroom (Pleurotus ostreatus DSM 1833) production using liquid inoculation by comparing the liquid method with the solid method.

The author used banana straw as a substrate, which was ground and dried at 60°C for 1h. After bagging the substrate, the bags were then submerged for 12h underwater. After eliminating the excess water, the substrate was weighed and supplemented with 5% (on a dry weight basis) with rice bran.

These bags were then sterilized in an autoclave for 1.5h (temperature assumable 121°C).

After the cool down the bags were then inoculated with 5, 10, 15, or 20% via solid or liquid inoculum.

To make the comparison a little bit easier, I used similar colors for each inoculation method (Figure 17). If we look at this diagram, we find that for the solid inoculation method, there isn’t much change at all. The range of biological efficiency is around 1% for both harvests. If we look at the liquid inoculation method, we see for both harvests an increase in biological efficiency with increasing the inoculation rate.

The best inoculation rate for solid inoculation would be between 5% and 20%.

The best inoculation rate for liquid inoculation would be between 15% and 20%.

Figure 17: Influence of the inoculation rate on biological efficiency[20]

The author then investigated the influence of the inoculation rate on productivity (Figure 18). Here we find for the solid inoculation method that the more we increase the inoculation rate, the lower the productivity gets. With the 15% mark the lowest point. This changes with an inoculation rate of 20%. Here the productivity jumps back.

If we analyze the results of the liquid inoculation method, then we see with increasing of the inoculation rate an increase in productivity.

The best inoculation rate for solid inoculation would be 5%.

The best inoculation rate for liquid inoculation would be 20%.

Figure 18: Influence of the inoculation rate on productivity[21]

Next on our list is the impact of the inoculation rate on the yield (Figure 19). Here we get a similar result as described with productivity. While it shrinks for the solid inoculation method, it plateaued for the liquid inoculation method.

The best inoculation rate for solid inoculation would be 5%.

The best inoculation rate for liquid inoculation would between 10% and 20%.

Figure 19: Influence of the inoculation rate on the yield[22]

Now let’s glance at the correlation factors for each inoculation method (Figure 20 to Figure 22). As surprisingly it is, the results of this science paper shown a negative correlation of the solid inoculation method for the biological efficiency, yield, and productivity. But a positive correlation for the liquid inoculation method.

These findings are the total opposite of what I read and heard while searching about how to grow mushrooms.

Figure 20: Correlation between the inoculation rate on biological efficiency[23]

Figure 21: Correlation between the inoculation rate on the yield[24]

Figure 22: Correlation between the inoculation rate on productivity[25]

Besides the spawn rate the spawn age will influence the yield of your mushrooms. In the following video I will talk exactly about this topic.

How to find an alternative Substrate for Oyster mushrooms

In our next science paper, the author wanted to find a cost-effective alternative substrate for the cultivation of Pleurotus cornucopiae 608. The author used 1) switchgrass (Panicum virgatum, 99%) as the primary substrate, chopped it into 5-10cm long pieces and added 1% (dry wt) of ground limestone to it. The moisture content was increased to 68%. The sterilized substrate was then supplemented at various levels (0% or 6%).

For the second run, the author used pasteurized cottonseed hulls (75% dry wt), 24% chopped wheat straw, and 1% ground limestone (all wt/wt). The sterilized substrate was then non-supplemented or supplemented with seven levels of delayed-release nutrient (Campbell’s S-41).

The mixed substrates were air-steam pasteurized at 65°C for 45min.

Now let’s look at the findings.

#Crop I

Table 4 gives us an overview of the results of the first crop. The side by side comparison of the two substrates as well as the different supplement levels and spawn rate gives us a first inside about the impact of them.

Table 4: Overview of the results from crop I[26]

The first figure (23, top) shows the influence of the spawn rate and the supplement level on the days to production (NDTP) if cottonseed hulls plus wheat straw were used as a substrate. The playing field starts dropping as soon as the spawn rate is increased. The fasted NDTP was achieved at the 5%/6% mark (orange area).

For switchgrass (Figure 23, bottom) the drop of the NDTP already starts at the 3.75% spawn rate mark for both 0% and 6% supplement level (orange area).

Figure 23: Influence of the spawn rate and supplement level on the days to production for cottonseed hulls plus wheat straw (top), and switchgrass (bottom)[27]

The next figure (24, top) gives us an inside into the influence of the spawn rate and supplement level on the mushroom size if cottonseed hulls plus wheat straw was used as a substrate. If you look at the graph, you will notice with the increase of both spawn rate and supplement level the mushroom size drops (orange). The biggest size for cottonseed hulls plus wheat straw was achieved at the 2.5%/6% mark.

The area for the mushroom size on switchgrass (Figure 24, bottom) looks quite even. Only on the edges 2.5%/0% and 5%/6% the size drops.

Figure 24: Influence of the spawn rate and supplement level on mushroom size for cottonseed hulls plus wheat straw (top), and switchgrass (bottom)[28]

The impact of the spawn rate and the supplement level on the yield can be seen in figure (25). As you notice, there is a large area (Figure 25, top, orange) in which the yield ranges from 2kg to 4kg. The highest yield for cottonseed hull plus cottonseed hulls was achieved at the 3.75%/6% and 5%/6% mark.

For switchgrass (Figure 25, bottom) the highest yield was achieved after the 3.75% spawn rate mark.

Figure 25: Influence of the spawn rate and supplement level on the yield for cottonseed hulls plus wheat straw (top), and switchgrass (bottom)[29]

The influence of the spawn rate and the supplement level on the biological efficiency (BE) for cottonseed hulls plus wheat straw (Figure 26, top) and switchgrass (Figure 26, bottom) are quite different. While for cottonseed hulls plus wheat straw the highest BE was with over 100% achieved at the 6% supplement level (Figure 26, top, grey area), the BE for switchgrass (Figure 26, bottom) doesn’t exceed 30%.

Figure 26: Influence of the spawn rate and supplement level on the biological efficiency for cottonseed hulls plus wheat straw (top), and switchgrass (bottom)[30]

To get a better idea of the influence, I calculated the correlation between each factor (Figure 27). For cottonseed hulls plus wheat straw (Figure 27, top) we see a positive correlation for yield and BE and a negative correlation for size and NDTP independent of the supplement level.

Switchgrass (Figure 27, bottom) shows a little bit of a different picture. Here we see for all factors a positive correlation at a supplement level of 0%. For the 6% supplement level, we get a positive correlation for yield and BE, and a negative correlation for size and NDTP.

Figure 27: Correlation factors of the spawn rate for different supplement levels on different quality factors for cottonseed hulls plus wheat straw as a substrate (top), and switchgrass (bottom)[31]

#Crop II

For the second crop, the author set the spawn rate to 3.75% and only varied the supplement level. This time in more steps. The results are shown for the cottonseed hulls plus wheat straw in table 5 and for the switchgrass in table 6.

For both substrates, we see an apparent increase in yield and biological efficiency. While the mushroom size doesn’t change that much for the first substrate (cottonseed hulls plus wheat straw), but for the second substrate (switchgrass).

Table 5: Overview of the results for cottonseed hulls plus wheat straw from crop II[32]

Table 6: Overview of the results for switchgrass from crop II[33]

These findings are also shown in the correlation factors (Figure 28). The only difference between the two substrates can be found in the NDTP. Here we see a negative correlation for the first substrate (Figure 28, top) and a positive correlation for the second substrate (Figure 28, bottom).

Figure 28: Correlation of the supplement level and … for cottonseed hulls plus wheat straw (top) and for switchgrass (bottom)[34]

The Influence of SEALING and Mixing

After all this deep dive-in into inoculation now its time that the bags or the bottles get sealed.

But is this necessary?

To answer this question, we must first address why we seal in the first place.

The main reason for sealing is, according to my findings, to avoid contamination. But if we seal the bags there will be no air exchange. As you know mushrooms produce a lot of CO2 and at a certain CO2 concentration the growth will stop.

For this reason, the high-end bags have a filter patch on them and even for the simpler bags they use cotton to cover the opening. While the high-end bags are not available everywhere and are expensive, and the low-end filter patch seams to work it raises the question, if we can avoid contamination without using filter patches at all?

After some research, I found these six steps which can reduce the contamination of the bags.

1. Make sure that the substrate is enough sterilized.
2. Work under a flow hood.
3. Use a higher spawn rate.
4. Do not mix after the inoculation.
5. Use fast-growing mushroom species.
6. Use smaller bags.

While some of these steps seem obvious others are not. Therefore, I will elaborate a little bit on each one of them.

The better you sterilize the substrate the lower the overall contamination level will be. This means the starting point (aka level of contamination) determines if the used mushroom species can outgrow the contaminations or not.

If you didn’t read so far, my article about sterilization, I highly recommend that you do it. In this article, I not only compared different sterilization techniques and methods but I analyzed them like I did for this article.

I will go over different research papers and their findings concerning the best sterilization method.

Next, by working under a flow hood you make sure that the air which will enter the open bags is as clean as possible. While working under a flow hood make sure that you personally wear clean clothes and sterilize everything.

For further reading I suggest that you go back to my article Things to know before starting a mushroom farm – Growing 101 and re-read chapter seven. In it, I focus specifically on the different vectors of contamination.

As we just learned a higher spawn rate increases the mycelium running rate (MRR). This helps your mushroom species to outgrow the competitors. If you want to learn more about the MRR you should check out my article How the substrate influences your mushroom yield – Substrate 201 (if you didn’t find time to read it so far). In it, I lay out the different factors which will impact the MRR.

The fourth steps seem to be counter-intuitive. But I came across this step while visiting two different mushroom farms. On the first, the owner switches from mixing to non-mixing. On the second, the farmer used only the non-mixing method. But what are the advantages of not mixing the substrate?

First and foremost, if you do it manually you save a lot of time if you don’t mix and therefore money.

Second, if the mycelium covers first the surface it will protect the substrate from contamination.

The use of a fast-growing mushroom species helps not only to suppress contamination but to save you time and therefore money. Consequently, if you can, choose always a fast-growing mushroom species if all the other factors are not influenced by this decision.

And lastly, if you use smaller bags you will support the mushroom species in outgrowing the competitor fungi.

Another interesting side note is that while in the US, the growers typically are using bags with filter patches and mix the substrate, in Asia the growers are using bags with plugs and no mixing at all.

I met a farmer back in Brazil who didn’t even was using a plug. He just folded the end several times and used a staple to close it. Is this the ultimate inoculation method? For him, it seems to work!

With that said I will leave you for now. If you are interested in the other two points you will find down the links to both articles.

• how we should document what we are doing
• and about scheduling, so we don’t run out of new spawn.

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[1] Own table based on Royse (2002)

[2] Own figure based on Royse (2002)

[3] Own figure based on Royse (2002)

[4] Own figure based on Royse (2002)

[5] Own figure based on Royse (2002)

[6] Own table based on Royse (2002)

[7] Own figure based on Royse (2002)

[8] Own figure based on Royse (2002)

[9] Own figure based on Royse (2002)

[10] Own figure based on Royse (2002)

[11] Own table based on Bhatti (2007)

[12] Own figure based on Bhatti (2007)

[13] Own figure based on Bhatti (2007)

[14] Own figure based on Bhatti (2007)

[15] Own figure based on Bhatti (2007)

[16] Own figure based on Bhatti (2007)

[17] Own figure based on Bhatti (2007)

[18] Own figure based on Bhatti (2007)

[19] Own calculation and figure based on Bhatti (2007)

[20] Own figure based on Silveira (2008)

[21] Own figure based on Silveira (2008)

[22] Own figure based on Silveira (2008)

[23] Own figure based on Silveira (2008)

[24] Own figure based on Silveira (2008)

[25] Own figure based on Silveira (2008)

[26] Own table based on Royse (2004)

[27] Own figure based on Royse (2004)

[28] Own figure based on Royse (2004)

[29] Own figure based on Royse (2004)

[30] Own figure based on Royse (2004)

[31] Own calculation and figure based on Royse (2004)

[32] Own table based on Royse (2004)

[33] Own table based on Royse (2004)

[34] Own figure based on Royse (2004)