Two mechanisms of mushroom feeding were considered in the implementation of the concept of controlled mushroom feeding. The first mechanism refers to a direct feeding by the enzymatic degradation of dead organic substances when the obtained nutrient ingredients are collected in the mycelium. Their amount plays a significant role in the production process and affects the yield in the first and second flush.

The second mechanism is the indirect feeding, consisting of the absorption from the compost easily soluble nutrients produced during the cold composting (commensalism). Thus, nutrients dissolved in water are transported through the mycelium to the fruiting bodies and therefore affect the yield in the third and fourth flush. The change of the feeding method results in substantial yield decrease. The average yields from these flushes do not exceed 5 kg/m². This situation raises the question as to whether the yields in these flushes, particularly the third one can be increased up to 10 kg/m². Two hypotheses were examined. The first one assumes that yield gain can be achieved through an increase of nutrient stocks during the direct feeding. It should guarantee the yield at the level of 40 kg/m² in two flushes and additional 5 kg/m², which in total could provide the yield of 50 kg/m² in the third flush. So far this concept has failed. An increase of nutrient stock could not be balanced efficiently with water, which was required to complete requirements of higher dosage of corn feeder. The compost was not able to absorb more water therefore the increase of water amount could have resulted in compost decay. Not balancing water needs caused a yield reduction due to the drying out of compost. The obtained result was opposite of the planned goal as the yield in the third flush decreased. As the second flush yield increased above 17 kg\m², a short-term retention of fruiting body growth occurred on the casing surface and caused a delay of their emergence from the deeper layers. This situation resulted due to the dying out mycelium on the casing surface during the second flush that was caused by the casing drying out. The production stabilized yielding (5-6 kg/m²) in the fourth flush when the fruiting bodies developed normally. Regarding this situation the second hypothesis of using a standard amount of corn feeder was tested. This approach assumes that the yield can be increased by an addition of monosaccharides and citric acid into casing. Also, in order to stimulate the growth of fruiting bodies an effect of changes of microclimate, mainly lowering an air temperature would enhance growth of fruiting bodies allow the obtained generations to be tested. The first results indicate that this approach might work and would help to collect additional yield above 5 kg/m² in the third and fourth flush up to a predetermined limit of 10 kg\m². It still requires some improvement and gradual implementation throughout the plant where the mushroom production and tests are carried out.

The development of this first concept has not been given up. These efforts to increase the compost water capacity and also using the possibility of soaking water placed in the bottom of the box lined with tightly foil have been undertaken.

Colonized substrate that contains sufficient volumes of water and nutrient ingredients will not guarantee high yields if casing does not support the feeding process. Several performed tests followed by the implementation of the concept regarding controlled mushroom feeding provided data that lead into the conclusions describing conditions that are required so that casing will not decrease expected yields. It is obvious that the casing must have a uniform composition and structure, and should be evenly applied; not higher than 5 cm. The intense production requires medium or heavy casing. Light casing is not recommended due to its low water holding capacity. The specific requirements supporting the process of feeding besides water holding capacity are the following:

1. Water shortage can not occur between casing application and end of the production process. Casing should be “shiny”, not matt. Water deficit causes mycelium drying and fruiting bodies, and in consequence a considerably decrease in the number of fruiting bodies particularly in the second and third flush. Water shortage worsens the quality of fruiting bodies, deprives their color and reduces yield by decreasing unitary weight: fruiting bodies are light. The mycelium should be white and alive on the casing surface during the entire production period. The addition of water into casing is easy, despite the growth stage of fruiting bodies irrigation will not worsen their quality as their nutritional requirements are fulfilled and their growth is controlled.
2. Dry bubble disease (or brown spot) develops more intense on weakened and yellow mycelium.
3. The casing must have a much higher salinity that is presently recommended and maintained, and it should be sustained at the same level throughout the entire period of cultivation. Appropriate salinity improves the quality of fruiting bodies and the yield by an increase of their mass and also pinning during flush is easier and more reproducible. Under these conditions the fruiting bodies are not exposed to short-term retention of their growth and respond better to activities aiming at the development of different generation stages. The salinity can easily be controlled, as there is a method, which allows taking direct measures from the casing. Effects of stable and higher salinity on mushroom yields in the third and fourth flush are expected.
4. Stable calcium content. Research studies indicate a significant role of calcium in the feeding. The performed tests confirm the usefulness of calcium chloride in the feeding process. First, the pH should be stabilized as its decrease can enhance a risk of green mold occurrence on the casing surface in the third and following flushes. It is probably easier to fulfill calcium requirements of fruiting bodies if its source is provided in the casing. The transport time is shorter than that from the substrate. Besides, often application of calcium chloride induces a reaction with concrete and causes sealing concrete pores. This significantly reduces the risk of survival of spores of causal agent of dry bubble disease, as the concrete is the main source of infection within the cultivation hall.

Two-layer casing

So far the individual tests with the two-layer casing were performed. The collected results indicate that it might be a potential useful solution. An application of a hydrogel filled with liquid nutrients is a new idea that will be soon examined. This should create conditions supporting more sufficient colonization of the casing and better connection of mycelium with the substrate, and also shorten transport of components into fruiting bodies and primordia.

The growing period during which the mycelium grows throughout the compost and the casing layer is known as the vegetative phase and must precede a feeding process. The vegetative growth refers to the colonization and enzymatic degradation of the substrate inhabited by a mushroom. The colonization occurs from the moment during which the mycelium contacts its surroundings, and ends when the colonized environment is fully overgrown. First, available environment structures i.e. compost and casing layer get covered by mushroom mycelium. This period is relatively short and usually takes about 3-5 days. The colonization rate depends mostly on the ratio of mycelium volume to the volume of the occupied environment, as well as the availability of usable carbohydrates, moisture, structure and temperature.  During colonization a gray mycelium develops covering substrate or casing layer and subsequently changing microflora within feeding environment.  The changes happen in two independent courses of action, the biosupression that is an elimination of unwanted microorganism, particularly antagonistic microorganism and the commensalism, which is a process favoring development of useful microorganisms, mainly Scytalidium thermophilum – in the substrate, and Pseudomonas putida – in casing. The entire process is dependent upon carbon dioxide content and the amount of hydrogen peroxide production. A feeding process begins when enzymes become active.  At the beginning of the process obtained nutrient ingredients are used to continue mycelium growth in order to control environment. Subsequently, nutrient ingredients that are obtained from enzymatic degradation and their transport to fruiting bodies cumulate in rhizomorphic mycelium (white, thick strings). This phase is considered complete with a transition into generative phase, and forming fruiting bodies stage begins.

The producers address the important question in regards to the duration of the time required for compost colonization that would provide the highest yields at well-balanced feeding conditions.

To achieve the best results the following conditions should be provided.

1. The colonization period should be short. Only fully colonized compost can be an effective source of substrate nutrients in enzymatic feeding. The duration of compost colonization by mycelium is a very important feature, which allows evaluation its selectivity. Quick colonization protects the compost against the development of unwanted, competitive organisms. Based on the conducted tests it has been concluded that the presence of soluble carbohydrates and the amount and form of used mycelium are the most significant factors. Shorter colonization time results in extended enzymatic feeding at the constant mycelium growing period. At the same time, loose and pliable straw creates a larger surface, which increases mushroom enzymatic activity.
2. Potential yield of fruiting bodies is determined by the mycelium mass developed in the compost. Consecutively the mycelium mass depends on a length of carbohydrate chains, that need to be degraded, amount of decay fungi in the compost and size of mycelium surface contacting compost. Cellulose, the main component in straw, is the most difficult element in enzymatic degradation. The process of starch degradation is much slower.  The increase in mass of rhizomorphic mycelium depends on the compost structure as well.
3. The temperature range of 23 – 27^oC and high CO₂ concentration support the quick compost overgrowth by mycelium.
4. The duration of the compost overgrowth process under production conditions is 12-18 days. Regardless of the unchanged length of overgrowing stage, this process can affect the yield level. In principle, a period that is too short will cause destruction in the process of biosuppresion and commensalism and there is a risk that none of them will complete. Therefore, the competing organisms will develop sooner and the temperature effect will occur. If the ingredients contained in the substrate are difficult to assimilate, the overgrowth period should be longer. Can this period be too long? A decision regarding when to end a digestion process and unwanted energy usage is difficult to make. Under the laboratory conditions mycelium mass has been growing up to 45 days.

Recolonization

It might be worthy to consider the introduction of a new term – recolonization, it is second growth of mycelium after breaking the compost removed from a tunnel or after mixing the compost on a shelf – at overgrowth phase II in production hall. This period is relatively short, 2 – 4 days and it depends on the existing temperature, water application and CO₂ concentration. Compost density is also very important. Excessive compaction of the substrate makes it difficult to increase its volume while too poor – makes it difficult to control the temperature in the substrate during shock (aeration).  Regarding further feeding, especially when introducing feeders, the control of the course of aeration is very important. Throughout this period, water is applied to the compost, and the thermal effect is controlled. Improperly applied water, along with air, low concentration of carbon dioxide in the compost and the absence or late reaction to the start of the thermal effect results in high substrate temperature increases. This causes a disturbance in feeding or in extreme cases leads to the overheating of compost.

Casing colonization

When fulfilled feeding is provided and water is well balanced in the compost, mycelium develops better and does not continue vegetative growth despite starting a shock. Thus pinning is easier. The colonization of casing and its role in feeding, particularly in cultivation without compost, and application of two layers will be examined after the establishment of a substrate content and an evaluation of substrate conditions without compost, based on fixed feeders.

In the past recent years it has been possible to balance the dose of a feeder in correlation to the water amount added into the substrate that would provide a full cover of nutrient requirements at a level of 35-37 kg/m2 in two flushes. However, it did not result in the stable and high yields.  Therefore a radical change in an approach to mushroom cultivation is in high demand. It means that there is a necessity to identify the causes of unstable yields in the current production practice and the findings that could help solve the existing issues. The substrate is not restricting a level and quality of mushroom yields but instead a production practice.

These are the identified problems that have been resolved so far.

1. Irregular air movement in cultivation room and above each shelf.
2. Significantly differentiated pH, calcium content and salinity of the casing layer.
3. Unrepeatable structure of casing layer at a time of shock initiation.
4. Simultaneous increase of large number of fruiting bodies in the second flush that resulted in the excessive harvest costs and worsening quality of collected mushroom caps.
5. Water shortage during harvest time resulting in the decay of mycelium and fruiting bodies on the casing layer surface particularly at the end of second flush.
6. Weakening of fruiting bodies in the second harvesting phase despite well-balanced nutrient requirements in both first and second flush.
7. Lack of reproducibility of fixed and sufficient number of fruiting bodies in the third flush.  In this case a yield was dependent on number of fruiting bodies not their weight. Besides, a substantial increase in the yield of third flush still poses a significant challenge.
8. Uncontrolled infections with Dactylium.

Despite unidentified problems, the final outcome was frequently affected by errors resulting from various quality of provided products, equipment failure etc. Currently one can introduce a concept of lost profits caused by sources within the company. Since it was determined that the substrate potential allows achieving high yields it has occurred that there is a necessity of defining second new definition regarding mushroom cultivation, which is a controlled feeding. The factors that can be controlled were already recognized. However, what procedure should be employed in regards of expected and sufficient nutrition effects is a new and difficult task.

The described difficulties have psychological nature as many rules of mushroom production have been recognized as inadequate in a new situation challenged by new issues. For instance, at present a producer needs to predict how fruiting bodies will perform within the next several hours and accordingly the microclimate parameters have to be adapted to meet the foreseen expectations. Generally this involves  “deterioration” and not “improving” a microclimate.

Human factor plays a significant role at expected increase of crops and currently used compost. This is due to the fact that at present the desirable results can be obtained without increases in costs and even with their reduction.  The elements that determine success are following: professional qualifications, ability to observe, correct conclusions regarding current happenings, commitment and time invest in production process.

Management of production with high yields will create a demand for an electronic control of mycelium conditions, fruiting bodies and primordia. This concept has been already developed. However, the potential producers are not convinced that the new technology and software would solve the existing problems. This production area is also looking to answer a question regarding if and how to introduce the robot operating systems.

At the end let me explain why it is so difficult to evaluate the quality of substrate and its production potential. Generally speaking, the producers own facilities, know technologies and have developed skills that would let them achieve yields at certain levels. Let’s consider a yield at the level of 30-32 kg/m2.  Usually the yield increases and improvement in compost efficiency become out of reach due to the lack of producer ability. In general, the high decrease in yields caused by worsening compost quality is observed than higher yields resulting from improved compost value. It explains the significant fluctuations in the yields noted for instance in the season of 2013/2014 due to the difficulties of adjustment of compost production technology to the variable quality of straw. Besides, investments in improving compost quality are ineffective if the water rate that would be required to utilize larger quantities of nutrition ingredients provided in better quality compost were not increased. There is a very strong resistance among mushroom producers against water application into the substrate and casing layer during harvesting. Thus, neither improvement of compost quality nor higher yields have been observed.

Two papers about the process of mushroom feeding provide new information and confirm findings that were presented in THE FUNDAMENTALS OF MUSHRROM FEEDING AND FARMING TECHNOLOGY DEVELOPMENTS.

Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus

Edita Jurak, Mirham A. Kabel, Harry Gruppen

Carbohydrate Polymers 101 (2014) 281-288  www.elsevier.com/locate/carbpol

I became interested in this paper because the presented data showed that water-insoluble xylans (for instance wheat flour, straw, stems, corn cobs and wheat oats) and glucans are degraded during mycelium growth into small particles soluble in water. During the mycelium growth, a process of degradation of these elements by mycelium was observed that resulted in increase of water-soluble products.  Xylans and glucans degraded by mushroom, according to literature, constitute the first source of carbohydrates for mushroom that later is utilized for primordia formation.

These results confirm that the established theory that the process of mushroom feeding might be improved by adding feeders that include corn meal after mycelium colonize the compost as well as by water-soluble carbohydrates, which are present in colonized composts.

Below I present a selection of the most practical information useful in the development of knowledge about the process of mushroom feeding that will also be an inspiration for further studies.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

Aleksandrina Patyshakuliyeva1†, Edita Jurak2†, Annegret Kohler3, Adam Baker4, Evy Battaglia1,5, Wouter de Bruijn2, Kerry S Burton6, Michael P Challen7, Pedro M Coutinho8, Daniel C Eastwood9, Birgit S Gruben1,5, Miia R Mäkelä10, Francis Martin3, Marina Nadal5, Joost van den Brink1, Ad Wiebenga1, Miaomiao Zhou1, Bernard Henrissat8, Mirjam Kabel2, Harry Gruppen2 and Ronald P de Vries1,5*

Patyshakuliyeva et al. BMC Genomics 2013, 14:663

http://www.biomedcentral.com/1471-2164/14/663

The most significant information that might be useful in educating about the process of mushroom feeding that was described in this article and that would become an inspiration for further research is presented below.

„Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist

mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies.

The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples.

In nature plant biomass is the main carbon source for many fungal species. A. bisporus (the white button mushroom) is commercially cultivated on a composted mixture of lignocellulose containing materials (mainly wheat straw and horse manure), which is highly selective for this fungus [2,3]. The major constituents of the lignocellulose fraction of compost are cellulose and the hemicellulose xylan (70% of the biomass) [4] and lignin [5-7]. Due to their diverse and complex polymeric nature, degradation of plant cell wall polysaccharides to their monomeric constituent requires a large range of enzymes [8,9]. Most of these enzymes have been divided into families in a classification system for Carbohydrate Active enZymes (CAZy, www.cazy.org) [10]. It has been shown that during mycelial growth and fruiting A. bisporus produces a range of extracellular enzymes, which are involved in the degradation of the lignocellulosic fraction in compost [11-14]. A shift in fungal metabolism takes place during development of the fruiting body of A. bisporus that is closely linked to an increased rate of cellulose and hemicellulose degradation [15]. The production of laccase and cellulase was suggested to be connected to the high rate and flow of carbon metabolism during fruiting body development [16,17]. Lignin degradation by A. bisporus decreases towards the end of the mushroom production cycle [18-20].

Mannitol functions as an osmolyte, which accumulates to high levels during fruiting body growth while after sporulation the level of mannitol decreases rapidly [35]. It might also serve as a post-harvest reserve carbohydrate [31,33,36]. Trehalose also serves as a reserve carbohydrate, which is present at lower levels than mannitol that decline during fruiting body development. It has been suggested that trehalose is synthesized in the mycelium and translocated to the fruiting body [16,32,34].

Expression of most PPP genes is similar in casing, compost and fruiting bodies compared to plate grown mycelium, while only some genes are slightly up- (in compost and casing layer) or down-regulated (in fruiting bodies) (Figure 1, Additional files 3 and 4). There Oxalic acid and citric acid are among the two most commonly produced organic acids by fungi [38]. No specific upregulation for oxalic acid metabolic genes was observed in any of the samples. In contrast, several of the citric acid metabolic genes were expressed at higher levels in fruiting bodies than in compost or the casing layer. These genes are expressed at significantly higher levels in compost than in the other samples. For xylan and cellulose related genes, 90% and 64%, respectively, were expressed in compost while in casing layer and fruiting bodies less than 15% of these genes were expressed. In compost, expression of genes encoding enzymesn targeting other polysaccharides (e.g. starch, pectin and xyloglucan) was also observed.

Fungal cell wall degrading and modifying enzymes have received less attention than plant cell wall degrading enzymes, resulting in a less well defined assignment of function. During growth A. bisporus needs to synthesize and modify its cell wall. As growth occurs in compost, casing layer and fruiting bodies, genes encoding fungal cell wall modifying enzymes need to be expressed in all growth stages. However, as the morphology of these stages is not identical, different genes may be expressed in compost and fruiting bodies. (czy rzeczywiście potrzebne są obumarłe komórki grzybni? Jeżeli tak to dodatek przykładowo drożdży powinien być przydatny) When the A. bisporus mushrooms have matured, compost consists of lignin (41% w/w) and ash (36% w/w), carbohydrates (17% w/w) and proteins (13%).

The casing layer is a mixture of calcium and peat that consists mainly of lignin (52% w/w) and ash (29% w/w). There are few carbohydrates present (14% w/w) and the main monosaccharides released after acid hydrolysis were xylose (1.4% w/w), mannose (0.6% w/w) and glucose (7.5% w/w) [41]. As mentioned above, the actual lignin amount is likely to be lower than measured due to calcium and sandy particles that remain on the filter after acid hydrolysis. Aqueous extraction of compost, casing layer and fruiting bodies revealed that more than 95% of carbohydrates are insoluble.

Changes in free soluble monosaccharides were observed in these samples. Concentrations of arabinose, galactose and xylose were high in compost, while only traces of these monosaccharides were found in casing layer and fruiting bodies (Table 3). High levels of glucose were observed in all samples. Mannitol and trehalose levels were significantly higher in fruiting bodies than in compost and casing layer (Table 3), as were the levels of citric acid (data not shown), while no oxalic acid was detected in the samples. The very high level of sorbitol in the compost samples could suggest a role as a transportable carbon compound from the vegetative mycelium to the fruiting body (Table 3).

Soluble oligosaccharides were detected in the compost, while none were detected in the casing layer or fruiting bodies (Figure 3).

Discussion

In this study, genes encoding carbon metabolic genes  were identified in the genome of A. bisporus and their expression in different growth stages was compared to the available carbohydrates and the expression of genes encoding carbohydrate modifying enzymes.

Analysis of the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome [37] revealed correlation between these genes and composition of carbohydrates.

A large decrease of carbohydrate content and, therefore, polysaccharides was revealed in the compost after growth of A. bisporus and fruiting body production.

Expression of genes encoding other plant polysaccharide degrading enzymes that are not normally associated with compost, e.g. starch, pectin and xyloglucan related genes, was also detected. In nature A. bisporus can grow on various substrates ranging from leaf litter and soil under cypress in coastal California to manured soil, composts of plant debris, and other horticultural and agricultural situations reported in Europe [43]. Growth on these different substrates is likely due to the ability of A. bisporus to produce a wide range of plant polysaccharide degrading enzymes and it may co-express genes aimed at different polysaccharides.

The casing layer serves as an intermediate phase In the casing layer, which is a mixture of peat and lime, it is likely that the detected glucose and mannose at least partially drive from the mycelial cell wall, in the form of glucans and mannoproteins, respectively. While some genes encoding putative plant cell wall degrading enzymes were expressed in the casing layer, the level of up-regulation compared to plate-grown mycelium is much smaller than that in compost. In addition, expression

of some chitinase encoding genes was detected. The casing layer seems to be an intermediate phase in which some genes related to plant biomass degradation are expressed, but also modification of the A. bisporus cell wall is an important process for the conversion to fruiting body morphology. The lack of soluble polysaccharides indicates that the role of the mycelium in the casing layer is mainly to supply carbohydrates to the fruiting body.

This suggests that A. bisporus has specific genes for mycelium development and growth and others for fruiting body formation and modification.

These results support the compositional and morphological differences found between mycelium and fruiting bodies [35]. Expression of different sets of genes encoding fungal cell wall modifying enzymes has also been described for other fungi. Enzymes from families GH5 and CE4 have several described activities, some of which are related to plant cell wall polysaccharides, while others are related to fungal cell wall polysaccharides (www.cazy.org). For some of the enzymes from these families upregulation in compost was observed, while others were upregulated in fruiting bodies.

Expression analysis demonstrated that the pentose catabolic pathway and galacturonic acid pathway were strongly upregulated in compost and moderately upregulated in the casing layer, while they were down regulated in fruiting bodies.

The concentration of mannitol in fruiting bodies was six times higher than in compost. However, expression of mannitol pathway genes was significantly lower in fruiting bodies than in compost, suggesting that Mannitol is synthesized in the vegetative mycelium and transported to the fruiting body. Earlier studies observed that mannitol functions as an osmoregulatory compound and facilitates a continuous influx of water from compost to the fruiting body to support turgor and fruiting body development [58,59]. This would suggest that mannitol is unlikely to be transported by diffusion from the mycelium. Therefore, it should either be transferred by active transport or alternatively, be synthesized in the fruiting body. If the latter is the case, a possible explanation for the observed expression of the genes could be that the encoded enzymes are transported into the fruiting body.

As citric acid is known to have preservative properties against bacteria in food [60], it is tempting to speculate that the accumulation of citric acid in fruiting bodies may also be involved in the defence mechanism of the mushroom against bacteria.

A. bisporus is highly dependent on obtaining carbon from its surroundings. In contrast, the mycorrhizae L. bicolor obtains carbon from its symbiotic partner in the form of sucrose, placing a much lower demand on a versatile carbon metabolism.

Conclusions

The data from our study demonstrates that overall there is a clear correlation between expression of genes related to plant and fungal polysaccharides and the ability of A. bisporus to degrade these polysaccharides.

We see a clear difference in genes expressed within mycelium grown compost and fruiting bodies supporting the hypothesis that different genes are expressed in A. bisporus mycelium and fruiting bodies. This supports previous results that this fungus produces different enzymes during its life cycle [64]. However, it should also be recognized that gene expression is likely to be dynamic and here we have examined it at the time point when first flush was harvested (approximately 34 days after compost was inoculated with spawn.

Moreover, our study demonstrates a clear correlation between the expression of genes encoding plant and fungal cell wall polysaccharides with the composition of carbohydrates in compost, casing layer and fruiting bodies. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium in the compost, but different gene sets were expressed in these samples. In the present study an in silico metabolic reconstruction of the central carbon metabolism in A. bisporus was performed and combined with expression analysis of the relevant genes in different growth stages of A. bisporus. The analysis of metabolic pathways in A. bisporus may provide information about the requirements of carbon source and energy metabolism during commercial growth of A. bisporus. We showed that during growth in compost and casing a much larger variety of carbon sources was used by A. bisporus than during growth on synthetic medium. In contrast, carbon metabolism in fruiting bodies appears to be mainly aimed at hexoses. This could indicate that only these sugars are transported towards the fruiting body from the vegetative mycelium, which implies that carbon transport to the fruiting bodies is a highly regulated and selective process. “

My comments:

1. The casing layer can participate in the process of mushroom feeding in addition to its basic role, which is providing moisture essential for high yields and environment transporting nutrients by rhizomorphic mycelium. The possibility of digestion of polysaccharides existing in a casing layer during its colonization signifies necessity of performing tests with double casing layer and the first conducted test confirms that approach. Forming nutritional sources within casing materials seem to be particularly interesting because of the short transport of nutrients to fruiting bodies and primordia. It creates opportunity of decreasing the amount of substrate in mushroom production and thus might become essential element of cultivation without compost.
2. Information provided in this paper confirms that the main role in mushroom feeding is degradation of polysaccharides into monosaccharides – digestible nutritional elements. The studies concentrate on function of Carbohydrate Active enzymes. The paper is missing information regarding the role of proteins in the feeding process, and their utilization when they are applied within a supplement based on soybean. This justifies the use of corn meal.
3. Attention is also drawn to the possibility of employing starch in the feeding process, the main component of corn kernel.
4. There is interesting data, which says that mushrooms contain enzymes degrading cell walls of fungi, which are present in compost. This fact makes usage of waste products in the feeding process possible, for instance from alcohol fermentation. The performed tests confirm that.
5. The paper provides explanation regarding a yield decrease in the third and fourth flush that is caused by a different set of enzymes being formed after harvesting first flush. It requires reevaluation of the mushroom feeding process from this phase, particularly in the cultivation model without compost and possibilities of increasing yields in the third and following flushes.
6. Mushroom mycelium has the capability of producing a range of enzymes depending on a carbon source. Therefore, the usage of components other than compost, soybeans and corn in the feeding process might have a much broader application.
7. Does introduction of mannitol into a diet make sense? If yes, it raises a question how much and when.
8. The presence of citric acid in the feeding process can be questioned as far as its usefulness in mushroom cultivation is considered. Its addition to the irrigation system might be improving mushroom appearance and also protect against bacterial diseases.
9. Both soluble carbohydrates adsorbed by mushroom and insoluble that require enzyme degradation are present in the compost. In the tested cultivation model without a compost, introduction of monosaccharides into a subtract significantly accelerated colonization of supplement and structural part that supports spawn structure and spawn construction of mycelium

The presented conclusions do not illustrate all benefits of the conducted experiments and it might be valuable to discuss this data once again.

This question constitutes initial changes in the mushroom production technology and indicates necessity of modifications in the paradigm (approach).

The basis for the search for new supplement is the following statement “

“”No other solutions were explored in spite of the fact that in “The Mushroom Cultivation (1988) „ you can find a long list of other products that can fulfil the role of supplements and which come from plants found in Europe such as corn, potato, wheat, sunflower, sugar beet”.

Search for the new supplements began at a mushroom farm where the tests have been carried out. Mushroom production technology based on a feeding process has also been developed and implemented. (Chelkowscy: innovative tray farm Poland), Mushroom Business 060 August 2013). Mushroom production is carried out in four flushes on an area of 14 000 m2 in trays with an average load of 85 kg/m2 of compost phase III. A special room was built for mushroom production during harvesting in 3 and 4 flush.

Over recent years, variable quality of compost combined with protein supplements caused periodically, particularly in summer, a strong term affect. It resulted in very high costs of cooling down overheated compost. As consequences of these happenings protein supplements provided in phase III were omitted within one year.  In the meantime, for a short time a compost producer substituted cereal straw with a maize straw for compost phase III that was provided to a farm.

During the evaluating a provided substrate a very strong outgrowth of corncobs by mushroom mycelium was noticed. This observation inspired the mushroom producer for independent use of maize at his farm.

Based on the noted outcome we can provide answers regarding usefulness of supplements containing soybean meal and eventually replacing them with products of different composition.

The starting point for this consideration is the fact that mushrooms require energy derived from dead, organic matter for their growth and development. Group of enzymes efficiently degrades polysaccharides such as cellulose, hemicellulose, lignin etc. and as recently reported (2013) dead, organic matter present in the environment colonized by mushrooms. This matter is being formed from dead fungi occurring in compost after a conditioning process. There is a lack of information about enzymes assimilating proteins, particularly animal. This situation raises a fundamental question regarding the energy value of soybean HP and maize meal. Post-extracted soybean meal can provide about 9.5 MJ metabolic energy while maize meal about 14 MJ. There is also a great difference in protein content. Soybean meal contains about 46% of protein in dry mass while maize meal only 9%. Supplements including soybean cannot be applied at a dose 0.5% -1, 0% as they can cause thermal shock and overheating that would be difficult to control.  The substrate temperature above 27⁰ C suppresses feeding while above 36⁰ C causes decay and dying of mycelium that is frequently associated with occurrence of green molds and red pepper mites.

Regarding current situation it can be stated that application of supplements reach in protein content results in yield relevant to its energy value and excess of proteins affects development of thermophilic organisms.

Lack of thermal effect can be achieved without providing maize meal treated with formaldehyde. It has additional significance as it was withdrawn from the food production processes.

Moreover, delay in assimilation of feeding components during their enzymatic degradation caused by using formaldehyde restricts the availability of ingredients provided in supplements. This delay is unfavorable due to the short duration of feeding time after introduction of supplements or feeders. Besides, starch, as the main component of soybean meal and low protein content does not favor development of green mold.

One condition needs to be achieved that maize meal will meet the requirements as a feeder for the bottom mushroom.

Summary

The collected yields and compost condition indicate that supplements based on soybean meal can be successfully replaced with feeders containing properly prepared maize meal or other products. It seems that maize meal is balanced the best according to mushroom requirements. Maize is also significantly cheaper. Feeders containing maize meal might be also applied at higher doses and thus results in obtaining higher yields. The maize meal is not only a traditional supplement but also a feeder as it meets all requirements expected from feeders.

2013 Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

Aleksandrina Patyshakuliyeva1†, Edita Jurak2†, Annegret Kohler3, Adam Baker4, Evy Battaglia1,5, Wouter de Bruijn2, Kerry S Burton6, Michael P Challen7, Pedro M Coutinho8, Daniel C Eastwood9, Birgit S Gruben1,5, Miia R Mäkelä10, Francis Martin3, Marina Nadal5, Joost van den Brink1, Ad Wiebenga1, Miaomiao Zhou1, Bernard Henrissat8, Mirjam Kabel2, Harry Gruppen2 and Ronald P de Vries1,5*

Patyshakuliyeva et al. BMC Genomics 2013, 14:663

http://www.biomedcentral.com/1471-2164/14/663